From owner-proteins@net.bio.net Sun Dec 01 22:00:00 1996 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsfeed.internetmci.com!in1.uu.net!158.250.39.26!Gamma.RU!srcc!news.phys.msu.su!mx.nsu.ru!usenet From: Veronika <472svv@cceco.nsu.ru> Newsgroups: bionet.molbio.proteins Subject: Angiogenin Date: Mon, 02 Dec 1996 11:31:31 -0800 Organization: NSU Lines: 78 Message-ID: <32A32C8C.39C8@cceco.nsu.ru> NNTP-Posting-Host: ws2f.cceco.nsu.ru Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win16; I) Scientists, businessmen and simply interested in research and promotion of new bioengineering materials, we can offer you angiogenin that is the result of our last successful research project. 1. The independent private enterprise "Biok" was founded by scientists and businessmen for the purpose of realization and commercial application of the intellectual property of the founders. We would like to offer you the angiogenin substance and biotechnological method for its production. 2.Angiogenin is a member of angiogenic proteins-substances, which stimulates the growth of the blood vessels. Angiogenin is a basic single-chain protein of about 14 kD. It consists of 123 amino acids. In contrast to other angiogenic substances angiogenin has no direct mitogenic effect on fibroblast and endothelial cells, although it binds to endothelial cells of blood vessels. Angiogenin possesses strong angiogenic activity in biological assays ( in choricallantoic membrane of developed chicken and on rabbit corneal ) at dose as low as 1-5 ng. Mechanisms of its actions and physiological roles are being investigated. Nevertheless it's clear now that thanks to its' remarkable effects angiogenin seems to be a good substance for development of efficient drug on its base. It can be used for healing of wounds, for treatment of the cerebral thrombosis and myocardial infarction and other pathological conditions which are caused by neovasoularization disturbances. More over, on the base of the substance new diagnostic kits for detection of angiogenin presence in human tissue and biological fluids may be constructed as well as radioreceptor assay. 3. We have developed the original technology for production of angiogenin. Human recombinant angiogenin obtained is synthesized by original recombinant bacterial strain. It is of high biochemical and immunochemical purity and possesses high biological activity. Substance could be used for research goals. At present the preclinical trials of produced angiogenin is being carried out. 4. Our firm has developed and produced a bioreactor of a new type for suspension cultivation of tissue cells and recombinant bacterial strain. The bioreactor embodies a new principle of stirring a cell suspension without a stirrer. The suspension is agitated by creating quasi-stationary three - dimensional motion similar to a otating vortex ring, generated by the aerating gas flow. We would like to offer you : 1. We can supply angiogenin as biomaterial for your laboratories and other needs. 2. "Biok" is interested in partners for organization joint ventures producing angiogenin substance. 3. "Biok" is interested in partners for organization joint ventures producing bioreactors mentioned above. We are ready to listen to your any idea upon that subject. Sincerely yours, Director of "Biok" Yuri A. Ramazanov Deputy Director of Institute of Chemistry Nikolyai P. Mertvecov From owner-proteins@net.bio.net Sun Dec 01 22:00:00 1996 Path: biosci!agate!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!coopnews.coop.net!boulder.earthnet.net!usenet From: James Stiehr Newsgroups: bionet.molbio.proteins Subject: Re: GR source Date: Mon, 02 Dec 1996 07:57:30 -0700 Organization: Affinity BioReagents, Inc. Lines: 22 Message-ID: <32A2EE5A.5771@bioreagents.com> References: NNTP-Posting-Host: slip9.earthnet.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Win16; I) To: Karl Eisele , reedan@global2000.net Karl Eisele wrote: > > On Sat, 23 Nov 1996, Daniela Sciaky wrote: > > > Is there a company that sells glucocoricoid receptor protein? If you have > > any ideas please send your suggestions to my Email > > address-reedan@global2000.net. Thanks for the help. > > Daniela We can help you out with the recombinant protein, as well as numerous monoclonal and polyclonal antibodies to GR and many other steroid receptors. For more information, please visit our web site (http://www.bioreagents.com/affinity) or call at any of the numbers below. Sincerely, James Stiehr Affinity BioReagents, Inc. jstiehr@bioreagents.com 800-527-4535 303-278-4535 303-278-2424 (Fax) From owner-proteins@net.bio.net Sun Dec 01 22:00:00 1996 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!news.dra.com!news.he.net!news.nacamar.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: How to do NATIVE PAGE? Date: 2 Dec 1996 14:06:43 GMT Organization: University of Wuerzburg, Germany Lines: 31 Message-ID: <57unpj$3cf@winx03.informatik.uni-wuerzburg.de> References: <01bbdee6$beb1fa80$43019386@Schmidt.rz.ruhr-uni-bochum.de> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Thorsten Schmidt (Thorsten.Schmidt@rz.ruhr-uni-bochum.de) wrote: > I want to separate my target protein from other proteins in low > concentration via native PAGE (poly acrylamide gel electrophoresis). > What do I have to think about when planning the experiment? > Where are the differences between native PAGE and SDS-PAGE? Don't heat your protein. Prepare separate loading buffer. Prepare for longer running times. > I heart that one has to calculate the isoelectric point. > But how can I do this with a 300 aa-protein? (the sequence is known) You can calculate the IP with various programs from the amino acid sequence but you won't necessarily get the IP of the native protein in solution. All the IP calculation programs calculate the IP from the IPs of all amino acid side chains of the protein (although I don't know how; I don't think it's the average). However, in the native protein some of the side chains are buried and thereby do not influence the IP. In other words, your securest bet is to try it out. You might also want to look into blue native PAGE where, to my knowledge, the charge of the protein is mainly determined by the amount of Coomassie molecules binding to it. Reference is Schaegger & von Jagow, somewhere in Analytical Biochemistry (I think). I've never done it myself, though. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Sun Dec 01 22:00:00 1996 Path: biosci!agate!howland.erols.net!news-peer.gsl.net!news.gsl.net!news-stkh.gsl.net!news.gsl.net!nntp-oslo.UNINETT.no!nntp-trd.UNINETT.no!nntp.uio.no!news.apfel.de!nntp.zit.th-darmstadt.de!fu-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!news From: Boris Steipe Newsgroups: bionet.molbio.proteins Subject: Re: How to do NATIVE PAGE? Date: Mon, 02 Dec 1996 14:51:09 +0000 Organization: Genzentrum Lines: 12 Distribution: world Message-ID: <32A2EB00.36BD@LMB.uni-muenchen.de> References: <01bbdee6$beb1fa80$43019386@Schmidt.rz.ruhr-uni-bochum.de> Reply-To: steipe@LMB.uni-muenchen.de NNTP-Posting-Host: bs_boris.lmb.uni-muenchen.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Macintosh; I; PPC) Thorsten Schmidt wrote: > I heard that one has to calculate the isoelectric point. > But how can I do this with a 300 aa-protein? (the sequence is known) Use SwissTools at ExPASy - they have a form where you can paste in your sequence and it will return the pI and MW. Greetings, Boris From owner-proteins@net.bio.net Sun Dec 01 22:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!news.bbnplanet.com!su-news-hub1.bbnplanet.com!arclight.uoregon.edu!news.bc.net!torn!resunix.sickkids.on.ca!michael.didonato From: michael.didonato@utoronto.ca (Michael DiDonato) Newsgroups: bionet.molbio.proteins Subject: Re: What is N-terminal blockage? Date: Mon, 02 Dec 1996 15:41:05 -0500 Organization: Hospital For Sick Children Lines: 13 Message-ID: References: <32A252DA.423C@inetnebr.com> NNTP-Posting-Host: 142.20.36.232 Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.4 In article <32A252DA.423C@inetnebr.com>, spanagio@inetnebr.com wrote: > Does anyone know what is N-terminal blockage? Please email answer to: > spanagio@inetnebr.com > Thank you. N-terminal blockage refers to any post-translational modification which leads to the modification of the N-terminus of a protein. Examples are acetylation, methylation, etc... These will interfere with N-terminal sequencing reactions. Mike From owner-proteins@net.bio.net Sun Dec 01 22:00:00 1996 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!insync!Gamma.RU!srcc!news.phys.msu.su!mx.nsu.ru!usenet From: Stanislav <374rsj@cceco.nsu.ru> Newsgroups: bionet.molbio.proteins Subject: Angiogenin Date: Mon, 02 Dec 1996 11:49:24 -0800 Organization: Biok Lines: 54 Message-ID: <32A332C4.39C4@cceco.nsu.ru> NNTP-Posting-Host: ws2f.cceco.nsu.ru Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win16; I) Scientists, businessmen and simply interested in research and promotion of new bioengineering materials, we can offer you angiogenin that is the result of our last successful research project. 1. The independent private enterprise "Biok" was founded by scientists and businessmen for the purpose of realization and commercial application of the intellectual property of the founders. We would like to offer you the angiogenin substance and biotechnological method for its production. 2.Angiogenin is a member of angiogenic proteins-substances, which stimulates the growth of the blood vessels. Angiogenin is a basic single-chain protein of about 14 kD. It consists of 123 amino acids. In contrast to other angiogenic substances angiogenin has no direct mitogenic effect on fibroblast and endothelial cells, although it binds to endothelial cells of blood vessels. Angiogenin possesses trong angiogenic activity in biological assays ( in choricallantoic membrane of developed chicken and on rabbit corneal ) at dose as low as 1-5 ng. Mechanisms of its actions and physiological roles are being investigated. Nevertheless it's clear now that thanks to its' remarkable effects angiogenin seems to be a good substance for development of efficient drug on its base. It can be used for healing of wounds, for treatment of the cerebral thrombosis and myocardial infarction and other pathological conditions which are caused by neovasoularization disturbances. More over, on the base of the substance new diagnostic kits for detection of angiogenin presence in human tissue and biological fluids may be constructed as well as radioreceptor assay. 3. We have developed the original technology for production of angiogenin. Human recombinant angiogenin obtained is synthesized by original recombinant bacterial strain. It is of high biochemical and immunochemical purity and possesses high biological activity. Substance could be used for research goals. At present the preclinical trials of produced angiogenin is being carried out. 4. Our firm has developed and produced a bioreactor of a new type for suspension cultivation of tissue cells and recombinant bacterial strain. The bioreactor embodies a new principle of stirring a cell suspension without a stirrer. The suspension is agitated by creating quasi-stationary three-dimensional motion similar to a otating vortex ring, generated by the aerating gas flow. We would like to offer you : 1. We can supply angiogenin as biomaterial for your laboratories and other needs. 2. "Biok" is interested in partners for organization joint ventures producing angiogenin substance. 3. "Biok" is interested in partners for organization joint ventures producing bioreactors mentioned above. We are ready to listen to your any idea upon that subject. Sincerely yours, Director of "Biok" Yuri A. Ramazanov Deputy Director of Institute of Chemistry Nikolay P. Mervecov P.S. the firs message has the wrong address but this one has correct. correct address is 374rsj@cceco.nsu.ru From owner-proteins@net.bio.net Sun Dec 01 22:00:00 1996 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.erols.net!feed1.news.erols.com!news.enteract.com!news.inetnebr.com!news From: spanagio@inetnebr.com Newsgroups: bionet.molbio.proteins Subject: Re: What is N-terminal blockage? Date: Mon, 02 Dec 1996 03:54:14 +0000 Organization: Internet Nebraska Lines: 3 Message-ID: <32A252DA.423C@inetnebr.com> References: NNTP-Posting-Host: lin-pm1-022.inetnebr.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Macintosh; U; PPC) Does anyone know what is N-terminal blockage? Please email answer to: spanagio@inetnebr.com Thank you. From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!daresbury!lyra.csx.cam.ac.uk!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: bifunctional proteins Date: 3 Dec 1996 16:22:45 GMT Organization: University of Leicester, UK (PCFS User) Lines: 10 Message-ID: <581k4l$ei3@falcon.le.ac.uk> References: <961203104553.2263ea79@XRAY.BMC.UU.SE> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) MOWBRAY@XRAY.BMC.UU.SE wrote: >I would be grateful for any examples of bifunctional proteins (that is, >ones with two active sites of different function), particularly ones for >which structural information is available. Thanks in advance for any help. Check any biochemistry text for Ribulose bisphosphate carboxylase (RUBISCO). This is arguably the most important enzyme with two functions on this planet. This enzyme is responsible for carbon fixation in C3 plants. From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!news.sgi.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!feed1.news.erols.com!news.bconnex.net!clicnet!news.clic.net!rcogate.rco.qc.ca!altitude!usenet From: "Achim Recktenwald, PhD" Newsgroups: bionet.molbio.proteins Subject: Re: How to show protein interaction? Date: Tue, 03 Dec 1996 07:48:20 -0500 Organization: IBEX Technologies, Inc., Biochemistry, 5485 Pare, Montreal, PQ, H4P 1P7, Canada Lines: 23 Message-ID: <32A42194.189E@ibex.ca> References: <01bbdcae$fdd474c0$37019386@Schmidt.rz.ruhr-uni-bochum.de> <57nomf$25d4@uvaix3e1.comp.UVic.CA> NNTP-Posting-Host: ibex.