Intramitochondrial control of the oxidation of hexadecanoate in skeletal muscle. A study of the acyl-CoA esters which accumulate during rat skeletal-muscle mitochondrial beta-oxidation of [U-14C]hexadecanoate and [U-14C]hexadecanoyl-carnitine.

1. We describe the acyl-CoA and acyl-carnitine esters which arise from the incubation of well-coupled State 3 rat skeletal-muscle mitochondrial fractions with [U-14C]hexadecanoate and [U-14C]hexadecanoyl-carnitine. 2. Acyl-CoA ester intermediates of chain length 16, 14, 12, 10 and 8 carbons were detected. 3. Although incubations were in steady state in respect of oxygen consumption, 14CO2 production and generation of acid-soluble radioactivity, quantitative analysis of acyl-CoA esters showed that steady state was not achieved in respect of all intermediates. 4. 3-Hydroxyacyl- and 2-enoyl-CoA and -carnitine esters were found under normoxic conditions. 5. Direct measurement of NAD+ and NADH shows that under identical incubation conditions our observations cannot be explained by gross perturbation of the [NAD+]/[NADH] ratio. 6. We hypothesize that there is a small pool of rapidly recycling NAD+ channelled between complex I of the respiratory chain and the newly described mitochondrial-inner-membrane-associated beta-oxidation trifunctional enzyme [Uchida, Izai, Orii and Hashimoto (1992) J. Biol. Chem. 267, 1034-1041].