Isolation of a malonyl-CoA-sensitive CPT/beta-oxidation enzyme complex from heart mitochondria.
The goal of this study was to establish conditions for solubilization and characterization of CPTo, the malonyl-CoA sensitive form of mitochondrial carnitine palmitoyltransferase. CPTo of heart mitochondria is soluble in 1% octyl glucoside with retention of malonyl-CoA sensitivity. The degree of malonyl-CoA sensitivity is dependent on both the concentration of octyl glucoside and the presence of salt (KCl). In mannitol-sucrose, 0.5-1% octyl glucoside solubilizes CPTo without loss of malonyl-CoA sensitivity; however, either increasing the detergent concentration or addition of KCl promotes loss of malonyl-CoA sensitivity. The immunoglobulin fraction from immune serum obtained from rabbits immunized with the malonyl-CoA-insensitive form of CPT (CPTi) purified from beef heart mitochondria was used for preparation of an affinity column. The antibody column retained both malonyl-CoA-sensitive and -insensitive CPT activity without apparent selectivity. In addition to CPT, several other major protein bands were detected when the antibody column eluates were subjected to SDS-PAGE; however, native gel electrophoresis gives a large, high molecular weight, diffuse band. After elution of the antibody-CPT column with salt, a 68,000-Da protein is retained by the column. The retained protein contains the CPT activity, but it is not inhibited by malonyl-CoA. Thus, salt elution separates catalysis from inhibition. When the salt eluate is subjected to affinity chromatography using agarose-CoA, two protein peaks are obtained; both bind malonyl-CoA. One of the two fractions contains beta-hydroxyacyl-CoA dehydrogenase, beta-ketothiolase, and crotonase activity. These data show that octyl glucoside solubilized CPTo and CPTi are associated with a complex that contains beta-oxidation enzymes.
Kerner
J
J
Department of Biochemistry, Michigan State University, East Lansing 48824.
Bieber
L
L
D000214:Acyl Coenzyme A / Q000494:pharmacology*; D000217:Acyltransferases / Q000302:isolation & purification*; D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D044943:Fatty Acid Desaturases / Q000302:isolation & purification / Q000378:metabolism; D008316:Malonyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats
D000214:Acyl Coenzyme A; D008316:Malonyl Coenzyme A; D044943:Fatty Acid Desaturases; D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase