Isolation and purification of mitochondrial carnitine octanoyltransferase activities from beef heart.

The purpose of this study was to characterize the mitochondrial carnitine octanoyltransferase of beef heart. Carnitine acyltransferase activities were solubilized from isolated beef heart mitochondria using KC1 and the nonionic detergent, Triton X-100, at final concentrations of 1 M and 2%, respectively. Upon fractionation of the solubilized protein on Cibacron Blue Sepharose, two protein peaks with carnitine octanoyltransferase were obtained. These two fractions accounted for all carnitine acyltransferase activity present in the original mitochondrial suspension. The first eluting peak was purified 400-fold by Sephadex G-100 gel filtration, CM-Sepharose ion exchange, and hydroxylapatite chromatography to a single protein of greater than 95% purity. This carnitine acetyltransferase shows highest activity with acetyl and butyryl carnitine and coenzyme A esters. It has a subunit molecular weight of 62,600 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, a native M, of 60,500 on Sephadex G-200 gel filtration, and a PI of 8.20 on sucrose density gradient isoelectric focusing. The second peak of carnitine acyltransferase activity from Cibacron Blue Sepharose was purified 1600-fold by fractionation on Sephadex G-100 gel filtration, QAESephadex ion exchange, and hydroxylapatite chromatography to a single protein of greater than 95% purity. This enzyme was carnitine palmitoyltransferase. It is most active with decanoyl and lauryl ester substrates, has subunit molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a PI of 8.05 on sucrose density gradient isoelectric focusing, and migrates as part of a detergent micelle of apparent M, 510,000 on Sephadex G-200 gel filtration. It is concluded that there are only two carnitine acyltransferase proteins present in beef heart mitochondria, one membrane-bound (carnitine palmitoyltransferase) and one membrane-associated (carnitine acetyltransferase). Each has significant activity toward hexanoyl, octanoyl, and decanoyl carnitine and coenzyme A esters. The presence of a separate medium chain length-specific carnitine acyltransferase in beef heart mitochondria is not confirmed by our results.