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) D.Lippert wrote: > If you want the simplest test for presence of dimers, I would have to > say non-denaturing PAGE. Both structures (monomer and dimer) should > have the same charge to mass ratio, but the dimer, being larger, will > migrate more slowly through the gel due to the sieving properties of > the acrylamide. This can be done easily in a day without the need for > large complicated equipment. If you want to prove that the bands you > see are dimers and trimers, run a 2D gel and do SDS-PAGE in the second > dimension. All of the bands that you see should migrate the same > distance in the second dimension. > I agree that the non-denaturing gel says nothing about molecular > weight, but it WILL separate a dimer from a monomer. [snip] Use a non-denaturing gradient-gel. If the interaction is strong enough, you'll get discrete bands. otherwise a smear in the range of interaction; e.g., I once worked on an octameric enzyme that disintegrated into tetramers and dimers. The smear in the gel was from the octameric band to the dimer band. Achim From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!XRAY.BMC.UU.SE!MOWBRAY From: MOWBRAY@XRAY.BMC.UU.SE Newsgroups: bionet.molbio.proteins Subject: bifunctional proteins Date: 3 Dec 1996 01:48:18 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <961203104553.2263ea79@XRAY.BMC.UU.SE> NNTP-Posting-Host: net.bio.net I would be grateful for any examples of bifunctional proteins (that is, ones with two active sites of different function), particularly ones for which structural information is available. Thanks in advance for any help. Sherry Mowbray mowbray@xray.bmc.uu.se From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!tulane.edu!rs5.tcs.tulane.edu!not-for-mail From: jayuziuk@rs5.tcs.tulane.edu (Jeffrey Yuziuk) Newsgroups: bionet.molbio.proteins Subject: Re: bifunctional proteins Date: 3 Dec 1996 15:07:09 GMT Organization: Tulane University Lines: 15 Distribution: world Message-ID: <581fmt$b2m@rs10.tcs.tulane.edu> References: <961203104553.2263ea79@XRAY.BMC.UU.SE> NNTP-Posting-Host: rs5.tcs.tulane.edu X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] MOWBRAY@XRAY.BMC.UU.SE wrote: : I would be grateful for any examples of bifunctional proteins (that is, : ones with two active sites of different function), particularly ones for : which structural information is available. Thanks in advance for any help. : Sherry Mowbray : mowbray@xray.bmc.uu.se Dear Sherry, I just attended a seminar yesterday on mRNA capping enzyme, by Dr. Stewart Shuman of Sloan-Kettering Institute. It has a guanidinyl-transferase and a methyl-transferase activity. Hope this helps. Jeff From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!rutgers!uwm.edu!spool.mu.edu!newspump.sol.net!news.mindspring.com!mindspring!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!su-news-hub1.bbnplanet.com!arclight.uoregon.edu!news.uoregon.edu!newsfeed.orst.edu!news.orst.edu!news.cs.indiana.edu!purdue!mozo.cc.purdue.edu!not-for-mail From: barani@deft.cc.purdue.edu (Raman) Newsgroups: bionet.molbio.proteins Subject: re: bifunctional enzymes Date: 3 Dec 1996 11:36:35 -0500 Organization: Purdue University Computing Center Lines: 35 Distribution: world Message-ID: <581kuj$hgc@deft.cc.purdue.edu> NNTP-Posting-Host: deft.cc.purdue.edu -Newsgroups: bionet.molbio.proteins -Subject: Re: bifunctional proteins -Date: 3 Dec 1996 15:07:09 GMT -Organization: Tulane University -Lines: 15 -MOWBRAY@XRAY.BMC.UU.SE wrote: -: I would be grateful for any examples of bifunctional proteins (that is, -: ones with two active sites of different function), particularly ones for -: which structural information is available. Thanks in advance for any help. -: Sherry Mowbray -: mowbray@xray.bmc.uu.se - - -Dear Sherry, - -I just attended a seminar yesterday on mRNA capping enzyme, by Dr. Stewart -Shuman of Sloan-Kettering Institute. It has a guanidinyl-transferase and a -methyl-transferase activity. Hope this helps. - -Jeff - - I believe it would be inappropriate to call dual role enzymes as bifunctional. A bifunctional enzyme mediates a reaction in either forward or backward direction. Enzymes that carry two entirely different active sites and reactions should be termed "di-functional enzymes". Raman From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!torn!resunix.sickkids.on.ca!michael.didonato From: michael.didonato@utoronto.ca (Michael DiDonato) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: HELP! TBS for GST? Date: Tue, 03 Dec 1996 14:29:05 -0500 Organization: Hospital For Sick Children Lines: 14 Message-ID: NNTP-Posting-Host: 142.20.36.242 Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.4 Xref: biosci bionet.molbio.methds-reagnts:52222 bionet.molbio.proteins:9474 I would like to know if anyone has carried out a purification of a GST fusion protein using Tris-buffered saline instead of PBS as the lysis buffer? If so, does the fusion still bind to the column using Tris? At what pH? Any suggestions/hints would be greatly appreciated. Mike -- Michael DiDonato Department of Biochemistry Research Hospital for Sick Children Toronto, Ontario, Canada From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!mr.net!newshub.tc.umn.edu!fu-berlin.de!news.coli.uni-sb.de!news-kar1.dfn.de!news-stu1.dfn.de!news-mue1.dfn.de!news-nue1.dfn.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: HELP! TBS for GST? Followup-To: bionet.molbio.methds-reagnts,bionet.molbio.proteins Date: 3 Dec 1996 22:33:54 GMT Organization: University of Wuerzburg, Germany Lines: 19 Message-ID: <5829si$dnj@winx03.informatik.uni-wuerzburg.de> References: NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Xref: biosci bionet.molbio.methds-reagnts:52230 bionet.molbio.proteins:9477 Michael DiDonato (michael.didonato@utoronto.ca) wrote: > I would like to know if anyone has carried out a purification of a GST > fusion protein using Tris-buffered saline instead of PBS as the lysis > buffer? If so, does the fusion still bind to the column using Tris? At > what pH? Well, I don't call it TBS but I routinely purify GST fusion proteins in 20 mM Tris, 200 mM NaCl, 2 mM DTT, 1 mM EDTA pH 7.6. Cells are lysed in a little less NaCl (100 mM NaCl). High pHs will denature GST. To clean my column I use 100 mM Tris 500 mM NaCl pH 8.5. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!ZOOL.UMD.EDU!goode From: goode@ZOOL.UMD.EDU ("Dennis Goode") Newsgroups: bionet.molbio.proteins Subject: Re: bifunctional proteins Date: 3 Dec 1996 08:56:22 -0800 Organization: University of Maryland Zoology Lines: 33 Sender: daemon@net.bio.net Distribution: world Message-ID: <89F2E81576@zool.umd.edu> NNTP-Posting-Host: net.bio.net MOWBRAY@XRAY.BMC.UU.SE Wrote on the subject: bifunctional proteins: >.I would be grateful for any examples of bifunctional proteins (that is, >ones with two active sites of different function), particularly ones for >which structural information is available. Thanks in advance for any help. >Sherry Mowbray >mowbray@xray.bmc.uu.se Sherry, Motor proteins like myosin and kinesin have two functional sites, an ATP-binding and hydrolysis site and an actin- or tubulin-binding site. Myosin structure has been solved both with and without actin bound. (Rayment et al Sci.261:50). G proteins also have a GTP-hydrolysis site and a site where they bind to and regulate the function of another protein. e.g. Ras has been solved. You also might look at a bifunctional kinase like MAP-kinase-kinase that can phosphorylate MAP kinase at two different sites, a threonine and a tyrosine. That may or may not require two different active sites. -Dennis Dr. M. Dennis Goode Phone (301) 405-6917 Department of Zoology Fax (301) 314-9358 University of Maryland e-mail goode@zool.umd.edu College Park MD 20742 ************************************************************* "If the Lord Almighty had consulted me before embarking upon the creation, I should have recommended something simpler." -Alphonso X of Castile, 15th Century From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!cs.utexas.edu!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsfeed.internetmci.com!mr.net!newshub.tc.umn.edu!fu-berlin.de!news-ber1.dfn.de!news-han1.dfn.de!news-koe1.dfn.de!news.rwth-aachen.de!news.ruhr-uni-bochum.de!news.uni-stuttgart.de!rz.uni-karlsruhe.de!news.fzk.de!usenet From: "David Moss" Newsgroups: bionet.molbio.proteins Subject: Re: bifunctional enzymes Date: 3 Dec 1996 21:17:07 GMT Organization: Forschungszentrum Karlsruhe Lines: 35 Distribution: world Message-ID: <01bbe157$58753020$5e09348d@ifiabio1> References: <581kuj$hgc@deft.cc.purdue.edu> NNTP-Posting-Host: ifiabio1.fzk.de X-Newsreader: Microsoft Internet News 4.70.1157 Raman schrieb im Beitrag <581kuj$hgc@deft.cc.purdue.edu>... > I believe it would be inappropriate to call dual role enzymes as > bifunctional. A bifunctional enzyme mediates a reaction in either > forward or backward direction. > > Enzymes that carry two entirely different active sites and reactions > should be termed "di-functional enzymes". Er... I think you're missing an important and elementary point of physical chemistry. An enzyme can't possibly change the equilibrium constant of the reaction it catalyzes, otherwise it would be breaking all of the laws of thermodynamics that I ever heard of. And since the equilibrium constant is the ratio of the rate constants for the forward and reverse reactions, it follows that an enzyme can't increase the forward reaction rate constant without a proportionate increase in the back reaction rate constant. Therefore all enzymes MUST mediate the reaction in both forward and backward directions. You provide the definition of "bifunctional enzyme" with an air of authority. Where did you get this definition from? If it's out of a textbook please tell me which one, so that I can extort lots of money from the publishers as the price for my silence. Hope that helps, David Moss From owner-proteins@net.bio.net Mon Dec 02 22:00:00 1996 Path: biosci!bmg.bhs.uab.edu!BMOORE From: BMOORE@bmg.bhs.uab.edu ("bryan") Newsgroups: bionet.molbio.proteins Subject: I need protein suggestions! Date: 3 Dec 1996 13:27:07 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <4AC32084B02@bmg.bhs.uab.edu> Reply-To: bryan NNTP-Posting-Host: net.bio.net I am looking for several proteins that meet most of the following: 1. Globular and between 10-60 KD 2. cDNA is available 3. One terminus is on the external of the folded protein, that is that it can attach to a linker peptide and not disrupt the native structure. 4. Significantly stable 5. To fulfill all my wishes it would be nice to have the crystal structure for it also. The protein does not need to have any enzymatic activity it simply needs to be able to be attached to a linker (possibly serine/alanine) and fold in it native structure. Any suggestions would be helpful. Thank You! Bryan Moore Department of Biochemistry and Molecular Genetics University of Alabama at Birmingham From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!su-news-hub1.bbnplanet.com!news.Stanford.EDU!not-for-mail From: ejohnson@leland.Stanford.EDU (Eric Ramon Johnson) Newsgroups: bionet.molbio.proteins Subject: Re: bifunctional proteins Date: 4 Dec 1996 12:59:45 -0800 Organization: Stanford University, CA 94305, USA Lines: 10 Message-ID: <584oo1$mti@cardinal1.Stanford.EDU> References: <961203104553.2263ea79@XRAY.BMC.UU.SE> NNTP-Posting-Host: cardinal1.stanford.edu In article <961203104553.2263ea79@XRAY.BMC.UU.SE>, wrote: >I would be grateful for any examples of bifunctional proteins (that is, >ones with two active sites of different function), particularly ones for >which structural information is available. Thanks in advance for any help. >Sherry Mowbray >mowbray@xray.bmc.uu.se How about chorismate mutase / prephenate dehydrogenase ?? From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!bmg.bhs.uab.edu!GMEACHAM From: GMEACHAM@bmg.bhs.uab.edu Newsgroups: bionet.molbio.proteins Subject: Re: I need protein suggestions! Date: 4 Dec 1996 11:53:29 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <4C2A33C5C4E@bmg.bhs.uab.edu> NNTP-Posting-Host: net.bio.net Date: 3 Dec 1996 13:27:07 -0800 I am looking for several proteins that meet most of the following: 1. Globular and between 10-60 KD 2. cDNA is available 3. One terminus is on the external of the folded protein, that is that it can attach to a linker peptide and not disrupt the native structure. 4. Significantly stable 5. To fulfill all my wishes it would be nice to have the crystal structure for it also. The protein does not need to have any enzymatic activity it simply needs to be able to be attached to a linker (possibly serine/alanine) and fold in it native structure. Any suggestions would be helpful. Thank You! Bryan Moore Department of Biochemistry and Molecular Genetics University of Alabama at Birmingham If you want a marker or a fusion, try GFP. MW is 22 kD. Clontech has cDNA.It's used to make fusion proteins frequently. It is rock solid (stable in 6M Urea). Ab's are available. The crystal was in Science <2 months ago. Even better, go upstairs two floors and ask somebody in room 643.It'll cost you a dollar.If it works the price jumps to five bucks. G.Meacham Univ. of Alabama at Birm. Dept. of Cell Biology From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!EU.net!Norway.EU.net!nntp.uio.no!nntp.uib.no!nntp-bergen.UNINETT.no!nntp-trd.UNINETT.no!daresbury!is.bbsrc.ac.uk!news From: Andy Phillips Newsgroups: bionet.molbio.proteins Subject: Re: bifunctional proteins Date: Wed, 04 Dec 1996 15:08:38 +0000 Organization: BBSRC, Institute of Arable Crops Research, LARS Lines: 26 Message-ID: <32A593F6.1BF0@bbsrc.ac.uk> References: <961203104553.2263ea79@XRAY.BMC.UU.SE> <581k4l$ei3@falcon.le.ac.uk> NNTP-Posting-Host: pc0628.lars.bbsrc.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02Gold (Win95; I) CC: andy.phillips@bbsrc.ac.uk Dr E. Buxbaum wrote: > > MOWBRAY@XRAY.BMC.UU.SE wrote: > >I would be grateful for any examples of bifunctional proteins (that is, > >ones with two active sites of different function), particularly ones for > >which structural information is available. Thanks in advance for any help. > > Check any biochemistry text for Ribulose bisphosphate carboxylase > (RUBISCO). This is arguably the most important enzyme with two functions > on this planet. This enzyme is responsible for carbon fixation in C3 > plants. Err, yes, except that the two functions of Rubisco (that is carboxylation and oxidation of ribulose 1,5-bisphosphate) are carried out at the same active site (see question) and the gaseous substrates (CO2 & O2) compete for the sugar phosphate substrate. Oh, and Rubisco also fixes CO2 in C4 plants, not just C3. Andy ------------------------------------------------------------------------ Email : andy.phillips@bbsrc.ac.uk : University of Bristol Home : andy@cycad.demon.co.uk : IACR Long Ashton Research Station Phone : +44-1275-549257 : Long Ashton Fax : +44-1275-394281 : Bristol, BS18 9AF, UK WWW : http://www.lars.bbsrc.ac.uk/plantsci/molbiol/andy.html ------------------------------------------------------------------------ From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!news2.digex.net!news5.digex.net!haven.umd.edu!purdue!mozo.cc.purdue.edu!not-for-mail From: barani@deft.cc.purdue.edu (Raman) Newsgroups: bionet.molbio.proteins Subject: re: bifunctional enzymes Date: 4 Dec 1996 22:46:57 -0500 Organization: Purdue University Computing Center Lines: 13 Distribution: world Message-ID: <585gjh$f4e@deft.cc.purdue.edu> NNTP-Posting-Host: deft.cc.purdue.edu The Enzyme Tryptophan Synthase is an Outstanding example which performs more than one activity in more than one site. Its E.C.Code is 4.2.1.20 I also recall that its molecular structure has been solved but don't remember where it was published. -Raman From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!news-peer.gsl.net!news.gsl.net!news.sprintlink.net!news-peer.sprintlink.net!EU.net!