continuing GST #2 story

Terry Hanzlik terryh at ento.csiro.au
Tue Jul 19 03:04:44 EST 1994


> >
> > On 15 Jul 1994 15:14:16 GMT, Stephen R. Lasky wrote:
> >
> > (some stuff deleted)
> >
> > >>In general though,  we use denaturation in the presence of SDS so that a
> > >>protein will migrate at a rate relative to its mass and not be effected by
> > >>amino acid composition.
> >
> > >That's the generally accepted therory and, no doubt, many if not most
> > >proteins fit nicely into that scheme. But there are exceptions, just one
> > >example (might be interesting for Jean-Marc and Emma): There is a protein
> > >called dTAF40 because on a SDS gel it has the apparent molecular mass of 40
> > >kd. Molecular cloning of the cDNA revealed a molecular mass of only 29 kd
> > >(Cell 75, 519-530, 1993). Could anybody kindly give an explanation for this
> > >unusual migration on a SDS gel?
> >
> > >Matthias Zeiner
> > >Inst. Biol. Chem.
> > >Heidelberg
> >
> > Dear Matthias,
> >
> > It is highly likely that the proteins in your question
> > that migrate as larger proteins on SDS-PAGE have a high proline
> > content.  This phenomenon involving proline has been noted many
> > times before; for
> > example see Pham and Sivasubarmanian in GENE 122, 345 (1993) or
> > Ziemer, Mason and Carlson in JBC 257, 11176 (1982).  My own
> > personal experience with such a protein is one from an insect
> > small RNA virus which is 17 kDa yet migrates at Mr = 24k (>140%
> > more apparent MW).
> > It is a PEST protein (a property correlated with rapid turnover
> > and possessed by many regulatory genes and oncogenes) and has a
> > 49% of P, E, S, and T.
> > However, to the nub of your question as to why proline does this,
> > I and the literature I've seen are still unaware.  Anybody else
> > have a handle on this?
> >
> > Terry Hanzlik, terryh at ento.csiro.au
> > CSIRO Division of Entomology
> > Box 1700
> > Canberra, ACT  2601
> > Australia
> >
> >
> >
> Dear Terry Hanzlik:
>
> Certain proteins, amoung them glycoproteins and possibly prolin as well,
> absorb SDS at a different ratio than other proteins.  Consequently,
> those proteins have a different surface net charge density and one
> cannot compare them with standard proteins by using just one SINGLE
> gel concentration.  A way out is the Ferguson plot technique which
> I have just explained 30 minutes ago in a detailed message to Matthias
> and to the methods-and-reagents group.
>
> If you have more questions or cannot find my detailed message on the
> network, please contact me.
>
> Best regards, Dietmar Tietz
>


Dear Dietmar,
Thanks for your note to me.  I thought those kind of plots were called
Smith-Hedrick plots.  That may be due to my having Hedrick as my
lab instructor at the University of California, however.  I must
say that your discussion is much more thorough than his handout
or old paper in Anal Biochem back in the '70s.  I think his paper came
out the same time as Fergeson's paper and it appears that
Fergeson won out in the immortality stakes.

 But your reply, like mine, does
not deal with the real question--why does SDS not bind at the
"proper" ratio to certain proteins?  Anybody doing work on the arcane subject
of SDS binding to proteins or has done an X-ray crystal structure an SDS
denatured protein (that last is a joke)?  Also, I think glycopoteins interaction
with acrylamide matrix and not with SDS is responsible for the
analomous migration of these kind of proteins.

Regards,

Terry Hanzlik, terryh at ento.csiro.au
CSIRO Division of Entomology
Box 1700
Canberra, ACT 2601
Australia

PS.  As I am getting some requests for refs on PEST proteins, I include
the following on this posting:
.
PEST proteins are defined as those proteins having regions rich
in P,E,S, and T bound by basic residues.  A correlation between this
property and rapid turnover in eukaryotic, but not prokaryotic, systems
was originally noted by Rodgers, Wells and Rechsteiner in
Science 234, 364-8 (1986).  Many highly regulated proteins have this
property such as Ornithine decarboxylase or oncogenes.  Since 1986,
 attempts to add or delete these sequences
have had mixed results in supporting their role in regulation.
It appears that PEST sequences do not act as a simple
tag that is recognized by the degrqadative machinery, but may
have a more general structural significance in a protein such as
folding in certain ways or to display flexibility in a way that
 contributes to metabolic instability.  I'm not sure how active
the field is in recent days.  My last ref is 1991.  My feeling is
that there is something to the PEST hypothesis.  These proteins do
funny things and a lot of them play key roles.  My personal experience
is as follows:

A virus I work with, the Helicoverpa armigera stunt virus (HaSV), is
a tetravirus and has only 3 genes, the 186 kDa replicase, the 71 kDa
coat protein and this wierd protein I call p17 which, as I noted
previously, migrates as 24 kDa on SDS-PAGE.  As predicted, the
protein was highly unstable when expressed in baculoviruses but
when expressed in E. coli, turned the poor bacteria into bee hives.
Let me hasten to explain, when I used the bacteria as controls for
bacteria expressing the coat protein, I happened to examine them on
sections by transmission EM.  They were full of flexible, hollow,
hexagonal tubes about 32 nm in diameter and had no specific length
(they looked like a bunch of bee hives).  The protein has no homology
to anything yet specific antisera made to the bacterially expressed
protein reacts strongly to an apparent host cell analog (?).
Examination of the host cell 36 kDa analog with an immunoflourescence
experiment on a confocal shows it to be present only in approx.
0.1% of the cells in discrete areas near or on the outer membrane
surface.  I can't tell what the virus does with the protein as it
doesn't grow well in cultured cells and experiments on live larvae
 are difficult.  My thinking on p17's functionality is that it is
an oncogene or a factor that regulates the transcriptase--thus my
interest in PEST proteins.  However, the tubes might be saying that
it is a protein analogous to certain plant virus movement proteins
 which have been shown to form tubes also (low proline howver)--see
Perbal, Thomas and Maule in Virology 195, 281-5 (1993).

I guess while I have posted this anecdote, I might as well do something
I should have done long ago:  anybody have any similar experiences with
these proteins or have aninput into what p17 is doing?


Thanks.



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