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.
Why not buffer your adenine nucleotide pools and clamp your ATP/ADP ratio
with 5 mM adenine nucleotide, 20 mM creatine plus 20 mM creatine phosphate,
plus excess creatine kinase? You can inhibit adenylate kinase specifically
by adding diadenosine pentaphosphate.
Martin Brand
Martin D. Brand
Department of Biochemistry
University of Cambridge
Tennis Court Road
Cambridge CB2 1QW
UK
phone +44 1223 333648
fax +44 1223 333345