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!maze.dpo.uab.edu!bmg146.cmc.uab.edu!user From: siyer@bmg.bhs.uab.edu (Sai) Newsgroups: bionet.molbio.proteins Subject: Re: Membrane Protein Folding Date: Wed, 04 Dec 1996 22:21:06 -0500 Organization: Biochem& Molec.genetics, UAB Lines: 26 Distribution: world Message-ID: References: <4C25E51199E@bmg.bhs.uab.edu> NNTP-Posting-Host: bmg146.cmc.uab.edu In article <4C25E51199E@bmg.bhs.uab.edu>, GMEACHAM@bmg.bhs.uab.edu wrote: > Does anyone have a reference on folding of cytostolic domains of > membrane proteins? References could include biochemical,biophysical, or > genetic techniques with no preference for substrates.Thanks in advance. > > > G.Meacham > Univ. of Alabama at Birmingham > Dept. of Cell Biology hey dude, i just got a recent paper on crystallization of membrane proteins...while it may not have much info on folding of the cytosolic domains, the references it has might be able to help you...the ref. is in "journal of bioenergetics and biomembranes", vol. 28, #1 (1996) and the article is "introduction:crystallization of membrane proteins- in need of a new focus?". you can either trek down here or go to lister hill...cheers... -- Sai Iyer siyer@bmg.bhs.uab.edu graduate student; dept of biochemistry and molecular genetics university of alabama at birmingham From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!bmg.bhs.uab.edu!GMEACHAM From: GMEACHAM@bmg.bhs.uab.edu Newsgroups: bionet.molbio.proteins Subject: Membrane Protein Folding Date: 4 Dec 1996 11:37:09 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <4C25E51199E@bmg.bhs.uab.edu> NNTP-Posting-Host: net.bio.net Does anyone have a reference on folding of cytostolic domains of membrane proteins? References could include biochemical,biophysical, or genetic techniques with no preference for substrates.Thanks in advance. G.Meacham Univ. of Alabama at Birmingham Dept. of Cell Biology From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!MANI.CBS.UMN.EDU!npv From: npv@MANI.CBS.UMN.EDU (Nora Plesofsky-Vig) Newsgroups: bionet.molbio.proteins Subject: re:bifunctional proteins Date: 4 Dec 1996 09:23:35 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <9612041723.AA01096@mani.cbs.umn.edu> Reply-To: nora@biosci.cbs.umn.edu NNTP-Posting-Host: net.bio.net Members of the AAA proteases (ATP-dependent metallopeptidase) in the inner mitochondrial membrane, Yta10p and Yta12p, function as proteases and also have chaperone-like activity for assembly of the ATP synthase complex. Mutation of these proteins to make them proteolytically inactive does not affect their chaperone-like activity (H. Arlt et al., Cell 85, 1996 875-885). Hope this helps. Nora Plesofsky-Vig From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!news.uh.edu!news.uth.tmc.edu!bmb155.med.uth.tmc.edu!user From: tliang@utmmg.med.uth.tmc.edu (T. Chyau Liang) Newsgroups: bionet.molbio.proteins Subject: Re: I need protein suggestions! Date: 4 Dec 1996 15:33:28 GMT Organization: U. Texas-Houston Lines: 53 Distribution: world Message-ID: References: <4AC32084B02@bmg.bhs.uab.edu> NNTP-Posting-Host: bmb155.med.uth.tmc.edu Mime-Version: 1.0 Content-Type: text/plain;charset=US-ASCII Content-Transfer-Encoding: 7bit In article <4AC32084B02@bmg.bhs.uab.edu>, bryan wrote: > I am looking for several proteins that meet most of the following: > 1. Globular and between 10-60 KD > 2. cDNA is available > 3. One terminus is on the external of the folded protein, that is that > it can attach to a linker peptide and not disrupt the native structure. > 4. Significantly stable > 5. To fulfill all my wishes it would be nice to have the crystal > structure for it also. > > The protein does not need to have any enzymatic activity it simply needs > to be able to be attached to a linker (possibly serine/alanine) and fold > in it native structure. > > Any suggestions would be helpful. Thank You! > Bryan Moore > Department of Biochemistry and Molecular Genetics > University of Alabama at Birmingham I think RNase A fulfills your criteria: 1. It is more or less globular and around 14 kDa (124 aa). 2. cDNA of RNase A should b available. 3. Its N-terminus is well exposed. In fact, the N-terminal fragment (S-peptide) can be cleaved by subtilisin and separated and put back on. Many N-terminal substitutions have been tried on S-peptide; most retain the ability to reassociate with the remainder of the protein and reconstitue activity. 4. Ask any molecular biologist. They will attest to the fact that RNase is TOUGH! 5. The X-ray and NMR structures are available. Added bonus (I know you probably don't care): 1. Activity measurement could be a handy means to monitor its structure/function. 2. Many mutants have been created for folding studies; i.e., losts of thermodynamic information. 3 Since S-peptide can be separated and put back with the remainder of the protein (S-protein), it can be handy as affinity ligand (much like biotin and avidin). 4. S-peptide or its shortened variant, C-peptide, can be conveniently synthesized (chemically). Think of all the possible biophysical studies... ;-) Hope this is helpful. T. Chyau Liang U Texas-Houston From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!www.nntp.primenet.com!nntp.primenet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsfeed.internetmci.com!csn!nntp-xfer-1.csn.net!csn!nntp-xfer-2.csn.net!yuma!purdue!mozo.cc.purdue.edu!not-for-mail From: barani@deft.cc.purdue.edu (Raman) Newsgroups: bionet.molbio.proteins Subject: re bifunctional enzymes Date: 4 Dec 1996 14:49:34 -0500 Organization: Purdue University Computing Center Lines: 46 Distribution: world Message-ID: <584kke$npo@deft.cc.purdue.edu> NNTP-Posting-Host: deft.cc.purdue.edu >From: "David Moss" >Subject: Re: bifunctional enzymes >Date: 3 Dec 1996 21:17:07 GMT > >Er... I think you're missing an important and elementary point of physical chemistry. An >enzyme can't possibly change the equilibrium constant of the reaction it catalyzes, otherwise it >would be breaking all of the laws of thermodynamics that I ever heard of. And since the >equilibrium constant is the ratio of the rate constants for the forward and reverse reactions, >it follows that an enzyme can't increase the forward reaction rate constant without a >proportionate increase in the back reaction rate constant. Therefore all enzymes MUST >mediate the reaction in both forward and backward directions. The forward and reverse reactions are controlled by concentration gradients, temperature, pH and other factors. While the equilibrium is the same, the reactions can proceed either in the forward or backward directions. Some enzymes are uni-directional (for all practical purposes): for example, DNA polymerase will only extend the DNA. It is very hard to detect any breaking of DNA by the polymerase. The term 'bifunctional' is used normally for enzymes which have TWO active sites and perform Two independent reactions. Enzymes with a single active site but two distinct reactions, or enzymes with single active site and show measureable reactions proceed in either forward or backward directions fall in the border line. No distinction has been made among them and different people call them differently. It may help to make such distinctions in future, along the lines similar to 'divalence' or 'bivalence' etc even if it requires renaming old conventions. >You provide the definition of "bifunctional enzyme" with an air of authority. Where did you >get this definition from? If it's out of a textbook please tell me which one, so that I can extort >lots of money from the publishers as the price for my silence. > >Hope that helps, >David Moss Newsgroups are the place where new ideas and opinions are discussed. If your interests are in EXTORTING lots of money, pl leave me out of your customer list! Thank You! -Raman From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!cs.utexas.edu!news.sprintlink.net!news-peer.sprintlink.net!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.idt.net!enews.sgi.com!news.sgi.com!news.msfc.nasa.gov!info.uah.edu!maze.dpo.uab.edu!bmg146.cmc.uab.edu!user From: siyer@bmg.bhs.uab.edu (Sai) Newsgroups: bionet.molbio.proteins Subject: Re: I need protein suggestions! Date: Wed, 04 Dec 1996 22:14:45 -0500 Organization: Biochem& Molec.genetics, UAB Lines: 26 Distribution: world Message-ID: References: <4C2A33C5C4E@bmg.bhs.uab.edu> NNTP-Posting-Host: bmg146.cmc.uab.edu In article <4C2A33C5C4E@bmg.bhs.uab.edu>, GMEACHAM@bmg.bhs.uab.edu wrote: > > > > > If you want a marker or a fusion, try GFP. MW is 22 kD. Clontech has > cDNA.It's used to make fusion proteins frequently. It is rock solid > (stable in 6M Urea). Ab's are available. The crystal was in Science <2 > months ago. Even better, go upstairs two floors and ask somebody in room > 643.It'll cost you a dollar.If it works the price jumps to five bucks. > > G.Meacham > Univ. of Alabama at Birm. > Dept. of Cell Biology hey, we use GFP stuff in our lab. so even better than G-off's advice would be to shlep down the hall and talk to us...we also undercut quite shamelessly...:) cheers... -- Sai Iyer siyer@bmg.bhs.uab.edu graduate student; dept of biochemistry and molecular genetics university of alabama at birmingham From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!cs.utexas.edu!howland.erols.net!newsfeed.internetmci.com!csn!nntp-xfer-1.csn.net!magnus.acs.ohio-state.edu!medmicro1.med.ohio-state.edu!pollack.1 From: pollack.1@osu.edu (J. Dennis Pollack) Newsgroups: bionet.molbio.proteins Subject: lysis of ecoli tansformants for enzy assay Date: Wed, 4 Dec 1996 20:26:21 GMT Organization: The Ohio State University Lines: 5 Message-ID: NNTP-Posting-Host: medmicro1.med.ohio-state.edu X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #4] Help! I want to lyse ecoli transformants (plasmid) in order to assay for enzymes. I need a technique that will not denature prots. I suspect lysozyme will work but what is the exact protocol. Maniatis and Current Protocols were not completly descriptive. I have hundreds to do, I'd rather not sonicate post lysozyme. Much thanks! Dennis From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Newsgroups: bionet.molbio.proteins Path: biosci!ihnp4.ucsd.edu!swrinde!news-peer.gsl.net!news.gsl.net!newsfeed.internetmci.com!news1.bellglobal.com!freenet-news.carleton.ca!cunews!nott!utnut!oci!at!fhoedem From: Flip Hoedemaeker Subject: Nickel columns and Arginine X-Sender: fhoedem@at Content-Type: TEXT/PLAIN; charset=US-ASCII Message-ID: Sender: news@oci.utoronto.ca Organization: Ontario Cancer Institute - U of Toronto Mime-Version: 1.0 Date: Tue, 3 Dec 1996 23:07:34 GMT Lines: 18 Today I tried to purify a protein on a nickel column in the presence of 1.5 M Arginine. I did not expect any problems, since Nickel purification works well in the presence of 6M Guanidine (= the side chain of Arg). Surprisingly, the arginine buffer seemed to strip off the nickel completely. I could recharge the column afterwards in 100 mM NiSO4. Has anybody seen this before? Could it be that Arg in combination with low amounts of imidazol (20 mM) is to blame? Flip ---------------------------------------------------------------------- | Flip Hoedemaeker * Tel: (416) 946-2000 EXT 5053 | | Ontario Cancer Institute | | 610 University Avenue * Fax: (416) 946-6529 | | Toronto Ont. | | Canada M5G 2M9 * Email: fhoedem@oci.utoronto.ca | ---------------------------------------------------------------------- From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!bunyip.cc.uq.oz.au!not-for-mail From: B.Morrison@botany.uq.edu.au (Belinda Morrison) Newsgroups: bionet.molbio.proteins Subject: radiolabelled DCMU Date: 5 Dec 1996 02:42:08 GMT Organization: Dept of Botany, Uni of Queensland Lines: 13 Message-ID: <585cq0$mkd$1@nargun.cc.uq.oz.au> NNTP-Posting-Host: biochem1.botany.uq.edu.au Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.6 Could someone please tell me where I could obtain some 14C-labelled DCMU or atrazine? Thanks in advance, Belinda Morrison. Botany Department University of Queensland AUSTRALIA B.Morrison@botany.uq.edu.au fax.: +61 7 3365 1699 From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!uwm.edu!newsspool.doit.wisc.edu!wiscnews.wiscnet.net!news From: J Potter Newsgroups: bionet.molbio.proteins Subject: Density gradient centrifugation Date: Wed, 04 Dec 1996 17:27:31 -0800 Organization: Medical College of Wisconsin Lines: 17 Message-ID: <32A62503.72FB@post.its.mcw.edu> NNTP-Posting-Host: d350-1.biochem.mcw.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win16; I) Dear Netters, I have an enzyme for which preliminary data suggests that it forms or associates with higher order structures. I have measured mws by gel filtration from 100 kDa (believed to be the subunit mw) to > 1 MDa (basically in the void volume of the column, a Superose 12). I am interested in density gradient centrifugation to separate the various sizes which contain activity...HOWEVER, I cannot use the standard glycerol gradient because glycerol inhibits the enzyme. I am looking for alternative gradients of sucrose or ? to separate around the mw range of 100 kDa - 26 S. I don't know if that broad range of resolution is possible, but any help would be greatly appreciated. Thanks in advance, Jennifer Potter jras@post.its.mcw.edu From owner-proteins@net.bio.net Tue Dec 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!spool.mu.edu!newshub.tc.umn.edu!fu-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!news From: Boris Steipe Newsgroups: bionet.molbio.proteins Subject: Re: Nickel columns and Arginine Date: Wed, 04 Dec 1996 13:13:24 +0000 Organization: Genzentrum Lines: 28 Distribution: world Message-ID: <32A57637.2F17@LMB.uni-muenchen.de> References: Reply-To: steipe@LMB.uni-muenchen.de NNTP-Posting-Host: bs_boris.lmb.uni-muenchen.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Macintosh; I; PPC) Flip Hoedemaeker wrote: > > Today I tried to purify a protein on a nickel column in the presence of > 1.5 M Arginine. I did not expect any problems, since Nickel purification > works well in the presence of 6M Guanidine (= the side chain of Arg). > Surprisingly, the arginine buffer seemed to strip off the nickel completely. > I could recharge the column afterwards in 100 mM NiSO4. That's an interesting observation. Assuming you are using the Quiagen material (NTA), the Log stability constant for the Ni complex is 11.5 . Arginines Log stability constant for Ni is 5.2; and 9.5 (!) for the 2:1 complex. So, indeed, at 1.5 M Arg you are stripping your column. It is the combination of NH and carboxylate which gives many aminoacids quite significant chelating properties. (Source: Data for Biocehmical Research, Dawson et al. eds., Clarendon, Oxford 1986) Best wishes, Boris -- +---Dr. Boris Steipe---------------+ | Genzentrum | | Feodor-Lynen Str. 25 Tel +49 (0)89 74017-417 | | 81377 Muenchen, Germany Fax - 448 | +------+ From owner-proteins@net.bio.net Wed Dec 04 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!agate!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!uunet!in3.uu.net!151.99.250.2!server-b.cs.interbusiness.it!usenet From: celli@cmns.mnegri.it (Nicola Celli) Newsgroups: bionet.molbio.proteins Subject: NB4 expression library Date: 5 Dec 1996 11:50:15 GMT Organization: Consorzio Mario Negri Sud Lines: 19 Sender: -Not-Authenticated-[8336] Message-ID: <586ctn$ep6@server-b.cs.interbusiness.it> NNTP-Posting-Host: 195.120.213.76 X-Posted-From: InterNews 1.0.5@envir01.cmns.mnegri.it Xdisclaimer: No attempt was made to authenticate the sender's name. Hi. We are working with NB4 cells (promyelocytic leukemia) and looking for a new gene. Now we would like to screen a NB4 expression library with antibodies obtained in our laboratory. It's obvious that should be easier (and faster !) for us to use a premade library than to make it up by ourself. Does anybody possess this library or knows if is commercially available ? Thank you very much for your attention. Ciao. Nicola Celli "G. Paone" Enviromental Healt Center" Consorzio Mario Negri Sud 66030 S. Maria Imbaro (CH) ITALY e-mail: celli@cmns.mnegri.it From owner-proteins@net.bio.net Wed Dec 04 22:00:00 1996 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!bonaire.ccit.arizona.edu!hes From: Harold E Smith Newsgroups: bionet.molbio.proteins Subject: Re: How to show protein interaction? Date: Thu, 5 Dec 1996 21:52:37 -0700 Organization: The University of Arizona Lines: 42 Message-ID: References: <01bbdcae$fdd474c0$37019386@Schmidt.rz.ruhr-uni-bochum.de> <57jkl9$c6d@winx03.informatik.uni-wuerzburg.de> <329D8B82.5439@ibex.ca> NNTP-Posting-Host: bonaire.ccit.arizona.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: hes@bonaire.ccit.arizona.edu In-Reply-To: <329D8B82.5439@ibex.ca> On Thu, 28 Nov 1996, Achim Recktenwald, PhD wrote: > Cornelius Krasel wrote: > > > > Thorsten Schmidt (Thorsten.Schmidt@rz.ruhr-uni-bochum.de) wrote: > > > I have a protein and I just want to test if it forms dimers, trimers etc.? > > > (The protein was purified by HisTag-Chromatographie and is in solution > > > (20mM Tris, 0,5 M NaCl, 1 M Imidazol)) > > > What is the simplest way to do this? > > > > I'd try analytical gel filtration if you have the equipment (i.e. an > > FPLC/HPLC). Alternatively, you may try analytical ultracentrifugation > > (I have absolutely no experience with this) or maybe non-denaturing > > gel electrophoresis. > > > > --Cornelius. > > > > -- > > /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ > > /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ > > /* "Science is the game we play with God to find out what His rules are." */ > > > I would also try native polyacrylamide-gel-electrophoresis. If the bond > between the subunits is strong, you'll get one single band, the multimer > form; if its very weak, you'll only see a sharp band at the mol. weight > for the single subunit. But if the bond is neither nor than you get a > smear on your gel, starting at the point of the highest multimer down to > the lowest multimer, or even the single subunit. In the latter case the > multimer form is constantly decomposing during the electrophoresis. > > Achim > > There are a few problems with native PAGE. First, the protein generally migrates at a position far from its predicted molecular weight. Second, this aberrant migration prevents easy determination of the number of subunits within the multimer. Accurate assessment the true molecular weight/number of subunits requires multiple gels of varying acrylamide concentrations. -Harold Smith From owner-proteins@net.bio.net Wed Dec 04 22:00:00 1996 Path: biosci!rutgers!uwm.edu!www.nntp.primenet.com!nntp.primenet.com!enews.sgi.com!news.sgi.com!sdd.hp.com!hamblin.math.byu.edu!news.Arizona.EDU!bonaire.ccit.arizona.edu!hes From: Harold E Smith Newsgroups: bionet.molbio.proteins Subject: Re: lysis of ecoli tansformants for enzy assay Date: Thu, 5 Dec 1996 22:09:57 -0700 Organization: The University of Arizona Lines: 16 Message-ID: References: NNTP-Posting-Host: bonaire.ccit.arizona.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: hes@bonaire.ccit.arizona.edu In-Reply-To: On Wed, 4 Dec 1996, J. Dennis Pollack wrote: > Help! I want to lyse ecoli transformants (plasmid) in order to assay for > enzymes. I need a technique that will not denature prots. I suspect lysozyme > will work but what is the exact protocol. Maniatis and Current Protocols were > not completly descriptive. I have hundreds to do, I'd rather not sonicate > post lysozyme. Much thanks! Dennis > > The quickest way to lyse hundreds might be to cotransform them with a plasmid that expresses the T7 lysozyme gene. A freeze/thaw cycle or addition of 0.1% Triton X-100 will efficiently lyse the cells. Novagen carries a pLysS plasmid that provides choramphenicol resistance and has no effect on cell growth/plasmid maintenance. Hope this helps. -Harold Smith From owner-proteins@net.bio.net Wed Dec 04 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!www.nntp.primenet.com!nntp.primenet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!portc02.blue.aol.com!newsfeed.pitt.edu!nntp.club.cc.cmu.edu!casaba.srv.cs.cmu.edu!das-news2.harvard.edu!oitnews.harvard.edu!purdue!mozo.cc.purdue.edu!not-for-mail From: barani@deft.cc.purdue.edu (Raman) Newsgroups: bionet.molbio.proteins Subject: re: bifunctional enzymes Date: 5 Dec 1996 14:00:40 -0500 Organization: Purdue University Computing Center Lines: 34 Distribution: world Message-ID: <58764o$f60@deft.cc.purdue.edu> NNTP-Posting-Host: deft.cc.purdue.edu :From: jayuziuk@rs5.tcs.tulane.edu (Jeffrey Yuziuk) : :Raman (barani@deft.cc.purdue.edu) wrote: :(snip, snip) :: The term 'bifunctional' is used normally for enzymes which have TWO :: active sites and perform Two independent reactions. Enzymes with :: a single active site but two distinct reactions, or enzymes with single :: active site and show measureable reactions proceed in either forward :: or backward directions fall in the border line. No distinction has been :: made among them and different people call them differently. :: :Raman Dude! : :Take a chill pill! Can't you recognize sarcasm? (And if you check your :earlier post, it appears to me like you've changed your definition of :"bi-functional" already.) : :Jeff Sorry, you are incorrect. My original reference covers what I believe to correctly represent bifuncational enzymes. My latter reference is about what is being used today and I have indicated that I do not agree with it. You have conveniently *snipped* it. I have repeated it for your memory: {It may help to make such distinctions in future, along the lines similar to 'divalence' or 'bivalence' etc even if it requires renaming old conventions. } You could also try a chill-pill and appreciate sarcasm in my response. -Raman From owner-proteins@net.bio.net Wed Dec 04 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!feed1.news.erols.com!news.idt.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!tulane.edu!rs5.tcs.tulane.edu!not-for-mail From: jayuziuk@rs5.tcs.tulane.edu (Jeffrey Yuziuk) Newsgroups: bionet.molbio.proteins Subject: Re: re bifunctional enzymes Date: 5 Dec 1996 16:25:31 GMT Organization: Tulane University Lines: 35 Distribution: world Message-ID: <586t1r$mec@rs10.tcs.tulane.edu> References: <584kke$npo@deft.cc.purdue.edu> NNTP-Posting-Host: rs5.tcs.tulane.edu X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Raman (barani@deft.cc.purdue.edu) wrote: (snip, snip) : The term 'bifunctional' is used normally for enzymes which have TWO : active sites and perform Two independent reactions. Enzymes with : a single active site but two distinct reactions, or enzymes with single : active site and show measureable reactions proceed in either forward : or backward directions fall in the border line. No distinction has been : made among them and different people call them differently. : : : >You provide the definition of "bifunctional enzyme" with an air of authority. Where did you : >get this definition from? If it's out of a textbook please tell me which one, so that I can extort : >lots of money from the publishers as the price for my silence. : > : >Hope that helps, : >David Moss : : Newsgroups are the place where new ideas and opinions are discussed. : : If your interests are in EXTORTING lots of money, pl leave me out of : your customer list! : : Thank You! : : -Raman : Raman Dude! Take a chill pill! Can't you recognize sarcasm? (And if you check your earlier post, it appears to me like you've changed your definition of "bi-functional" already.) Jeff From owner-proteins@net.bio.net Thu Dec 05 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!panix!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!aars!not-for-mail From: lluis@aars.mit.edu (Lluis Ribas) Newsgroups: bionet.molbio.proteins Subject: Re: How to show protein interaction? Date: 6 Dec 1996 20:26:35 GMT Organization: Massachvsetts Institvte of Technology Lines: 48 Message-ID: <589vhr$8gb@senator-bedfellow.MIT.EDU> References: <01bbdcae$fdd474c0$37019386@Schmidt.rz.ruhr-uni-bochum.de> <57jkl9$c6d@winx03.informatik.uni-wuerzburg.de> <329D8B82.5439@ibex.ca> <329EADA4.7A74@bbsrc.ac.uk> NNTP-Posting-Host: aars.mit.edu X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Andy Phillips (andy.phillips@bbsrc.ac.uk) wrote: : Achim Recktenwald, PhD wrote: : : > > Thorsten Schmidt (Thorsten.Schmidt@rz.ruhr-uni-bochum.de) wrote: : > > > I have a protein and I just want to test if it forms dimers, trimers etc.? : > > > (The protein was purified by HisTag-Chromatographie and is in solution : > > > (20mM Tris, 0,5 M NaCl, 1 M Imidazol)) : > > > What is the simplest way to do this? : : > I would also try native polyacrylamide-gel-electrophoresis. If the bond : > between the subunits is strong, you'll get one single band, the multimer : > form; if its very weak, you'll only see a sharp band at the mol. weight : > for the single subunit. But if the bond is neither nor than you get a : > smear on your gel, starting at the point of the highest multimer down to : > the lowest multimer, or even the single subunit. In the latter case the : > multimer form is constantly decomposing during the electrophoresis. : > : > Achim : : Non-denaturing PAGE is not to be trusted for estimation of molecular weights: in : electrophoresis, the rate at which proteins move is in inverse relationship to : their mol. weight, because the bound SDS makes all proteins have roughly the same : charge/mass ratio. In the absence of SDS, the charge on the protein is a function : of its pI, so two proteins with the same mass but different pIs will move at : different rates: the protein with low pI have a higher net negative charge than : that with a high pI, and so the former will move faster in the gel. As an extreme : example, cytochrome c (pI ~10) in a non-denaturing PAGE gel at pH8 will move : towards the cathode! : A good way to avoid all these problems is by chemically cross-linking you protein solution. THen you can run your sample in a denaturing gel and use normal molecular weight standarts. You'll find many cross-linking protocols normally available protein chemistry books. Good luck, Lluis . ps. Analytical centrifugation and/or gel filtration are easier options, however. : Andy From owner-proteins@net.bio.net Thu Dec 05 22:00:00 1996 Path: biosci!CS.SANDIA.GOV!scistra From: scistra@CS.SANDIA.GOV (Sorin C. Istrail) Newsgroups: bionet.molbio.proteins Subject: RECOMB 97: Final Program, Stud Financial Support, Babysitting, Travel Date: 6 Dec 1996 19:19:37 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 496 Sender: daemon@net.bio.net Distribution: world Message-ID: <199612062347.QAA06432@frodo2.cs.sandia.gov> NNTP-Posting-Host: net.bio.net RECOMB97 NEWS December 6, 1996 **************** recomb97@hto.usc.edu http://www.cs.sandia.gov/recomb97 http://www.eldoradohotel.com Reminder: Early registration deadline (extended to Dec 20, 1996) is approaching fast!! THE ELDORADO HOTEL IS FULL!!! However, there are a few extra rooms available at the RECOMB 97 rate. Please hurry up. The overflow will go to the Hilton of Santa Fe Hotel (1-800-336-3676 or fax 505-986-6439 ask for the "RECOMB97 Conference") where we have a block of rooms reserved at identical rates. 1. RECOMB'97 Final Program 2. Available Financial support for students/graduate students/postdocs. Thanks to the generous help from the Sloan Foundation and the U.S. Department of Energy. 3. Babysitting and Recreational Activities 4. Travel to Santa Fe --------------------------------------------------------- 1. RECOMB'97 Final Program FIRST ANNUAL INTERNATIONAL CONFERENCE ON COMPUTATIONAL MOLECULAR BIOLOGY January 20-23, 1997 Eldorado Hotel Santa Fe, New Mexico Sponsored by ACM SIGACT with support from SLOAN Foundation US Department of Energy recomb97@hto.usc.edu http://www.cs.sandia.gov/recomb97 RECOMB 97 SCHEDULE ****************** *************************************** Sunday January 19, 1996 *************************************** 7:00-10:00 p.m. Reception Eldorado Hotel Anasazi South *************************************** Monday January 20, 1996 *************************************** 9:00 a.m.- 5:00 p.m. Poster Session, Zia B&C General Session, Anasazi Ballroom 8:45 a.m. Opening Remarks: Michael Waterman, General RECOMB Chair 1997 Program Committee Chair Sorin Istrail, General RECOMB Vice-Chair 1997 Conference Chair Pavel Pevzner, General RECOMB Chair 1998 Program Committee Chair --------------------------------------- 9:00 Distinguished Conference Lecture Session Pavel Pevzner, Chair Rich Roberts Hunting for new restriction enzymes in GeneBank --------------------------------------- 10:00 Break Session on Mapping Chair: Phil Green 10:15 Shili Lin, Terence P. Speed An Algorithm for Haplotype Analysis 10:40 Donna Slonim, L. Kruglyak, L. Stein, E. Lander Building Human Genome Maps with Radiation Hybrids Break 11:05 Session on Genomic Rearrangements Chair: Richard Karp 11:15 Haim Kaplan, Ron Shamir, Robert Tarjan Faster and Simpler Algorithm for Sorting Signed Permutations by Reversals 11:40 David Sankoff, V. Ferretti, Joe Nadeau Conserved segment identification 12:05 Lunch, Eldorado Court --------------------------------------- 1:30 Invited Lecture Session Pavel Pevzner, Chair Rob Lipshutz DNA Probe Arrays - Accessing the Genome --------------------------------------- 2:30 Break Session on New Technologies Chair: Gene Myers 2:45 J. Richard Bradley, Steven Skiena Fabricating Arrays of Strings 3:10 Dan Fasulo, Tao Jiang, Richard M. Karp, R. Settergren, E. C. Thayer An Algorithmic Approach to Multiple Complete Digest Mapping 3:45 S. Muthukrishnan, Laxmi Parida A highly effective simple combinatorial approach for constructing physical maps by optical mapping 4:10 Break --------------------------------------- 4:25 Invited Lecture Session Sam Karlin, Chair Terry Speed Variations on a theme of Lander and Waterman --------------------------------------- 8:00-10:00 p.m. Business Meeting, Anasazi Ballroom *************************************** Tuesday January 21, 1996 *************************************** 9:00 a.m.- 5:00 p.m. Poster Session, Zia B&C General Session, Anasazi Ballroom --------------------------------------- 8:30 Invited Lecture Session Rich Roberts, Chair David Botstein Expression Arrays: An Open Problem for Mathematicians in Post-Genome Sequence Biology --------------------------------------- 9:30 Break Session on Sequence Alignment Chair: Steven Altschul 9:45 Gene Myers, Sanford Selznick, Zheng Zhang, Webb Miller Progressive Multiple Alignment with Constrains 10:10 George A. Komatsoulis, Michael Waterman Chimeric alignment by dynamic programming: Algorithm and biological uses 10:35 Break Session on Sequence Alignment Chair: Temple Smith 10:45 Gary Benson Sequence Alignment with Tandem Duplication 11:10 K. Reinert, Hans-Peter Lenhof, P. Mutzel, K. Mehlhorn, J. D. Kececioglu A Branch-and-Cut Algorithm for Multiple Sequence Alignment 11:35 Ralf Zimmer, Tom Lengauer Fast and Numerically Stable Parametric Alignment of Biosequences 12:00 Lunch, Eldorado Court --------------------------------------- 1:30 Invited Lecture Session Mike Waterman, Chair Sam Karlin Assesing Inhomogeneities in Bacterial Long Genomic Sequences --------------------------------------- 2:30 Break Session on Gene Recognition Chair: Gary Stormo 2:45 Ying Xu, Edward Uberbacher Reference-based Gene Model Prediction on DNA Contigs 3:10 Sing-Hoi Sze, Pavel Pevzner Las Vegas Algorithms for Gene Recognition: Suboptimal and Error-Tolerant Spliced Alignment 3:35 Break Session on Protein Folding Chair: Martin Karplus 3:45 Ken Dill, Andrew T. Phillips, J. Ben Rosen, Protein Structure Prediction and Potential Energy Landscape Analysis using Continuous Global Minimization 4:10 William Hart, S. Istrail Lattice and Off-Lattice Side Chain Models of Protein Folding: Linear Time Structure Prediction Better than 86% of Optimal 4:10 Break --------------------------------------- 4:25 Invited Lecture Session Martin Karplus, Chair Temple Smith The threading approach to the inverse protein folding problem --------------------------------------- 7:00 p.m. Cocktails, Anasazi Ballroom 7:30-10:00 Banquet, Anasazi Ballroom --------------------------------------- 7:30 Stanislaw Ulam Computational Biology Address Session Mike Waterman, Chair Eric Lander The Human Genome and Beyond --------------------------------------- *************************************** Wednesday January 22, 1996 *************************************** 9:00 a.m.- 5:00 p.m. Poster Session, Zia B&C General Session, Anasazi Ballroom --------------------------------------- 8:30 Invited Lecture Session Sorin Istrail, Chair Martin Karplus Protein Dynamics: From the Native to the Unfolded State and Back Again --------------------------------------- 9:30 Break Session on Protein Folding Chair: Bonnie Berger 9:45 William Hart On the Computational Complexity of Sequence Design Problems 10:10 Tatsuya Akutsu, S. Miyano On the Approximation of Protein Threading 10:45 Break Session on Mapping Chair: Ron Shamir 10:55 Amir Ben-Dor, Benny Chor On Constructing Radiation Hybrid Maps 11:20 Fengzhu Sun, Gary Benson, Mike Waterman Pooling Strategies for Establishing Physical Genome Maps Using FISH 11:45 Mutida Jain, Gene Myers Algorithm for Computing and Integrating Physical Maps Using Unique Probes 12:10 Lunch, Eldorado Court --------------------------------------- 1:30 Invited Lecture Session Sorin Istrail, Chair Jonathan King Problems in Understanding the Structure and Assembly of Viruses --------------------------------------- 2:30 Break Session on Protein Folding Chair: Bonnie Berger 2:45 Richa Agarwala, Serafim Batzogloa, V. Dancik, Scott E. Decatur, M. Farach, S. Hannenhalli, S. Muthukrishnan, S. Skiena Local Rules for Protein Folding on Triangular Lattice and generalized Hydrophobicity in the HP Model 3:10 Hans-Peter Lenhof New Contact Measures for the Protein Docking Problem 3:35 Break Session on Proteins Chair: Thomas Lengauer 3:45 Bonnie Berger, Mona Singh An Iterative Method for Improved Protein Structural Motif Recognition 4:10 Erich Bornberg-Bauer Chain Growth Algorithms for HP-Type Lattice Proteins 4:35 Hiroshi Mamitsuka Supervised Learning of Hidden Markov Models for Sequence Discrimination *************************************** Thursday January 23, 1996 *************************************** General Session, Sweeney Convention Center Session on Sequence Alignment Chair: Martin Farach 8:30 Zheng Zhang, William R. Pearson, Webb Miller Aligning a DNA Sequence with a Protein Sequence 8:55 Eric L. Anson, Gene Myers ReAligner: A Program for Refining DNA Sequence Multi-Alignments 9:20 Lusheng Wang, Tao Jiang, Dan Gasfield A More Efficient Approximation Scheme for Tree Alignment 9:20 Break Session on Genomic Rearrangements Chair: David Sankoff 9:35 B. DasGupta, T. Jiang, S. Kannan, M. Li, Z. Sweekyk On the Complexity and Approximation of Syntenic Distance 10:00 Alberto Caprara Sorting by Reversals is Difficult 10:25 Break Session on Statistics Chair: Bruce Weir 10:40 Simon Heath The application of Markov Monte Carlo Methods to Radiation Hybrid Mapping 11:05 M. G. Reese, F. H. Eeckman, D. Kulp, D. Haussler Improved Splice Site Detection in Genie 11:30 Gary A. Churchill Monte Carlo Sequence Alignment 11:55 Lunch, Sweeney Convention Center Session on Sequence Alignment Chair: Webb Miller 1:30 Benno Schwikowski, Martin Vingron The Deferred Path Heuristic for the Generalized Tree Alignment Problem 1:55 Tetsuo Shibuya, Hiroshi Imai New Flexible Approaches for Multiple Sequence Alignment 1:55 Break Session on Mapping Chair: Maynard Olson 2:05 T. Christof, M. J\"unger, John Kececioglu, P. Mutzel, G. Reinel A Branch-and-cut Approach to physical mapping with end-probes 2:30 David Wilson, David Greenberg, Cynthia Phillips Beyond Islands: Runs in Clone-Probe Matrices 2:55 Break Session on Evolutionary Trees and DNA Computing Chair: Martin Vingron 3:05 Jamie Cohen, Martin Farach Numerical Taxonomy on Data: Experimental Results 3:30 W. Cai, Anne Condon, R.Mm Corn, E Glasser, Z. Fei, T. Frutos, Z. Guo, MG Lagally, Q. Lui, L.M. Smith, A. Theil The Power of Surface-Based DNA Computing 3:55 M. Ogihara, A. Ray Simulating Boolean Circuits on a DNA Computer End of RECOMB 97 -------------------------------------------------- 2. Financial support for students/graduate students/postdocs Limited support for students is available. We anticipate being able to make about 22 financial awards of $400 each for 22 students. Please send us a one-page request to via email to scistra@cs.sandia.gov including your affiliation, the name of your Ph.D. adviser (if appropriate) and the area of research that you are working on. We would like to select for support the students that will benefit the most from our meeting as well as those that will be able to make valuable contributions to the conference. 3. Babysitting and Recreational Activities "Santa Fe Destinations" is a company located in the Eldorado Hotel. They provide professional services for: babysitting, airport transportation, entertainment, recreational activities, spouse programs etc. Please call Tom Williams the Director of the company for services related to your visit in Santa Fe. 4. Travel to Santa Fe http://www.eldoradohotel.com/getting_there.html By plane to the Albuquerque International Airport. All major airlines use the airport. From there "Shuttlejack" ((505) 988-2441; $20 one-way trip; about one hour long) buses pick people up and drop them directly at the Eldorado Hotel nine times a day. Please call Shuttlejack and make reservations. The complete information about arriving at the Eldorado Hotel is available from their web page http://www.eldoradohotel.com/getting_there.html We hope to see you soon in Santa Fe. -- Sorin Istrail Conference Chair From owner-proteins@net.bio.net Thu Dec 05 22:00:00 1996 Path: biosci!biosci!not-for-mail From: "E. Kolker" Newsgroups: bionet.general,bionet.info-theory,bionet.molbio.proteins,sci.med.pharmacy,bionet.molbio.embldatabank,bionet.population-bio,bionet.molbio.evolution,sci.bio.systematics,bionet.software Subject: New deadline & Web site Date: 6 Dec 1996 05:44:32 -0800 Organization: University of Washington Lines: 88 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <327E21BF.167E@bioinformatics.weizmann.ac.il> NNTP-Posting-Host: net.bio.net Xref: biosci bionet.general:24391 bionet.info-theory:4414 bionet.molbio.proteins:9500 sci.med.pharmacy:37773 bionet.molbio.embldatabank:729 bionet.population-bio:2126 bionet.molbio.evolution:5408 sci.bio.systematics:1449 bionet.software:17299 ANNOUNCEMENT AND CALL FOR PAPERS Computational Biology Session: "Computing in the Genome Era" March 31 - April 1, 1997 Eleventh International Conference on Mathematical and Computer Modelling and Scientific Computing March 31 - April 3, 1997 Georgetown University Conference Center Washington, DC, USA The Eleventh International Conference on Mathematical and Computer Modelling and Scientific Computing is scheduled to take place March 31 - April 3, 1997 at the Georgetown University Conference Center, Washington, DC, U.S.A. The objective of the Computational Biology Session "Computing in the Genome Era" (March 31 - April 1, 1997) is to discuss the current state of computational biology, its approaches, methods, general problems, achievements, and future developments with emphasis on sequence research and analysis for the Genome Projects. SPEAKERS of the session include: S. Altschul (Natl Center for Biotechnology Iinformation, Bethesda), A. Bairoch (Geneva University, Switzerland), T. Clark (Millenium Pharmaceuticals, Cambridge), W. Gish (Washington University, St. Louis), T. Gojobori (Natl Institute of Genetics, Mishima, Japan), P. Green (University of Washington, Seattle), S. Henikoff (Fred Hutchinson Cancer Research Center, Seattle), L. Hood (University of Washington, Seattle), A. Kerlavage (Institute for Genomic Research, Rockville), E. Koonin (Natl Center for Biotechnology Iinformation, Bethesda), O. Ritter (German Cancer Research Center, Heidelberg, Germany), D. Searls (SmithKline Beecham Pharmaceuticals, King of Prussia), E. Shpaer (Perkin-Elmer Applied Biosystems, Foster City), E. Trifonov (Weizmann Institute of Science, Israel). STEERING COMMITTEE: S. Altschul, A. Bairoch, W. Gish, P. Green, S. Henikoff, E. Koonin, E. Trifonov Papers (Abstracts) are invited on all relevant aspects of computational biology for presentation at the session (a new deadline is DECEMBER 23, 1996), to be selected on competitive basis by the steering committee (notification of acceptance is JANUARY 4, 1997). One-page abstracts should clearly describe the work and its conclusions. Full length manuscripts (limited to six pages) of papers presented at the conference will be published in the Conference Proceedings, in a special issue of the journal MATHEMATICAL MODELLING AND SCIENTIFIC COMPUTING, Vol. 8, 1997 (ISSN 1067-0688). The manuscripts for the special issue are due JUNE 15, 1997. The special issue of the journal will be published by SEPTEMBER 1997. ALL participants shall pay the registration fee. Abstracts (ONE PAGE, about 200 words, PLAIN JUSTIFIED TEXT) may be submitted in hard copy or via fax or by e-mail (PREFERRED) under subject "Abstract". The abstracts must be formatted to fit on 8-1/2 x 11 inch (approximately 21.5 cm x 28 cm or European Standard A-4 size) paper, typed in single space. The title must be capitalized and centered followed by the author's name(s), institution, and full address, including fax and e-mail. Send two copies (ONE copy if sent by fax or e-mail) of the abstract to the session organizer before DECEMBER 23, 1996: Eugene Kolker Dept of Molecular Biotechnology and Genome Center Box 357730, University of Washington Seattle, WA 98195-7730, USA Fax: +1-206-685-7301 E-mail: egn@u.washington.edu The Computational Biology Session is proudly sponsored by SMITHKLINE BEECHAM and MILLENIUM. You can find additional info on our Web site: http://www.genome.washington.edu/~eugene/meeting.html From owner-proteins@net.bio.net Fri Dec 06 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!howland.erols.net!newsfeed.internetmci.com!news.microagewny.com!usenet From: Douglas Easton Newsgroups: bionet.molbio.proteins Subject: Genetics Position Date: Sat, 07 Dec 1996 11:23:55 -0500 Organization: State University College at Buffalo (Biology) Lines: 40 Message-ID: <32A99A1B.7458@wzrd.com> Reply-To: dpeaston@wzrd.com NNTP-Posting-Host: ppp15.microagewny.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) GENETICIST STATE UNIVERSITY (SUNY) COLLEGE AT BUFFALO Applications are invited for a tenure-track ASSISTANT PROFESSOR to begin September 1997. We are seeking a colleague who is enthusiastic about teaching undergraduates. Teaching responsibilities will include Genetics, Introductory Biology for non-majors, and either Botany or Cell Biology. The successful candidate will be expected to develop an active research program involving undergraduate and master's students. Applicants must have a Ph.D., and postdoctoral teaching and research experience is preferred but not required. The State University College at Buffalo (Buffalo State College) is the largest arts and science college in the SUNY system with an enrollment of 13,000 students (including 2000 in graduate programs). The campus is located in a residential district in the city of Buffalo. The Department of Biology enrolls about 250 undergraduate and about 30 graduate students in MA and MSEd programs. Of our 16 faculty, one-half have received their doctorates within the last 13 years. We maintain greenhouse facilities, environmental chambers and research laboratories. The College's newly renovated Aquatic Research Laboratory provides laboratories and research vessels to faculty and students. Applicants should send a curriculum vitae with a thoughtful statement of teaching philosophy, research goals and three letters of recommendation by January 20, 1997 to: Dr. Randal Snyder, Chair of Search Committee, Department of Biology, Buffalo State College, 1300 Elmwood Avenue, Buffalo, NY 14222. Email: SNYDERRJ@SNYBUFAA.CS.SNYBUF.EDU Telephone: 716- 878-4314. We especially encourage applications from women and minorities. From owner-proteins@net.bio.net Sat Dec 07 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!insync!Gamma.RU!srcc!news.uni-stuttgart.de!rz.uni-karlsruhe.de!news.fzk.de!usenet From: "David Moss" Newsgroups: bionet.molbio.proteins Subject: Re: re bifunctional enzymes Date: 8 Dec 1996 14:48:20 GMT Organization: Forschungszentrum Karlsruhe Lines: 42 Distribution: world Message-ID: <01bbe50e$d702ac00$5e09348d@ifiabio1> References: <584kke$npo@deft.cc.purdue.edu> NNTP-Posting-Host: ifiabio1.fzk.de X-Newsreader: Microsoft Internet News 4.70.1157 Raman says: > >From: "David Moss" > >Subject: Re: bifunctional enzymes > >Date: 3 Dec 1996 21:17:07 GMT > > > >Er... I think you're missing an important and elementary point of physical chemistry. An > >enzyme can't possibly change the equilibrium constant of the reaction it catalyzes, otherwise it > >would be breaking all of the laws of thermodynamics that I ever heard of. And since the > >equilibrium constant is the ratio of the rate constants for the forward and reverse reactions, > >it follows that an enzyme can't increase the forward reaction rate constant without a > >proportionate increase in the back reaction rate constant. Therefore all enzymes MUST > >mediate the reaction in both forward and backward directions. > > The forward and reverse reactions are controlled by concentration gradients, > temperature, pH and other factors. While the equilibrium is the same, > the reactions can proceed either in the forward or backward directions. > Some enzymes are uni-directional (for all practical purposes): for example, > DNA polymerase will only extend the DNA. It is very hard to detect any > breaking of DNA by the polymerase. This is not an answer to the point I made. Sure, some equilibria are so far to the right that the back reaction is negligible even when it's accelerated by an enzyme. You still can't treat enzymes that catalyze in both directions as a special group, and propose a name for this group, because ALL enzymes MUST catalyze equally in both directions. Regards, David Moss From owner-proteins@net.bio.net Sat Dec 07 22:00:00 1996 Path: biosci!news.alaska.edu!ftjrd From: ftjrd@aurora.alaska.edu (John Demboski) Newsgroups: bionet.molbio.proteins Subject: 2 job announcements - U Alaska Fairbanks Date: Sun, 08 Dec 1996 16:26:09 -0800 Organization: University of Alaska Computer Network Lines: 108 Message-ID: NNTP-Posting-Host: lab3.uafbio.alaska.edu Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.1 The University of Alaska Fairbanks is seeking qualified applicants for two tenure-track Assistant Professor positions: Cellular/Molecular Biologist and Vertebrate Nutritional Physiologist/Ecologist. Descriptions of both vacancies follow below and you can see these announcements at this URL: http://zorba.uafadm.alaska.edu/iab/index.html. Please respond to the appropriate Chair of each search committee for more information. -------------------------------------------------------------------------- CELL & MOLECULAR BIOLOGY DEPARTMENT OF BIOLOGY & WILDLIFE INSTITUTE OF ARCTIC BIOLOGY UNIVERSITY OF ALASKA FAIRBANKS ALASKA The Department of Biology and Wildlife and Institute of Arctic Biology at the University of Alaska Fairbanks seek qualified applicants for the tenure-track position of Assistant Professor of Cellular/Molecular Biology. The appointee is expected to establish a productive research program at the cellular or molecular level that ideally will integrate with existing faculty interests including: high latitude physiology, neurobiology, endocrinology, or chronobiology. The successful applicant will be expected to teach two courses/year including cell biology and an additional course such as developmental biology or molecular biology, and to supervise graduate students. An earned Ph.D. in biology or related field is mandatory; post-doctoral and teaching experience are preferred. Initial support for graduate student teaching assistants, laboratory space, and start-up funds are included in this appointment which is expected to begin in fall of 1997. Qualified women and minorities are encouraged to apply. Send applications including: statements of research interest, teaching philosophy, curriculum vitae, and three letters of reference by January 15, 1997 to: Dr. Gerald Shields Chair, Cellular/Molecular Search Department of Biology and Wildlife and Institute of Arctic Biology, University of Alaska Fairbanks Fairbanks AK 99775 (907) 474-7656, Fax (907) 474-6967 E-mail: gshields@redback.lter.alaska.edu The University of Alaska Fairbanks is the Land Grant, Sea Grant, and Space Grant institution in Alaska. The University of Alaska is an Educational Institution and an EEO and Affirmative Action Employer. Your Application for Employment with The University of Alaska May Be Subject to Public Disclosure ........................................................................... Tenure Track Position Vertebrate Nutritional Physiologist/Ecologist The Department of Biology and Wildlife and the Institute of Arctic Biology at the University of Alaska Fairbanks seek a faculty member for a tenure-track position at the level of ASSISTANT PROFESSOR. Candidates are expected to have an earned doctorate in Biology or closely related field with expertise in both laboratory and field research, and interests in working at levels from molecular to the whole animal. The successful candidate will be a critical member of a group of researchers by bridging the disciplines of wildlife ecology, physiology, and molecular biology. This individual also will be expected to: develop a strong externally funded research program, mentor graduate students, and teach two courses per year, including Nutritional and Physiological Ecology of Wildlife. Extensive facilities are available including: captive research herds of muskox and caribou (Large Animal Research Station), a remote field station north of the Arctic circle (Toolik Field Station), animal vivarium equipped to conduct research in environmental physiology, and a Core DNA Sequencing Facility. We encourage candidates with the potential for forming collaborative research programs with applied resource agencies and with existing faculty having interests in physiological and ecological adaptations to northern environments and ruminant physiological ecology. We especially encourage the application of qualified women and minority candidates. The possibility exists for a shared tenure-track position forcandidates with spouses. Preference will be given to candidates with postdoctoral and university teaching experience, and a strong publication record. To apply, send curriculum vitae, statements of research interests and teaching philosophy, copies of pertinent reprints, and have three letters of reference sent to: Dr. James Sedinger Search Chair Institute of Arctic Biology University of Alaska Fairbanks Fairbanks, AK 99775-7000 (907) 474-6598 E-mail: ffjss@aurora.alaska.edu Closing date: 15 December 1996 From owner-proteins@net.bio.net Sat Dec 07 22:00:00 1996 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!arclight.uoregon.edu!news.bc.net!info.ucla.edu!news.ucdavis.edu!boris.ucdavis.edu!not-for-mail From: ez076055@boris.ucdavis.edu (Anh-Truc Dang) Newsgroups: bionet.molbio.proteins Subject: help on electronegativity Date: 9 Dec 1996 01:28:17 GMT Organization: University of California, Davis Lines: 2 Message-ID: <58fpvh$28u@mark.ucdavis.edu> NNTP-Posting-Host: boris.ucdavis.edu X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] I am writing a paper and was wondering if anyone can answer my question on the many ways in which electronegativity is important in life. From owner-proteins@net.bio.net Sat Dec 07 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!www.nntp.primenet.com!nntp.primenet.com!uwm.edu!msunews!news From: (shahvika) Newsgroups: bionet.molbio.proteins Subject: HIV gp120 Date: Sun, 08 Dec 1996 18:15:00 GMT Organization: Michigan State University Lines: 6 Message-ID: <32ab059b.2657366@news.msu.edu> NNTP-Posting-Host: pm247-15.dialip.mich.net X-MSUnetID: shahvika X-Newsreader: Forte Agent .99e/32.227 I am an undergraduate at Michigan State University interested in the molecular structure of gp120. Does anybody know where I might find the molecular coordinates for use in RasMol or another visualization package (I have Babel.) Thanks for your help. Vikas Shah From owner-proteins@net.bio.net Sat Dec 07 22:00:00 1996 Path: biosci!agate!howland.erols.net!news.sgi.com!news.cs.indiana.edu!purdue!mozo.cc.purdue.edu!not-for-mail From: barani@deft.cc.purdue.edu (Raman) Newsgroups: bionet.molbio.proteins Subject: re: bifunctional enzymes Date: 8 Dec 1996 11:56:20 -0500 Organization: Purdue University Computing Center Lines: 68 Distribution: world Message-ID: <58ervk$540@deft.cc.purdue.edu> NNTP-Posting-Host: deft.cc.purdue.edu |From: "David Moss" | |Jeffrey Yuziuk asserts that Raman changed his |definition of "bifunctional enzyme". Raman denies |this. |I quote: | |Raman wrote in posting Raman posted his view on how the enzymes should be termed: |> I believe it would be inappropriate to call dual role enzymes as |> bifunctional. A bifunctional enzyme mediates a reaction in either |> forward or backward direction. |> |> Enzymes that carry two entirely different active sites and reactions |> should be termed |> |> "di-functional enzymes". | Raman posted on how the term is being used today: | |Raman wrote in posting |> The term 'bifunctional' is used normally for enzymes which have TWO |> active sites and perform Two independent reactions. Enzymes with |> a single active site but two distinct reactions, or enzymes with single |> active site and show measureable reactions proceed in either forward |> or backward directions fall in the border line. No distinction has been |> made among them and different people call them differently. Raman continued in the same article on what should be correctly used: Er... David and Jeff authoritatively and conveniently snip the vital portions of Raman's article! (a case for error by omission?) That portion is reproduced below: {It may help to make such distinctions in future, along the lines similar to 'divalence' or 'bivalence' etc even if it requires renaming old conventions. } |Game, set and match to Jeff! Er...LOTS of money helped Jeff, I guess! | The prefix "bi" is reserved for "partitioning into two" "two halves" "mutual" "bothways" "paired" etc. e.g. - bisect, bilateral, bifid, bisexual, bidentate The prefix "di" is reserved for "double" "twice" as in multiplicity. e.g. - divalent, diphyllous, dibranchiate Use of the term "difunctional" is most appropriate for enzymes with TWO different active sites and TWO different functions, notwithstanding old conventions or money and games! Hope that helps! -Raman From owner-proteins@net.bio.net Sat Dec 07 22:00:00 1996 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!insync!Gamma.RU!srcc!news.uni-stuttgart.de!rz.uni-karlsruhe.de!news.fzk.de!usenet From: "David Moss" Newsgroups: bionet.molbio.proteins Subject: Re: bifunctional enzymes Date: 8 Dec 1996 15:00:14 GMT Organization: Forschungszentrum Karlsruhe Lines: 33 Distribution: world Message-ID: <01bbe510$80f30bf0$5e09348d@ifiabio1> References: <58764o$f60@deft.cc.purdue.edu> NNTP-Posting-Host: ifiabio1.fzk.de X-Newsreader: Microsoft Internet News 4.70.1157 Jeffrey Yuziuk asserts that Raman changed his definition of "bifunctional enzyme". Raman denies this. I quote: Raman wrote in posting <581kuj$hgc@deft.cc.purdue.edu>: > I believe it would be inappropriate to call dual role enzymes as > bifunctional. A bifunctional enzyme mediates a reaction in either > forward or backward direction. > > Enzymes that carry two entirely different active sites and reactions > should be termed > > "di-functional enzymes". Raman wrote in posting <584kke$npo@deft.cc.purdue.edu>: > The term 'bifunctional' is used normally for enzymes which have TWO > active sites and perform Two independent reactions. Enzymes with > a single active site but two distinct reactions, or enzymes with single > active site and show measureable reactions proceed in either forward > or backward directions fall in the border line. No distinction has been > made among them and different people call them differently. Game, set and match to Jeff! Regards, David Moss From owner-proteins@net.bio.net Sat Dec 07 22:00:00 1996 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!news.enteract.com!news.inetnebr.com!crcnews.unl.edu!manager From: 00191792@bigred.unl.edu (The E Man) Newsgroups: bionet.molbio.proteins Subject: What is N-terminal blockage? Date: 9 Dec 1996 03:02:02 GMT Organization: University of Nebraska--Lincoln Lines: 28 Message-ID: <58fvfa$9n9@crcnis3.unl.edu> NNTP-Posting-Host: bigred.unl.edu Summary: Biochemistry 433 lab Keywords: N-terminal X-Newsreader: TIN [UNIX 1.3 950621BETA PL0] N-terminl blockage is the interaction that certain amino acids have with enzymes that degrade the protein. An example is the (E3) Ubiquitin-protein ligase reaction. Upon binding a protein substrate, E3 catalyses the transfer of ubiquitin from ES-2 ~ ubiquitin to free amino groups (usually Lys E-NH2) on the protein. Once the protein has been conjugated with ubiquitin, it is degraded by a large 26S ATP-dependent protease complex. The exact mechanism is unknown. It liberates peptide products from the ubiquitin-protein conjugates and cleaves the isopeptide bonds joining the conjugaes, allowing ubiquitin to be recycled. The blockage comes into effect by the role in which E3 reconizes and selects proteins for degradation. E3 selects proteins by the nature of the N-terminal amino acid. Proteins must have a free alpha-NH2 to be susceptible. However, proteins with either Met, Ser, Ala, Thr, Val, Gly, or Cys at the amino terminus are resistant to the ubiquitin-mediated degradation pathway. It is this resistance to degradative pathways that illustrates the N-terminal blockage effect. If a protein has one of the aforementioned amino acids as its N-terminus then the degradation of the enzyme has been blocked. Reference: Garrett, Reginald & Grisham, Charles; Biochemistry (textbook) 1995; pgs1072 From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!agate!howland.erols.net!Frankfurt.Germany.EU.net!Germany.EU.net!main.Germany.EU.net!news-koe1.dfn.de!news-kar1.dfn.de!news-stu1.dfn.de!news-mue1.dfn.de!news-nue1.dfn.