Membrane lipids have multiple effects on interfacial catalysis by a phosphatidic acid-preferring phospholipase A1 from bovine testis.
We previously purified a cytosolic phospholipase A1 that could catalyze the preferential hydrolysis of phosphatidic acid in mixed-micelle assays. Here we studied the enzyme's interactions with unilamellar lipid membranes and examined effects of the lipids on enzyme binding, stability, and catalysis. A major finding was that membrane lipids could influence the stability, activity, and specificity of the enzyme under conditions where enzyme binding to the membranes was likely to be saturated. Thus, the enzyme was unstable at 37 degrees C in the absence of membranes but bound to membranes that contained anionic phosphoglycerides and could be stabilized by these membranes in the presence of albumin. The overall activity of the bound enzyme toward membrane phosphoglycerides, assayed in the presence of albumin, increased when phosphatidylethanolamine was substituted for phosphatidylcholine. Furthermore, the enzyme's catalytic preference for phosphatidic acid increased when cholesterol and diacylglycerol were included in the membranes, sn-1-stearoyl-2-arachidonoylphosphatidylethanolamine was substituted for sn-1-palmitoyl-2-oleoylphosphatidylethanolamine, and the concentration of phosphatidic acid was increased from 0 to 10 mol % of the total membrane phosphoglycerides. Finally, changes in the relative contents of phosphatidylcholine and phosphatidylserine in the membranes influenced the enzyme's catalytic preference for different molecular species of phosphatidic acid. These results provide the first available information about the enzyme's ability to interact with membranes and identify conditions that yield high enzyme activity toward membrane-associated phosphatidic acid.
Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, University of Washington, Seattle 98195-7370, USA.
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Higgs
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D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D004075:Diglycerides / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D020404:Glycerophospholipids / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010712:Phosphatidic Acids / Q000737:chemistry* / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D054466:Phospholipases A1; D011485:Protein Binding; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*
C011439:1,2-diacylglycerol; D004075:Diglycerides; D005227:Fatty Acids; D020404:Glycerophospholipids; D008563:Membrane Lipids; D010712:Phosphatidic Acids; D010714:Phosphatidylethanolamines; D002784:Cholesterol; D010741:Phospholipases A; D054466:Phospholipases A1