Rabbit myocardial lysophospholipase-transacylase. Purification, characterization, and inhibition by endogenous cardiac amphiphiles

R. W. Gross, R. C. Drisdel, B. E. Sobel

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Abstract

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 (M(r) = 63,000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It had a specific activity of 4 μmol/mg/min for fatty acid release and 2 μmol/mg/min for phosphatidylcholine synthesis. Both its hydrolase and transacylase activities were saturated at lysophosphatidylcholine concentration of 20 μM and transacylation was prominent at submicellar concentrations of substrate (2 μM). Fatty acid release obeyed Michaelian kinetics, but Lineweaver-Burk plots of transacylase activity were parabolic. In contrast, plots of the reciprocal of the initial reaction velocity of phosphatidylcholine formation (1/V) verus 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 μM) 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.

Original languageEnglish
Pages (from-to)15165-15172
Number of pages8
JournalJournal of Biological Chemistry
Volume258
Issue number24
StatePublished - 1983

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