Purification and properties of peroxisomal carnitine palmitoyltransferase in chick embryo liver.
Peroxisomal carnitine palmitoyltransferase was purified by solubilization using Tween 20 and KCl from the large granule fraction of the liver of clofibrate-treated chick embryo, DEAE-Sephacel and blue Sepharose CL-6B column chromatography. The peroxisomal carnitine palmitoyltransferase was an Mr 64,000 polypeptide; the mitochondrial carnitine palmitoyltransferase had a subunit molecular weight of 69,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carnitine acetyltransferase was an Mr 64,000 polypeptide. Antibody against purified peroxisomal carnitine palmitoyltransferase reacted only with peroxisomal carnitine palmitoyltransferase, but not with mitochondrial carnitine palmitoyltransferase or carnitine acetyltransferase. In addition, anti-peroxisomal carnitine palmitoyltransferase reacted only with the protein in peroxisomes purified from chick embryo liver by sucrose density gradient centrifugation. Thus, it was confirmed that purified peroxisomal carnitine palmitoyltransferase was a peroxisomal protein. Compared with mitochondrial carnitine palmitoyltransferase, peroxisomal carnitine palmitoyltransferase was extremely resistant to inactivation by trypsin. The pH optimum of peroxisomal carnitine palmitoyltransferase was 8.5, differing from that of mitochondrial carnitine palmitoyltransferase. The Km value of peroxisomal carnitine palmitoyltransferase for palmitoyl-CoA (32 microM) was similar to that of the mitochondrial one, whereas those values for L-carnitine (140 microM), palmitoyl-L-carnitine (43 microM) and CoA (9 microM) were lower than those of mitochondrial carnitine palmitoyltransferase. Peroxisomal carnitine palmitoyltransferase exhibited similar substrate specificities in both the forward and reverse reactions, with the highest activity toward lauroyl derivatives. Furthermore, this enzyme showed relatively high affinities for long-chain acyl derivatives (C10-C16) and similar Km values (30-50 microM) for acyl-CoAs, acylcarnitine and CoA, and a constant Km value (approximately 150 microM) for carnitine. These results indicate that peroxisomal carnitine palmitoyltransferase played a role in the modulation of the intracellular CoA/long-chain acyl-CoA ratio at the hatching stage of chicken when long-chain fatty acids are actively oxidized in peroxisomes.
D000217:Acyltransferases / Q000302:isolation & purification*; D000818:Animals; D002332:Carnitine O-Acetyltransferase / Q000302:isolation & purification; D002334:Carnitine O-Palmitoyltransferase / Q000302:isolation & purification*; D002642:Chick Embryo; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007700:Kinetics; D008099:Liver / Q000196:embryology / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D012995:Solubility; D013379:Substrate Specificity; D014357:Trypsin
D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase; D002332:Carnitine O-Acetyltransferase; D014357:Trypsin