Rabbit myocardial lysophospholipase-transacylase. Purification, characterization, and inhibition by endogenous cardiac amphiphiles.
Rabbit myocardial lysophospholipase-transacylase was purified 69,000-fold to near homogeneity by ammonium sulfate precipitation, DEAE-Sephacel, hydroxylapatite chromatography, and high precision liquid chromatography. The purified protein was a single band (Mr = 63,000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It had a specific activity of 4 mumol/mg/min for fatty acid release and 2 mumol/mg/min for phosphatidylcholine synthesis. Both its hydrolase and transacylase activities were saturated at a lysophosphatidylcholine concentration of 20 microM and transacylation was prominent at submicellar concentrations of substrate (2 microM). Fatty acid release obeyed Michaelian kinetics, but Line-weaver-Burk plots of transacylase activity were parabolic. In contrast, plots of the reciprocal of the initial reaction velocity of phosphatidylcholine formation (1/V) versus 1/[S]2 were linear. Computer simulations of a reaction mechanism in which two molecules of substrate formed a ternary complex with the enzyme resulted in linear Lineweaver-Burk plots for fatty acid release and linear 1/V versus 1/[S]2 plots for phosphatidylcholine synthesis. Low concentrations of long chain acylcarnitine (5-20 microM) markedly inhibited both fatty acid release and phosphatidylcholine synthesis. Inhibition of lysophospholipase-transacylase by L-palmitoylcarnitine was reversible by dilution or dialysis. Since long chain acylcarnitines increase in the cytosolic compartment of ischemic myocardium, these results suggest that inhibition of lysophospholipase-transacylase by long chain acylcarnitines contributes to the accumulation of lysophosphoglycerides in ischemic myocardium with consequent deleterious effects on membrane function.
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D003201:Computers; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008245:Lysophospholipase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008956:Models, Chemical; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D009206:Myocardium / Q000201:enzymology*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000494:pharmacology; D010172:Palmitoylcarnitine / Q000494:pharmacology; D010740:Phospholipases / Q000302:isolation & purification*; D011817:Rabbits; D013379:Substrate Specificity
D008244:Lysophosphatidylcholines; D009097:Multienzyme Complexes; D010169:Palmitic Acids; D010172:Palmitoylcarnitine; D019308:Palmitic Acid; D000217:Acyltransferases; D010740:Phospholipases; D008245:Lysophospholipase; C021782:lysophospholipase-transacylase