GTPase assay for low molecular weight GTP binding proteins

Patti W. Jareo pjareo at creighton.edu
Mon Jun 23 15:18:12 EST 1997


I am attempting to determine whether the low molecular weight
GTP-binding protein I have detected is also exhibiting GTPase activity. 
I have found numerous references for assays, mostly measuring activity
in subunit-type G proteins, however.  I am still following up some leads
in the literature, but if anyone could point me at a specific assay, it
would help tremendously!

I do have another question, as well.  I repeatedly see that App(NH)p
(adenylyimidophosphate) and Gpp(NH)p (guanylylimidophosphate) are used
in these assays, and I cannot, for the life of me, figure out WHY one is
used rather than the other, or exactly what they are supposed to do! 
The Gpp... is a nonhydrolyzable GTP analog, and the App... has a similar
relationship to ATP.  If a nonhydrolyzable ATP analog "plugs up" any
ATPases present, I can understand how that would eliminate the potential
contribution of any ATPases to the hydrolysis of labeled GTP added as
substrate.  The GTP analog is supposed to be a Gs activator... but if it
is a nonhydrolyzable analog, I'm confused as to what it is supposed to
be doing in this reaction!  Does anyone know WHY these compounds are
used in GTP hydrolysis assays, and how would that impact an assay
specifically intended to measure hydrolysis by low molecular weight G
proteins rather than the larger subunit type G-proteins??????  

I have Sigma fact sheets.  I have a ream of papers.  ANY help would be
greatly appreciated!  And I will continue to look, as well.. I'll hit
the library again tomorrow morning.....

Thanks, folks.... ::sigh:: 

Patti W. Jareo, Ph.D.
Creighton University
Omaha NE  68178



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