> S.J. 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.
Is there a particular reason for using palmitate as your substrate?
Unless you need to know the rate of flux through acyl-CoA synthase, it
is much easier to measure the rate of beta-oxidation in isolated
mitochondria using palmityl-carnitine as substrate. If this substrate
would suffice for the scientific question you are trying to answer, it
would be a way to avoid the problem of ATP utilization/regeneration
entirely.
M.E. Martens
