On 1 Jun 1995, Simon Eaton wrote:
> I have a problem in my incubations with heart mitochondria in that I'm
> doing incubations looking at beta-oxidation flux from palmitate; hence
> I need ATP (for acyl-CoA synthase) and ADP (for oxidative
> phosphorylation). This doesn't cause a problem in muscle or liver mitos,
> but in heart I run out of ATP very quickly in the absence of added
> substrate; most of it appears to end up in AMP rather than ADP so it
> doesn't appear to be myofibrillar ATPase, unless it's going via adenylate
> kinase. I can't think of a way of regenerating ATP, apart from using
> pyuruvate kinase/ PEP, which regenerates ATP, but also eventually yields
> acetyl-CoA, which could confuse the issue of beta-oxidation itself.
>>Simon,
It sounds you've got a strong activity of acyl-CoA synthetase
Acyl-OH + Mg.ATP <--> Acyl-AMP + Mg.PPi
Acyl-AMP + CoASH ---> Acyl-CoA + AMP
Your ATP is used up but transformed into AMP rather than ADP. The
pyruvate kinase reaction recycles ADP to ATP. On the other hand Ox-Phos
does the same thing. Thus you may start your reaction with ADP only, it
will turn to ATP (Ox-Phos and/or Pyruvate kinase) and be all depleted by
acyl-CoA synthetase, to get a net reaction:
Mg.ADP + Pi --> AMP + Mg.PPi.
For recycling nucleotides, you could poise the system by INCREASING the
adenylate kinase activity (add some more to help the mitochondrial
intermembrane or myofibrillar adenylate kinase), so that the following
equilibrium is rapidly achieved, relative to your reactions (Ox-Phos and
acyl-CoA synthase):
AMP + Mg.ATP <--> ADP + Mg.ADP
or even better
AMP + Mg.GTP <--> ADP + Mg.GDP
The latter will allow you to use CATALYTIC AMOUNTS of adenine nucleotides
while GTP (added in excess) will be consumed.
Cheers,
Claude
Claude Aflalo ###########################################################
Dept. of Life Sciences Phones: Office 972-7-472118
Ben Gurion University of the Negev Lab 972-7-472119
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Beer Sheva 84105 Israel email: aflaloc at bgumail.bgu.ac.il