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Protein Purification Question Date: 9 Dec 1996 18:42:01 GMT Organization: University of Wuerzburg, Germany Lines: 70 Distribution: world Message-ID: <58hmhp$6t6@winx03.informatik.uni-wuerzburg.de> References: <1.5.4.32.19961209171606.00701d2c@lehigh.edu> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Jim Campanella (jjc3@LEHIGH.EDU) wrote: > The problem is this. In a class I have been > teaching, we have been purifying an enzyme > as one of the projects. In the past, the > last purification step is a desalting step > using dialysis. The enzyme is dissolved in > the following common buffer: > > 20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, > 0.5% Tween20, 0.5% NP-40, and 0.5 mM PMSF. > > The dissolved enzyme is then dialyzed > overnight against the following buffer: > > 20 mM Tris-HCl, pH 8, 100 mM KCl, 0.1 mM > EDTA, 50% glycerol, 0.5% NP-40, 0.5% > Tween20, and 1mM DTT. > > This works well to get a clean enzyme that > has activity, but I thought it might be > nice to modernize and speed up the > protocol for the class. So, I thought > a desalting column (eg. Bio-Gel P-6DG) > might be a nice replacement for the > dialyzing step. > > My problems: > > A) I forgot that the dialyzing > step serves the secondary purpose of > getting glycerol into the buffer with > the enzyme. So how do I get the glycerol > into the buffer if I use the desalting > column? Two possibilities: Either you run the column in exactly the same buffer you dialyse against (i.e. the composition you gave above). Problem: the glycerol will make the buffer very viscous, and it's probably not much fun to run a column in 50% glycerol (although I've never tried). The other solution would be to use as a running buffer for the desalting column a buffer which has all components at twice the concentration you gave above (except the glycerol) and dilute the protein solution 1:1 with glycerol afterwards. (Slowly.) > B) It seems to me that if I use the > column I'll be losing some salts > that will be needed to help buffer > the enzyme-- salts that are still > present but equilibrated with dialysis. Why? (You will, dependent on the volumes, still have residual amounts of HEPES and PMSF in your buffer, but you can neglect those.) A general problem with gel filtration columns is that the volume which can be applied is rather limited. This problem is not severe in desalting but can get really tricky if you want to purify a larger volume of protein by gel filtration. But still, if you have 10 ml of enzyme solution, it's probably easier (and less work) to dialyze the stuff, especially because you can do it overnight. Hope that helps, --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!LEHIGH.EDU!jjc3 From: jjc3@LEHIGH.EDU (Jim Campanella) Newsgroups: bionet.molbio.proteins Subject: Protein Purification Question Date: 9 Dec 1996 09:16:00 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 69 Sender: daemon@net.bio.net Distribution: world Message-ID: <1.5.4.32.19961209171606.00701d2c@lehigh.edu> NNTP-Posting-Host: net.bio.net Hi, I have a problem that may be fairly simple to solve, but it's not obvious to me at the moment-- probably because I admit I'm not much of a biochemist. Perhaps someone out there could help. The problem is this. In a class I have been teaching, we have been purifying an enzyme as one of the projects. In the past, the last purification step is a desalting step using dialysis. The enzyme is dissolved in the following common buffer: 20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 0.5% Tween20, 0.5% NP-40, and 0.5 mM PMSF. The dissolved enzyme is then dialyzed overnight against the following buffer: 20 mM Tris-HCl, pH 8, 100 mM KCl, 0.1 mM EDTA, 50% glycerol, 0.5% NP-40, 0.5% Tween20, and 1mM DTT. This works well to get a clean enzyme that has activity, but I thought it might be nice to modernize and speed up the protocol for the class. So, I thought a desalting column (eg. Bio-Gel P-6DG) might be a nice replacement for the dialyzing step. My problems: A) I forgot that the dialyzing step serves the secondary purpose of getting glycerol into the buffer with the enzyme. So how do I get the glycerol into the buffer if I use the desalting column? B) It seems to me that if I use the column I'll be losing some salts that will be needed to help buffer the enzyme-- salts that are still present but equilibrated with dialysis. Again, how do I solve the problem of this loss? It seems to me that I may be creating more problems that I am solving by trying to "simplify" this protocol. On the other hand, this seems like a problem that protein chemists would run into every day-- one for which there should be a simple solution. Any suggestions would be greatly appreciated. Thanks, Jim Campanella Dept. of Biological Sciences Lehigh University Bethlehem, PA USA From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!mr.net!news.mid.net!crcnews.unl.edu!manager From: 00191792@bigred.unl.edu (The E Man) Newsgroups: bionet.molbio.proteins Subject: What is N-terminal Blockage? Date: 9 Dec 1996 14:27:19 GMT Organization: University of Nebraska--Lincoln Lines: 28 Message-ID: <58h7k7$a9q@crcnis3.unl.edu> NNTP-Posting-Host: bigred.unl.edu Summary: Keywords: N-terminal X-Newsreader: TIN [UNIX 1.3 950621BETA PL0] N-terminal blockage is the interaction of the N-terminal amino acid with enzymes that degrade proteins. Certain amino acids are susceptible to these degradative enzymes, while other are not. An example is the E3 (ubiquitin-protein) ligase. E3 plays a central role in the recognition and selection of proteins for degradation. E3 selects proteins by the nature of the N-terminal amino acid. Proteins must first have a free alpha-NH2 to be susceptible. Proteins that have either Met, Ser, Ala, Thr, Val, Gly, or Cys at the amino terminus are resistant to the ubiquitin-mediated degradation pathway. While other amino acids at the N-terminus have half lives of 2-30 seconds. The effect the N-terminus amino acid has on its own degradation is an example of N-terminal blockage. If the N-terminus is one of the aforementioned amino acids then the protein is resistant to ubiquitin-mediated degradation pathways. In effect the N-terminal amino acid has blocked degradation of itself. This clearly demonstrates N-terminal blockage. Eric Vander Woude Reference: Garrett, Reginald and Grisham, Charles; Biochemistry (textbook), 1995, pgs. 1072-1073 From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!news.bconnex.net!clicnet!news.clic.net!rcogate.rco.qc.ca!altitude!usenet From: "Achim Recktenwald, PhD" Newsgroups: bionet.molbio.proteins Subject: Re: How to show protein interaction? Date: Mon, 09 Dec 1996 07:49:29 -0500 Organization: IBEX Technologies, Inc., Biochemistry, 5485 Pare, Montreal, PQ, H4P 1P7, Canada Lines: 48 Message-ID: <32AC0AD8.6B01@ibex.ca> References: <01bbdcae$fdd474c0$37019386@Schmidt.rz.ruhr-uni-bochum.de> <57jkl9$c6d@winx03.informatik.uni-wuerzburg.de> <329D8B82.5439@ibex.ca> NNTP-Posting-Host: ibex.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) To: Harold E Smith Harold E Smith wrote: > > On Thu, 28 Nov 1996, Achim Recktenwald, PhD wrote: > > > Cornelius Krasel wrote: > > > > > > Thorsten Schmidt (Thorsten.Schmidt@rz.ruhr-uni-bochum.de) wrote: > > > > I have a protein and I just want to test if it forms dimers, trimers etc.? > > > > (The protein was purified by HisTag-Chromatographie and is in solution > > > > (20mM Tris, 0,5 M NaCl, 1 M Imidazol)) > > > > What is the simplest way to do this? > > > > > > I'd try analytical gel filtration if you have the equipment (i.e. an > > > FPLC/HPLC). Alternatively, you may try analytical ultracentrifugation > > > (I have absolutely no experience with this) or maybe non-denaturing > > > gel electrophoresis. > > > > > > --Cornelius. > > > > > > -- > > > /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ > > > /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ > > > /* "Science is the game we play with God to find out what His rules are." */ > > > > > > I would also try native polyacrylamide-gel-electrophoresis. If the bond > > between the subunits is strong, you'll get one single band, the multimer > > form; if its very weak, you'll only see a sharp band at the mol. weight > > for the single subunit. But if the bond is neither nor than you get a > > smear on your gel, starting at the point of the highest multimer down to > > the lowest multimer, or even the single subunit. In the latter case the > > multimer form is constantly decomposing during the electrophoresis. > > > > Achim > > > > > There are a few problems with native PAGE. First, the protein generally > migrates at a position far from its predicted molecular weight. Second, > this aberrant migration prevents easy determination of the number of > subunits within the multimer. Accurate assessment the true molecular > weight/number of subunits requires multiple gels of varying acrylamide > concentrations. > -Harold Smith Or a gradient gel; of what I was thinking, but forgot to mention. Achim From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!worldnet.att.net!uunet!in1.uu.net!160.45.4.4!fu-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!news From: Boris Steipe Newsgroups: bionet.molbio.proteins Subject: Re: IMAC Problem/De-inhibiting enzyme Date: Tue, 10 Dec 1996 08:48:29 +0000 Organization: Genzentrum Lines: 16 Distribution: world Message-ID: <32AD23B3.2947@LMB.uni-muenchen.de> References: <1996Dec9.153343.90409@cc.usu.edu> Reply-To: steipe@LMB.uni-muenchen.de NNTP-Posting-Host: bs_boris.lmb.uni-muenchen.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Macintosh; I; PPC) Phil Harrison wrote: > I am assuming that the enzyme either > came off the column early, taking some cobalt with it, or was washed > off when the cobalt was. Can anyone suggest a way to remove the > (presumably) bound Co from the enzyme? In most cases metals will prefer a proper chelating agent to your protein and can thus be dialyzed away eg. with 10mM EDTA. This is very likely true for your protein since it binds a number of divalent cations. In some cases metals may be bound tightly to thiols. Then adding some reducing agent will help. Good luck, Boris From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!news.idt.net!enews.sgi.com!news.sgi.com!mr.net!newshub.tc.umn.edu!fu-berlin.de!news.belwue.de!news.uni-stuttgart.de!rz.uni-karlsruhe.de!news.fzk.de!usenet From: "David Moss" Newsgroups: bionet.molbio.proteins Subject: Re: bifunctional enzymes Date: 9 Dec 1996 21:15:19 GMT Organization: Forschungszentrum Karlsruhe Lines: 28 Distribution: world Message-ID: <01bbe60e$0ffd74e0$5e09348d@ifiabio1> References: <58ervk$540@deft.cc.purdue.edu> NNTP-Posting-Host: ifiabio1.fzk.de X-Newsreader: Microsoft Internet News 4.70.1157 Can I try to roll up this "who said what" argument? I interpreted the sentence from your original posting >A bifunctional enzyme mediates a reaction in either >forward or backward direction. As a statement of the way things are (I still think that's how it sounds), and couldn't accept the assertion. As merely your view of how the enzymes should be termed, no problem. Much more interesting to me is the question of whether a designation for such a class of enzymes is necessary at all, in view of the following argument (from another of my postings): >Sure, some equilibria are so far to the right that the back >reaction is negligible even when it's accelerated by an >enzyme. You still can't treat enzymes that catalyze in both >directions as a special group, and propose a name for >this group, because ALL enzymes MUST catalyze equally >in both directions. I'm still interested to hear Raman's response to that. Regards, David Moss From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!enews.sgi.com!news.sgi.com!news.cs.indiana.edu!purdue!mozo.cc.purdue.edu!not-for-mail From: barani@deft.cc.purdue.edu (Raman) Newsgroups: bionet.molbio.proteins Subject: Re: bi- vs di - my 2 cents... Date: 9 Dec 1996 13:40:53 -0500 Organization: Purdue University Lines: 70 Distribution: world Message-ID: <58hmfl$om2@deft.cc.purdue.edu> NNTP-Posting-Host: deft.cc.purdue.edu |Reply-To: smb@bioch.ox.ac.uk |Subject: Re: bi- vs di - my 2 cents... |Date: Mon, 09 Dec 1996 12:07:55 +0000 | |Hi all, | Not sure that this helps...but I'll say it anyway... | | I could be totally wrong [although it's never been known to |happen yet!! ;-)], and I'm not sure if anyone has already said this or |not, but I always thought the choice of either bi- or di- as a prefix |was governed by the root of the word you want to prefix i.e. bi- is |for words with a Latin root (bi- comes from the Latin bis, meaning |twice), di- for words with a Greek root (di from the Greek from dis, |meaning twice). | | So since "functional" has a Latin root, I'd say that "difunctional", |isn't actually a word. Having said that, technical jargon is often a Disagreed. Hybrid words are very common. From your own example: dys (greek) + funct (latin) + ion = dysfunction is a hydrid. Some other examples of hybrids are automobile, neonatal, hypertension, dehydrate, exenterate ... |law unto itself - all sorts of people coin new terms, and new |meanings for existing words... sometimes even people who don't know |their Latin from their Greek (shocking eh?!?). That don't make it |right though... | | Raman, I'd be interested to hear where your definition of a general |distinction between bi- and di- came from - your examples, didn't |illustrate it that well e.g. your "bi-" words have Latin |roots etc etc... | |-- Simon | |Simon M. Brocklehurst |Dept. Biochemistry |Univeristy of Oxford |UK Whether one uses 'bi' or 'di' has often been a matter of semantics. Fortunately, looking at the examples one finds that Latinos had a flair for dividing and Greeks preferred to multiply! :-) If one wishes to remain a purist, then we shall disregard the use of the term 'funct' and use a greek equivalent along with 'di' since we are indeed discussing about MULTIPLE functions and domains! Problem with "bi" and "di" arose earlier while naming many compounds. For example some compounds were named "diphosphate" while they should have been named "bisphosphate" and so on. Later corrections were applied in some cases and I am not certain about others. Atleast now it is known that questions have been raised earlier about the correct use of "bi" and "di". Multiple functional domains are only assembled to form an enzyme with multiple functions. When there is no relationship between the two functions, it is misleading to use the term 'bifunctional'. Just my humble opinion. If I come across more precise discussions about these differences, I will gladly bring them up for notice. Thanks and Best Regards -Raman From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!howland.erols.net!news-peer.gsl.net!news.gsl.net!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Protein Purification Question Date: Tue, 10 Dec 96 18:13:16 GMT Organization: UW-Madison Lines: 57 Distribution: world Message-ID: <58iv4k$1gee@news.doit.wisc.edu> References: <199612092152.OAA12534@NMSU.Edu> NNTP-Posting-Host: f182-217.net.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-No-Archive: Yes In article <199612092152.OAA12534@NMSU.Edu>, hroychow@NMSU.EDU (Hiranya Roychowdhury) wrote: #At 09:16 AM 12/9/96 -0800, Jim Campanella wrote: #>Hi, # #>snip< #>The problem is this. In a class I have been #>teaching, we have been purifying an enzyme #>as one of the projects. In the past, the #>last purification step is a desalting step #>using dialysis. The enzyme is dissolved in #>the following common buffer: #> #>20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, #>0.5% Tween20, 0.5% NP-40, and 0.5 mM PMSF. #> #>The dissolved enzyme is then dialyzed #>overnight against the following buffer: #> #>20 mM Tris-HCl, pH 8, 100 mM KCl, 0.1 mM #>EDTA, 50% glycerol, 0.5% NP-40, 0.5% #>Tween20, and 1mM DTT. # #>snip< #>My problems: #> #>A) I forgot that the dialyzing #>step serves the secondary purpose of #>getting glycerol into the buffer with #>the enzyme. So how do I get the glycerol #>into the buffer if I use the desalting #>column? #> #>B) It seems to me that if I use the #>column I'll be losing some salts #>that will be needed to help buffer #>the enzyme-- salts that are still #>present but equilibrated with dialysis. #>Again, how do I solve the problem of #>this loss? # # #In fact, the desalting column, when equilibrated with the second buffer, should # also 'change' the buffer (including glycerol) for the protein sample. The rule # of thumb to achieve desalting and buffer change simultaneously through a # column is not to load the protein sample in a volume exceeding 10% of the # column (bed) volume. But, it is better add the glycerol to the eluent (I # think) after the buffer change has been accomplished. Absolutely right. Potentially significant difference between dialysis against glycerol vs gel-filtration + dilution 1:1 with glycerol is that in the former case a sample will be significantly concentrated, while in the latter - diluted 2-fold. If this is of no concern, I'd go for gel-filtration and use spin columns which are incredibly easy to make from syringes (an added advantage of sample not been diluted at all; gravity columns result in 1.5-2-fold dilution). - Dima From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.erols.net!news3.cac.psu.edu!news.math.psu.edu!scramble.lm.com!news.psc.edu!nntp.sei.cmu.edu!bb3.andrew.cmu.edu!casaba.srv.cs.cmu.edu!das-news2.harvard.edu!fas-news.harvard.edu!newspump.wustl.edu!mitzi.rsmas.miami.edu!miasun.med.miami.edu!newssun!tguettou From: Toumy Guettouche Newsgroups: bionet.molbio.proteins Subject: Re: HIV gp120 Date: Mon, 9 Dec 1996 14:25:19 -0500 Organization: University of Miami, School of Medicine Lines: 20 Message-ID: NNTP-Posting-Host: newssun.med.miami.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <32ab059b.2657366@news.msu.edu> On Sun, 8 Dec 1996, it was written: > I am an undergraduate at Michigan State University interested in the > molecular structure of gp120. Does anybody know where I might find > the molecular coordinates for use in RasMol or another visualization > package (I have Babel.) Thanks for your help. > > Vikas Shah > > Hi Vikas, if you find that data, let me now. As far as I know, gp120 has successfully resisted any attempts to crystallize it. Good Luck Toumy From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!www.nntp.primenet.com!nntp.primenet.com!usenet.eel.ufl.edu!newsout1.alt.net!news1.alt.net!news.aros.net!news.cs.utah.edu!cc.usu.edu!cc.usu.edu!nntp Newsgroups: bionet.molbio.proteins Subject: IMAC Problem/De-inhibiting enzyme Message-ID: <1996Dec9.153343.90409@cc.usu.edu> From: arsphys@cc.usu.edu (Phil Harrison) Date: Mon, 09 Dec 1996 22:44:35 GMT Nntp-Posting-Host: rm16.frrl.usu.edu X-Newsreader: Forte Free Agent 1.0.82 Lines: 24 I am trying to purify an enzyme away from a contaminating enzyme with very similar properties (size, pI, substrate). I thought an IMAC (metal chelating) column might work, since my enzyme is inhibited by several divalent metal cations to a different degree (more) than the contaminating enzyme is. I assumed that indicated a different degree of binding to the metal ions, and might provide the basis of a separation technique. I tried cobalt first, since it inhibits less strongly than copper or zinc. However, I have not been able to recover any activity from the column. One major peak came off without any inducement (the pH gradient hadn't started yet). The rest of the protein didn't come off in the pH gradient, and had to be washed off with 50 mM EDTA. I can't find activity in either peak assaying with 20 or 100 mM EDTA. I am assuming that the enzyme either came off the column early, taking some cobalt with it, or was washed off when the cobalt was. Can anyone suggest a way to remove the (presumably) bound Co from the enzyme? Since copper and zinc bind more tightly to the IMAC column than cobalt does, might it be easier to get the enzyme away from copper or zinc bound to the IMAC? Thanks a lot for any suggestions. Phil Harrison arsphys@cc.usu.edu From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!NMSU.EDU!hroychow From: hroychow@NMSU.EDU (Hiranya Roychowdhury) Newsgroups: bionet.molbio.proteins Subject: Re: Protein Purification Question Date: 9 Dec 1996 13:52:55 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 61 Sender: daemon@net.bio.net Distribution: world Message-ID: <199612092152.OAA12534@NMSU.Edu> NNTP-Posting-Host: net.bio.net At 09:16 AM 12/9/96 -0800, Jim Campanella wrote: >Hi, >snip< >The problem is this. In a class I have been >teaching, we have been purifying an enzyme >as one of the projects. In the past, the >last purification step is a desalting step >using dialysis. The enzyme is dissolved in >the following common buffer: > >20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, >0.5% Tween20, 0.5% NP-40, and 0.5 mM PMSF. > >The dissolved enzyme is then dialyzed >overnight against the following buffer: > >20 mM Tris-HCl, pH 8, 100 mM KCl, 0.1 mM >EDTA, 50% glycerol, 0.5% NP-40, 0.5% >Tween20, and 1mM DTT. >snip< >My problems: > >A) I forgot that the dialyzing >step serves the secondary purpose of >getting glycerol into the buffer with >the enzyme. So how do I get the glycerol >into the buffer if I use the desalting >column? > >B) It seems to me that if I use the >column I'll be losing some salts >that will be needed to help buffer >the enzyme-- salts that are still >present but equilibrated with dialysis. >Again, how do I solve the problem of >this loss? In fact, the desalting column, when equilibrated with the second buffer, should also 'change' the buffer (including glycerol) for the protein sample. The rule of thumb to achieve desalting and buffer change simultaneously through a column is not to load the protein sample in a volume exceeding 10% of the column (bed) volume. But, it is better add the glycerol to the eluent (I think) after the buffer change has been accomplished. > snip< > >Any suggestions would be greatly >appreciated. > >Thanks, > >Jim Campanella > > >Dr. Hiranya Sankar Roychowdhury Plant Genetic Engineering Lab. New Mexico State University Las Cruces, NM 88003 Ph. (505) 646-5785 hroychow@nmsu.edu From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!rutgers!uwm.edu!news.cse.psu.edu!news3.cac.psu.edu!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!su-news-hub1.bbnplanet.com!arclight.uoregon.edu!usenet.eel.ufl.edu!warwick!lyra.csx.cam.ac.uk!news.ox.ac.uk!news From: Simon Brocklehurst Newsgroups: bionet.molbio.proteins Subject: Re: bi- vs di - my 2 cents... Date: Mon, 09 Dec 1996 12:07:55 +0000 Organization: University of Oxford, UK Lines: 29 Message-ID: <32AC011B.61DE@bioch.ox.ac.uk> References: <58ervk$540@deft.cc.purdue.edu> Reply-To: smb@bioch.ox.ac.uk NNTP-Posting-Host: max17.public.ox.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Hi all, Not sure that this helps...but I'll say it anyway... I could be totally wrong [although it's never been known to happen yet!! ;-)], and I'm not sure if anyone has already said this or not, but I always thought the choice of either bi- or di- as a prefix was governed by the root of the word you want to prefix i.e. bi- is for words with a Latin root (bi- comes from the Latin bis, meaning twice), di- for words with a Greek root (di from the Greek from dis, meaning twice). So since "functional" has a Latin root, I'd say that "difunctional", isn't actually a word. Having said that, technical jargon is often a law unto itself - all sorts of people coin new terms, and new meanings for existing words... sometimes even people who don't know their Latin from their Greek (shocking eh?!?). That don't make it right though... Raman, I'd be interested to hear where your definition of a general distinction between bi- and di- came from - your examples, didn't illustrate it that well e.g. your "bi-" words have Latin roots etc etc... -- Simon Simon M. Brocklehurst Dept. Biochemistry Univeristy of Oxford UK From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!agate!howland.erols.net!vixen.cso.uiuc.edu!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!daemon From: klenchin@facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: IMAC Problem/De-inhibiting enzyme Date: Tue, 10 Dec 96 18:18:32 GMT Organization: UW-Madison Lines: 20 Message-ID: <58iveg$1gee@news.doit.wisc.edu> References: <1996Dec9.153343.90409@cc.usu.edu> NNTP-Posting-Host: f182-217.net.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=KOI8-R Content-Transfer-Encoding: 8bit X-No-Archive: Yes In article <1996Dec9.153343.90409@cc.usu.edu>, arsphys@cc.usu.edu (Phil Harrison) wrote: #I am trying to purify an enzyme away from a contaminating enzyme #with very similar properties (size, pI, substrate). I thought an IMAC #(metal chelating) column might work, since my enzyme is inhibited by #several divalent metal cations to a different degree (more) than the #contaminating enzyme is. I assumed that indicated a different degree #of binding to the metal ions, and might provide the basis of a #separation technique. I tried cobalt first, since it inhibits less #strongly than copper or zinc. However, I have not been able to #recover any activity from the column. One major peak came off #without any inducement (the pH gradient hadn't started yet). The #rest of the protein didn't come off in the pH gradient, and had to be #washed off with 50 mM EDTA. I can't find activity in either peak #assaying with 20 or 100 mM EDTA. Your enzyme dies on the column irreversibly. Happens. Forget about IMAC. - Dima From owner-proteins@net.bio.net Sun Dec 08 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!visi.com!mr.net!news.mid.net!crcnews.unl.edu!manager From: 00049265@bigred.unl.edu (David Brian Frank) Newsgroups: bionet.molbio.proteins Subject: N-terminal blockage Date: 9 Dec 1996 21:33:47 GMT Organization: University of Nebraska--Lincoln Lines: 6 Message-ID: <58i0jr$b0o@crcnis3.unl.edu> NNTP-Posting-Host: bigred.unl.edu Summary: N-terminal blockage X-Newsreader: TIN [UNIX 1.3 950621BETA PL0] Keywords: N-terminal blockage occurs when a certain group is added on to the N-terminus. Such groups such as a methyl or acetyl group provide sufficient blockage. By adding these groups, other amino acids are no longer able to add on the N-terminal amino acid. From owner-proteins@net.bio.net Mon Dec 09 22:00:00 1996 Path: biosci!KRISHNA.DCRT.NIH.GOV!geetha From: geetha@KRISHNA.DCRT.NIH.GOV ("Geetha Vasudevan") Newsgroups: bionet.molbio.proteins Subject: kringle domain proteins... Date: 10 Dec 1996 09:59:39 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <9612101252.ZM7836@krishna.dcrt.nih.gov> NNTP-Posting-Host: net.bio.net Hello netters, I am interested in knowing about the kringle domain proteins. I learnt, they help in membrane binding...But also, some of the proteases, with catalytic triad activity, have kringle domains... What exactly are these domains made up of??? Why are they called so?? What is their role in catalysis??? Any info. would be highly appreciated. Thanks in advance. --GV -- ************************************************************************ *Geetha Vasudevan Ph: (301)402-0506 * *Post Doctoral Researcher Fax:(301)496-2172 * *ABS/LSB, Bldg.12A/Rm.2041 e-mail:geetha@helix.nih.gov* *DCRT, NIH, Bethesda, MD 20892-5626 * *Interested in protein folding-protein function-disease from proteins * ************************************************************************ From owner-proteins@net.bio.net Mon Dec 09 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!www.nntp.primenet.com!nntp.primenet.com!usenet.eel.ufl.edu!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: re bifunctional enzymes Date: 10 Dec 1996 14:16:24 GMT Organization: University of Leicester, UK (PCFS User) Lines: 13 Message-ID: <58jrbo$ht0@falcon.le.ac.uk> References: <584kke$npo@deft.cc.purdue.edu> <01bbe50e$d702ac00$5e09348d@ifiabio1> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) "David Moss" wrote: > ALL enzymes MUST catalyze equally in both directions. But that still means that they catalyse only one reaction, between two sets of substrates, forward and backward as termodynamics commands. The term multi-functional enzyme, if it has to have any meaning, can refer only to enzymes which catalyse different, independent reactions like A + B <-> C + D, E + F <-> G + H Several examples of this kind have been posted in this thread. From owner-proteins@net.bio.net Mon Dec 09 22:00:00 1996 Path: biosci!rutgers!uwm.edu!news.he.net!www.nntp.primenet.com!nntp.primenet.com!news-feed.inet.tele.dk!news.daimi.aau.dk!not-for-mail From: Morten Andreas Geday Newsgroups: bionet.molbio.proteins Subject: Methyltransferase inhibitors Date: Tue, 10 Dec 1996 13:26:44 +0100 Organization: IMSB, Aarhus University, Denmark Lines: 23 Message-ID: <32AD5704.E62@kemi.aau.dk> NNTP-Posting-Host: beauty.kemi.aau.dk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; HP-UX A.09.01 9000/715) Hi All. I am working on crystallisation of a Methyltransferase, and I am looking for a commercial available potent competetive innibitor of the bindingsite of S-Adenosyl-Methionine. So far I know of Sinefungin (delta-Adenosyl-ornithine), but the previous suppliers Serva, CalBiochem and Sigma have all stopped selling it. Any suggestions? Who sells it? Or do You have any leftovers? Morten Andreas Geday Laboratory of Macromolecular Crystallography Institute of Molecular and Structural Biology Aarhus Denmark From owner-proteins@net.bio.net Mon Dec 09 22:00:00 1996 Path: biosci!agate!spool.mu.edu!uwm.edu!www.nntp.primenet.com!nntp.p