Purification of rabbit myocardial cytosolic acyl-CoA hydrolase, identity with lysophospholipase, and modulation of enzymic activity by endogenous cardiac amphiphiles

Abstract

Rabbit myocardial cytosolic acyl CoA hydrolase activity was purified to near-homogeneity by ammonium sulfate precipitation and ion-exchange, gel filtration, chromatofocusing and hydroxylapatite chromatographies. Kinetic analysis of the purified protein demonstrated a maximum velocity of 24 .mu.mol/(mg.cntdot.min) and an apparent Km of 50 .mu.M. Cytosolic acyl-CoA hydrolase and lysophospholipase activities cochromatographed in every fraction of every step. The purified protein was a single band (MW 23,000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Cytosolic lysophospholipase and palmitoyl-CoA hydrolase activities apparently are catalyzed by a single polypeptide with dual activities. Palmitoyl-CoA competitively inhibited lysophospholipase activity (Ki = 4 .mu.M). Low concentrations (20 .mu.M) of lysophosphatidylcholine or L-palmitoylcarnitine increased palmitoyl-CoA concentrations but had little effect at high concentrations of palmitoyl-CoA. In contrast, high concentrations (100 .mu.M) of lysophosphatidylcholine or L-palmitoylcarnitine inhibited palmitoyl-CoA hydrolase activity. Evidently, interactions between endogenous cardiac amphiphiles and palmitoyl-CoA hydrolase contribute to the regulation of intracellular long-chain acyl-CoA concentrations and therefore potentially modulate fluxes of fatty acid through several biochemical pathways.