Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes.
1. 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. 2. 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 (KD = 0.03 microM), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. 3. 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. 4. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 microM) decreased the affinity of malonyl-CoA binding by an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. 5. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (greater than 80%) inhibition of high-affinity binding, concurrently with greater than 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. 6. 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.
Kolodziej
M P
MP
Hannah Research Institute, Ayr, Scotland, U.K.
Zammit
V A
VA
D000214:Acyl Coenzyme A / Q000494:pharmacology*; D000217:Acyltransferases / Q000037:antagonists & inhibitors*; D000818:Animals; D001384:Azetidines; D001665:Binding Sites; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology*; D008297:Male; D008316:Malonyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology*; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000648:ultrastructure; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D013217:Starvation / Q000201:enzymology
D000214:Acyl Coenzyme A; D001384:Azetidines; D010171:Palmitoyl Coenzyme A; D008316:Malonyl Coenzyme A; C042420:2-tetradecylglycidyl-coenzyme A; D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase