pyrophoshatasing cell extract for nucleotide depletion?

D.K. dk at no.email.thankstospam.net
Mon Aug 5 20:38:07 EST 2002


Johannes Graumann <graumann at sue.caltech.edu> wrote:
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>>D.K. <dk at no.email.thankstospam.net>                                           
>                                                          
>>Johannes Graumann <graumann at clyde.caltech.edu> wrote:
>>>Hello,
>>>
>>>If I were to make a cell extract and throw in pyrophosphatase - would 
>>>I manage to deplete it from nucleotide triphosphates?
>>
>>Very likely. Same is regularly done with non-specific phosphatases
>>and apyrase (said to be adenine-specific but happily chews up 
>>everythting else, it seems).
>>
>>>Also, am I right
>>>that g-S-ATP wouldn't be affected by this?
>>
>>I don't think so. As a rule, kinases have really hard time using NTPgS
>>analogs but phosphatases work kinda OK, since kcat is generally only
>>only several-fold lower than native substrates (this has been used 
>>widely in cell biology to distinguish between phosphorylation/
>>dephosphorylation pathways).
>>
>
>Thanks for the reply! Do you have any idea how I could combine a 
>depletion for triphosphates with the providing of g-S-ATP in a cell 
>extract?

The best I can come up with is to use hexokinase with glucose.
It depletes ATP levels incredibly fast, given a ~ mM glucose. 
Following this an excess of ATPgS should do the job I assume you 
need. Any residual ATP synthesized from other NTPs and/or ADP
should not be a problem as hexokinase will remain active.
(Doing all the right controls still applies). Hexokinase is dirt cheap
from Sigma, BTW. 

If you can provide a more detailed description of the experiment 
you are trying to do, odds are good there is a better solution. 

DK







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