Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes

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Abstract
The interaction of malonyl-CoA with the outer carnitine palmitoyltransferase (CPT) system of rat liver mitochondria was re-evaluated by using preparations of highly purified outer membranes, in the light of observations that other subcellular structures that normally contaminate crude mitochondrial preparations also contain malonyl-CoA-sensitive CPT activity. In outer-membrane preparations, which were purified about 200-fold with respect to the inner-membrane-matrix fraction, malonyl-CoA binding was largely accounted for by a single high-affinity component (K1D = 0.03 .mu.M), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. There was no evidence that the decreased sensitivity of CPT to malonyl-CoA inhibition observed in outer membranes obtained from 48-h-starved rats (compared with those from fed animals) was due to a decreased ratio of malonyl-CoA binding to CPT catalytic moieties. Thus CPT specific activity and maximal high-affinity [14C]malonyl-CoA binding (expressed per mg of protein) were increased 2.2- and 2.0-fold respectively in outer membranes from 48 h-starved rats. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 .mu.M) decreased the affinity of malonyl-CoA binding be an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (> 80%) inhibition of high-affinity binding, concurrently with > 95% inhibition of CPT activity. These treatments also unmasked an effect of subsequent treatment with palmitoyl-CoA to increase low-affinity [14C]malonyl-CoA binding. These data are discussed in relation to the possible mechanism of interaction between the malonyl-CoA-binding site and the active site of the enzyme.

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