Purification of rabbit myocardial cytosolic acyl-CoA hydrolase, identity with lysophospholipase, and modulation of enzymic activity by endogenous cardiac amphiphiles.
Rabbit myocardial cytosolic acyl coenzyme A (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 mumol/(mg . min) and an apparent Michaelis constant of 50 microM. Cytosolic acyl-CoA hydrolase and lysophospholipase activities cochromatographed in every fraction of every step. The purified protein was a single band (Mr 23 000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. These results suggest that cytosolic lysophospholipase and palmitoyl-CoA hydrolase activities are catalyzed by a single polypeptide with dual activities. Palmitoyl-CoA competitively inhibited lysophospholipase activity (Ki = 4 microM). Low concentrations (20 microM) of lysophosphatidylcholine or L-palmitoylcarnitine increased palmitoyl-CoA hydrolase activity at low palmitoyl-CoA concentrations but had little effect at high concentrations of palmitoyl-CoA. In contrast, high concentrations (100 microM) of lysophosphatidylcholine or L-palmitoylcarnitine inhibited palmitoyl-CoA hydrolase activity. The results suggest that 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.
D000818:Animals; D007700:Kinetics; D008055:Lipids / Q000494:pharmacology*; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008245:Lysophospholipase / Q000302:isolation & purification*; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D010170:Palmitoyl-CoA Hydrolase / Q000302:isolation & purification*; D010740:Phospholipases / Q000302:isolation & purification*; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology; D013869:Thiolester Hydrolases / Q000302:isolation & purification*
D008055:Lipids; D008244:Lysophosphatidylcholines; D010171:Palmitoyl Coenzyme A; D010740:Phospholipases; D008245:Lysophospholipase; D013869:Thiolester Hydrolases; D010170:Palmitoyl-CoA Hydrolase