Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria.
A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.
Ramsay
R R
RR
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Derrick
J P
JP
Friend
A S
AS
Tubbs
P K
PK
D000217:Acyltransferases / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008830:Microbodies / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000031:analogs & derivatives / Q000494:pharmacology; D013379:Substrate Specificity
C046844:2-bromopalmitoyl coenzyme A; D000596:Amino Acids; D010171:Palmitoyl Coenzyme A; D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase