Characterization of hepatic carnitine palmitoyltransferase. Use of bromoacyl derivatives and antibodies.
Carnitine palmitoyltransferase (CPT) is a mitochondrial-inner-membrane enzyme, with activities located on both the outer and inner sides of the membrane. The inhibition of CPT by bromopalmitate derivatives was studied in intact hepatic mitochondria (representing CPT-A activity, the outer enzyme), in inverted submitochondrial vesicles (representing CPT-B, the inner enzyme), and in purified hepatic CPT. Bromopalmitoyl-CoA had an I50 (concentration giving 50% inhibition of CPT activity) of 0.63 +/- 0.08 microM in intact mitochondria and 2.44 +/- 0.86 microM in inverted vesicles. Preincubation of mitochondria with bromopalmitoyl-CoA decreased V max. for both CPT-A and CPT-B. Sonication decreased sensitivity to bromopalmitoyl-CoA, and solubilization with Triton abolished sensitivity at the concentrations used (0-10 microM). Purified CPT had a bromopalmitoyl-CoA I50 of 353 microM in aqueous buffer, 67 microM in 20% dimethyl sulphoxide, 45 microM in phosphatidylcholine liposomes and 26 microM in cardiolipin liposomes. Increasing [carnitine] at constant bromopalmitoyl-CoA concentrations or increasing [bromopalmitoyl-CoA] in the preincubation resulted in increased inhibition of purified CPT. 2-Tetradecylglycidyl-CoA and malonyl-CoA did not offer measurable protection against bromopalmitoyl-CoA inhibition of the purified CPT, suggesting a different site of interaction of bromopalmitoyl-CoA with CPT. The data suggest that the sensitivity of CPT to bromopalmitoyl-CoA may be modulated by membrane environment and assay conditions.
D000214:Acyl Coenzyme A / Q000031:analogs & derivatives*; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002331:Carnitine / Q000031:analogs & derivatives*; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D017830:Octoxynol; D010171:Palmitoyl Coenzyme A / Q000031:analogs & derivatives* / Q000494:pharmacology; D010172:Palmitoylcarnitine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013010:Sonication
C046844:2-bromopalmitoyl coenzyme A; D000214:Acyl Coenzyme A; D000906:Antibodies; D003902:Detergents; D008081:Liposomes; D010171:Palmitoyl Coenzyme A; D010172:Palmitoylcarnitine; C052814:2-bromopalmitoylcarnitine; D011092:Polyethylene Glycols; D017830:Octoxynol; D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine