Abstract
Lysophospholipase transacylase was purified 214,360-fold to homogeneity from the rat liver 100,000 x g supernatant. After DEAE chromatography, total activity increased 12.9-fold, due to the removal of endogenous inhibitors. The inhibitors were isolated and identified as phosphatidic acid and fatty acid. The final preparation showed a single band on SDS-polyacrylamide electrophoresis with an M(r) of 60,000. Gel filtration through Sephacryl S-200 gave a similar value, suggesting that the enzyme exists as a monomer. Activity was highest at pH 6.0 and was not affected by Ca2+, Mg2+, and EDTA. The enzyme produced glycerophosphocholine (GPC), palmitic acid, and dipalmitoyl-GPC on incubation with 1-palmitoyl-GPC, indicating that the enzyme catalyzed both deacylation and transacylation. The relative rates of deacylation and transacylation were 1:0.3 under standard assay conditions. Km for 1-palmitoyl-GPC and Vmax of hydrolase activity were 91 microM and 12.9 mumol/min/mg, respectively. The enzyme was selective for choline lysophospholipid. Ethanolamine, inositol, and serine lysophospholipids were not good substrates of the enzyme. Phosphatidic acid was a potent, competitive inhibitor of the enzyme with Ki of about 10 microM as determined with 1-stearoyl-2-arachidonoyl glycerophosphate. Although less potent, lysophosphatidic acid, palmitoyl-L-carnitine, and fatty acid were also inhibitory to the enzyme.
Highlights
Of deacylation and transacylation were 1:0.3 under stan- the transacylase activityof lysophospholipase was dard assay conditionsK., for 1-palmitoyl-GPC anV,d first described in rat liver supernatant by Erbland and Mariof hydrolase activity were91 p~ and 12.9 pmollmidmg, netti [16],neither the small or largfeorm enzyme
Phosphatidic acid was a potent, competitive inhibitorcaloizfed in cytosol and mitochondria, but lysophospholipase I1 the enzyme with Kiof about 10 p~ as determined with was in microsomes
If we assume that the transacylase reaction waspurified to homogeneity for the the accumulatedPA is uniformly distributed within the stimufirst time from rat liver supernatant
Summary
Purification of Lysophospholipasefrom Rat Liver-Livers (70.1g wet Purification of Lysophospholipase from Rat liver Supernaweight) were obtained from 7 male Wistar rats weighing about 250 g tant-% avoid artifacts arising from proteolysis, we used pro-. X-100, applied to a column(1.5 x 14 cm) of octyl-Sepharose CL-4B comitantly increasingTriton X-100and ethylene glycol concenequilibrated with solution A containing 0.5 M NaCl and 0.01% Triton X-100, and theneluted with a linear gradient made from 75 mleach of. The yield was ammonium sulfate and centrifuged.The precipitate was dissolvedin 3.4 140%, probably dueto the removal of ammonium sulfate which ml of solutionA containing 0.05% Triton X-100 and applied to a column (2.5 X 45 c m )of Sephacryl S-400equilibrated with solutionAcontaining 0.05%Triton X-100 and eluted with the same solution. The fractions indicated by the bars were used for further purification (A-D), or as the purified enzyme ( E ) .The thin linesrepresent the concentrations of NaCl (A,0-1 M; E , 1-0 M), potassium phosphate (C, 2& 500 m), and ammonium sulfate and Triton X-100 Arrows in E indicate the locations of two molecular mass markers, bovine serum albumin (67 kDa) and cytochrome c (12.5 kDa)
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