Need help with CAT assays in bacteria

yugtuyg firstname.surname at bbsrc.ac.uk
Fri Aug 23 07:06:33 EST 1996


In article <321397B9.69D7 at bcm.tmc.edu>, "Sarah K. Highlander, Ph.D." <sarahh at bcm.tmc.edu> says:
>
>I am trying to assay cat activity in bacterial extracts. This is my 
>first go-round and could use some help. I am seeing activity using a 
>plasmid-borne cat gene but the background for non-plasmid containing 
>strains seems to be very high. I am concerned that the DMP-acetyl CoA 
>premix is very yellow to begin with and is obscuring the development of 
>color as the reaction proceeds. Is this mixture supposed to be bright 
>yellow? I believe that I made it correctly following the method of Shaw 
>(100 mM Tris, pH 7.8, 0.1 mM acetyl-CoA from yeast, 0.4 mg/ml DTNB).
>My reaction volume is 5 ml of this mixture to which I add 0.1 ml of a 
>clarified sonicated extract from a 10x concentrated overnight culture. I 
>am allowing the reaction to proceed for about an hour at 37°C.
>

Dear Sarah,
 I've recently finished a post-doc in Bill Shaws lab where quite a lot of
CAT work still goes on! Sounds to me like your AcCoA has degraded ie. DTNB
only goes yellow in the presence of free thiols (spontaneous hydrolysis
of AcCoA is also accelerated at a higher pH - try pH7.0-7.5 and reduce
the tris conc. to 20-50mM). As bacteria contain lots of free thiols and
the cytosol is reducing you are bound to get a high background when
using crude extracts.  CAT is a disgustingly efficient enzyme with a very
high Kcat/Vmax and so you can probably assay much less extract if you
believe you have good expression. Also, it's VERY thermostable (pure 
enzyme can be heated to 70 degrees for 10 mins with no loss of activity)
so try heating your clarified extracts to about 50-55 degrees for a few
minutes (a lot of the E.coli proteins will precipitate) and reclarify. This
may help with background problem. Also, perform your assays in real time
in a spec. at 412nm (1ml total volume in disposable plastic cuvettes) as
it's easy to read off the blank/background rates. You may well be able to
identify CAT-positive extracts with only a 5-10 minute assay period.
BUT CHECK THAT AcCoA! If it's degraded you are simply adding a free thiol
(CoA) to your DTNB!! Hope this helps,
Steve.
steven.dunn at bbsrc.ac.uk
Biochem. & Physiol. Dept,
IACR-Rothamsted,
Harpenden
Herts. AL5 2JQ
UK.
+44 1582 763133 x2785


 



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