Abstract
Metabolism of lysophosphatidylcholine (LPC), recently implicated in arrhythmogenesis, was characterized in rabbit ventricular homogenates. Activities of four enzymatic pathways were distinguishable after subcellular fractionation and DEAE-Sephacel chromatography including microsomal lysophospholipase, microsomal acyl coenzyme A/LPC acyltransferase, cytosolic lysophospholipase, and cytosolic lysophospholipase-transacylase. Microsomal lysophospholipase activity was attenuated 81% by acidosis comparable to that in ischemic myocardium (pH 6.5) and was inhibited by substrate. LPC acyltransferase was identified in the microsomal fraction based on CoA-dependent phosphatidyl choline synthesis, the positional specificity of acylation of LPC, and identical reaction velocities with both of its labeled co-substrates. LPC acyltransferase had a Vmax of 5.1 nmol/mg/min, a broad pH optimum centered at pH 7, and an apparent Km for LPC and palmitoyl-CoA of 14 microM and 7 microM. Cytosolic lysophospholipase was separated from lysophospholipase-transacylase by DEAE-Sephacel chromatography and distinguished from microsomal lysophospholipase by its broad pH activity curve, Michaelis-Menten kinetics (Vmax = 9.5 nmol/mg/min, Km = 7.5 microM), and lack of substrate inhibition. Lysophospholipase-transacylase was identified in the cytosolic fraction by CoA-independent phosphatidyl choline synthesis and purified 4885-fold from homogenate by ammonium sulfate precipitation, DEAE-Sephacel, hydroxylapatite, gel filtration, and polylysine chromatography. The partially purified enzyme had a transacylase/lysophospholipase activity ratio of 0.6, and transacylation of LPC was prominent at submicellar concentrations of substrate.
Highlights
Microsomal lysophospholipase activity at pH 6.0 was less than 10% that at pH 7.0 at every substrate and protein concentration shown in Fig. 4.Similar pH profiles wereobtained with TrisC1 buffer substituted for phosphate
The maximum velocity was independent of the total concentration of substrate in the incubation medium but was relatedtothesubstrate/protein ratio
This suggests that therelevant kinetic parameter isthe density of LPC in the membrane that is potentially capable of interacting
Summary
CHARACTERIZATION OF METABOLIC PATHWAYS AND PARTIAL PURIFICATION OF MYOCARDIAL LYSOPHOSPHOLIPASE-TRANSACYLASE*. Amphiphiles, including long chain acylcarnitine and lyso- specificity of acylation, and identical reaction velocities when phosphatides, have been implicated as biochemical mediators either of its co-substrates was labeled 2) lysophospholipase of electrophysiologicalderangements in ischemic myocardium (EC 3.1.1.5) (Fig. 1, bottom left) in the microsomal fraction, potentially contributing to malignant ventricular dysrhythmia characterized by kinetic parameters, pH profile, and substrate (1, 2) Such compounds alter electrophysiological behavior of inhibition; 3) lysophospholipase in the cytosolic fraction sepcanine Purkinje fibers and of ventricular muscle in a fashion arated from lysophospholipase-transacylaseactivity by ion closely analogous to myocardial ischemia when as littleas 2% exchange chromatography and characterized by its pH profile, of membrane phospholipid is constituted with amphiphiles maximum velocity, and substrate dependence; and 4) lysoacquired from exogenous sources (3).
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