Solubilization and separation of two distinct carnitine acyltransferases from hepatic microsomes: characterization of the malonyl-CoA-sensitive enzyme.
Conditions have been developed for the solubilization of hepatic microsomal carnitine acyltransferase activity in good yield, with excellent long-term stability and with retention of malonyl-CoA sensitivity. Solubilized microsomal carnitine acyltransferase activity can be separated into malonyl-CoA-sensitive and -insensitive activities either by gel filtration on Superdex 200 or by anion-exchange chromatography on Resource Q. On gel filtration the apparent molecular masses of the malonyl-CoA-sensitive and -insensitive activities are approx. 300 kDa and 60 kDa respectively. The malonyl-CoA-sensitive and -insensitive activities have different fatty-acyl-chain-length specificities and different stabilities in the detergent octyl glucoside. Together these findings indicate that the malonyl-CoA-sensitive and -insensitive activities are due to different enzymes. The malonyl-CoA sensitivity of the inhibitable enzyme is markedly increased on reconstitution into soybean L-alpha-lecithin liposomes, demonstrating that phospholipids play a crucial role in the inhibition by this metabolite. Evidence is also provided that the malonyl-CoA-sensitive microsomal carnitine acyltransferase is a different enzyme from the malonyl-CoA-sensitive carnitine palmitoyltransferase found in the mitochondrial outer membrane. The possible physiological role of the two microsomal acyltransferases is discussed.
Broadway
N M
NM
Department of Biochemistry and Molecular Biology, University College London, U.K.
Saggerson
E D
ED
D000214:Acyl Coenzyme A / Q000494:pharmacology; D000818:Animals; D002333:Carnitine Acyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008316:Malonyl Coenzyme A / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility
D000214:Acyl Coenzyme A; D007527:Isoenzymes; D011480:Protease Inhibitors; C077864:etomoxiryl-coenzyme A; D008316:Malonyl Coenzyme A; D002333:Carnitine Acyltransferases