Studies on carnitine palmitoyl transferase: The similar nature of CPT I (inner form) and CPT O (outer form)

Abstract

Rat liver mitochondrial carnitine palmitoyl transferase (CPT) was found to reside in two mitochondrial locations. Twenty to twenty-five percent of the total CPT activity was easily released and solubilized by digitonin. This activity appeared to be the outer form of CPT (CPT O). The remainder of the activity or the inner CPT (CPT I) was tightly membrane bound. Trypsin digestion of the digitonin prepared mitoplast did not affect residual CPT activity indicating that this activity probably resided on the inner side of the membrane. Following their separation by digitonin treatment, CPT O and CPT I were partially purified 14.7-and 16.7-fold, respectively. The purification of each enzyme involved extraction from the membrane with Tween 80, ammonium sulfate fractionation, gel filtration, and another ammonium sulfate fractionation. The partially purified CPT O and CPT I were found to have identical elution volumes from a G-200 column corresponding to a molecular weight of 430,000. Also they both were found to have nearly identical K m values for palmitoyl-CoA, palmitoyl-carnitine, CoA, and carnitine suggesting they were identical enzymes. The V values could not be compared due to differences in purity, but the ratio of V in the forward direction to V in the reverse direction was identical for CPT I and CPT O again suggesting enzyme identity. Assay of the CPT system “ in situ” by following the reduction of the acyl-CoA dehydrogenase, a flavoprotein, suggested that the activity of CPT I was 450-fold greater than the activity of CPT O when both were present in the intact membrane. These data suggest that “ in situ” factors exist which greatly change the catalytic properties of CPT I compared to CPT O.