ACTIVITY OF CARNITINE PALMITOYLTRANSFERASE IN MITOCHONDRIAL OUTER MEMBRANES AND PEROXISOMES IN DIGITONIN-PERMEABILIZED HEPATOCYTES - SELECTIVE MODULATION OF MITOCHONDRIAL ENZYME-ACTIVITY BY OKADAIC ACID

A procedure is described for the rapid measurement of the activity of mitochondrial-outer-membrane carnitine palmitoyltransferase (CPT(o)) and peroxisomal carnitine palmitoyltransferase (CPT(p)) in digitonin-permeabilized hepatocytes. CPT(o) activity was determined as the tetradecylglycidate (TDGA)-sensitive malonyl-CoA-sensitive CPT activity, whereas CPT(p) activity was monitored as the TDGA-insensitive malonyl-CoA-sensitive CPT activity. Under these experimental conditions, the respective contributions of CPT(o) and CPT(p) to total hepatocellular malonyl-CoA-sensitive CPT activity were 74.6 and 25.4%, which correlated well with the values of 76.9 and 23.1% for the respective contributions of the mitochondrial and the peroxisomal compartment to total hepatocellular palmitate oxidation. The sensitivity of CPT(o) to inhibition by malonyl-CoA was very similar to that of CPT(p); thus 50% inhibition of CPT(o) and CPT(p) activities was achieved with malonyl-CoA concentrations of 2.6 +/- 0.5 and 3.0 +/- 0.4 muM respectively. Short-term incubation of hepatocytes with the phosphatase inhibitor okadaic acid (i) increased the activity of CPT(o) and the rate of mitochondrial palmitate oxidation, (ii) decreased the affinity of CPT(o) for palmitoyl-CoA substrate, and (iii) decreased the sensitivity of CPT(o) to inhibition by malonyl-CoA. By contrast, neither the properties of CPT(p) nor the rate of peroxisomal palmitate oxidation were changed upon incubation of cells with okadaic acid. Results indicate therefore that CPT(o), but not CPT(p), may be regulated by a mechanism of phosphorylation/dephosphorylation. The physiological relevance of these findings is discussed.