Purification and characterization of phosphatidylcholine phospholipase D from pig lung.
Phospholipase D, which mediates phosphatidylcholine hydrolysis in response to agonist stimulation, is an important component of signal transduction. We now report the purification of this enzyme to homogeneity from pig lung microsomes. The enzyme was solubilized with heptylthioglucoside and purified 2,200-fold by successive chromatography on sulfate-Cellulofine, ether-Toyopearl, chelate-Toyopearl, Q-Sepharose, heparin-Toyopearl, and hydroxyapatite. The final enzyme preparation gave a single protein band of M(r) = 190,000 on SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed phosphatidylcholine but not lysophosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Optimum pH was 6.6. Half-maximal activity was obtained at 0.8 mM dipalmitoylglycerophosphocholine. The products were identified as phosphatidic acid and choline, but in the presence of ethanol, phosphatidylethanol was produced at the expense of phosphatidic acid. Ethanolamine and serine were not utilized as the phosphatidyl acceptor. Although not obligatory, Ca2+ and Mg2+ were stimulatory at high concentrations. The enzyme was markedly stimulated by unsaturated fatty acids in the presence of Mg2+ but not in its absence or by saturated fatty acids. N-Ethylmaleimide and detergents were inhibitory. Sucrose monolaurate had an aberrant effect on enzyme activity.
Okamura
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Department of Biochemistry, Gunma University School of Medicine, Maebashi, Japan.
Yamashita
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D000818:Animals; D002413:Cations, Divalent; D002850:Chromatography, Gel; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005227:Fatty Acids / Q000494:pharmacology; D006868:Hydrolysis; D008055:Lipids / Q000494:pharmacology; D008168:Lung / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D002413:Cations, Divalent; D003902:Detergents; D005227:Fatty Acids; D008055:Lipids; D010713:Phosphatidylcholines; D010739:Phospholipase D