Characterization of a solubilized malonyl-CoA-sensitive carnitine palmitoyltransferase from the mitochondrial outer membrane as a protein distinct from the malonyl-CoA-insensitive carnitine palmitoyltransferase of the inner membrane

Abstract

By using octyl glucoside in the presence of glycerol, it is possible to obtain a solubilized malonyl-CoA-sensitive carnitine palmitoyltransferase (CPT(o)) from the outer membranes of rat liver mitochondria. H.p.l.c. on hydroxyapatite column has now allowed a clear separation of the CPT(o) from the malonyl-CoA-insensitive CPT activity of the inner membranes (CPT(i)). The seprated CPT(o) activity showed inhibition by low micromolar concentrations of malonyl-CoA, 2-tetradecylglycidyl-CoA and etomoxir-CoA. On solubilization and fractionation, the CPT(o) rapidly lost activity, unlike the relatively stable CPT(i) activity. Reconstruction into asolectin liposomes enhanced the activity and the malonyl-CoA-sensitivity of the CPT(o) fractions, whereas it had no such effect on the activity or malonyl-CoA insensitivity of the CPT(i) fractions. A polyclonal antibody raised against the malonyl-CoA-insensitive enzyme, purified from the inner membrane, precipitated the CPT(i) activity, but showed no reactivity with the CPT(o) fractions. In Western blots, the above antibody did not react with any polypeptide of the CPT(o) fractions. Incubation of the outer-membrane preparations with [3H]etomoxir, in the presence of ATP and CoA, led to labelling of a 90 kDa polypeptide that in the above hydroxyapatite chromatography was eluted in the same region as the CPT(o). No such polypeptide labelling was seen in the CPT(i) fractions. With heart and skeletal-muscle mitochondria, the correspondingly labelled polypeptide was of about 86 kDa. These results show that the CPT(o) and CPT(i) are distinct proteins, that a subunit of 90 kDa for liver and 86 kDa for muscle constitutes a component of their respective CPT(o) systems, and that the 66 kDa subunit of the CPT(i) does not constitute a part of the CPT(o) system.