Fatty acid and phospholipid selectivity of different phospholipase A2 enzymes studied by using a mammalian membrane as substrate.
Previous studies using phospholipid mixed vesicles have demonstrated that several types of phospholipase A2 (PLA2) enzymes exhibit different selectivity for fatty acids at the sn-2 position, for the type of chemical bond at the sn-1 position or for the phosphobase moiety at the sn-3 position of phospholipids. In the present study, we have utilized natural mammalian membranes from U937 monocytes to determine whether two purified 14 kDa PLA2 isoenzymes (Type I, Type II) and a partially purified 110 kDa PLA2 exhibit substrate selectivity for certain fatty acids or phospholipids. In these studies, arachidonic acid (AA) release from membranes was measured under conditions where the remodelling of AA mediated by CoA-independent transacylase (CoA-IT) activity has been eliminated. In agreement with the mixed-vesicle models, AA was the major unsaturated fatty acid hydrolysed from membranes by the 110 kDa PLA2, suggesting that this PLA2 is selective in releasing AA from natural membranes. By contrast, Type I and Type II PLA2s were less selective in releasing AA from phospholipids and released a variety of unsaturated fatty acids at molar ratios that were proportional to the ratios of these fatty acids in U937 microsomal membranes. Examination of AA release from phospholipid classes indicated that all three enzymes released AA from the major AA-containing phospholipid classes (phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol) of U937 membranes. The 110 kDa PLA2 released AA from phospholipid subclasses in ratios that were proportional to the AA content within phospholipid classes and subclasses of U937 membranes. These data suggested that the 110 kDa PLA2 shows no preference either for the sn-1 linkage or for the sn-3 phosphobase moiety of phospholipids. By contrast, Type I and Type II PLA2s preferentially released AA from ethanolamine-containing phospholipids and appeared to prefer the 1-acyl-linked subclass. Taken together, these data indicate that the 110 kDa PLA2 selectively releases AA from U937 membranes, whereas Type I and Type II PLA2 release a variety of unsaturated fatty acids. Furthermore, the 110 kDa PLA2 releases the same molar ratios of AA from all major phospholipid subclasses, whereas Type I and Type II PLA2s show some specificity for phosphatidylethanolamine when these enzymes are incubated with a complex mammalian membrane substrate.
Diez
E
E
Department of Cell Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Chilton
F H
FH
Stroup
G
G
Mayer
R J
RJ
Winkler
J D
JD
Fonteh
A N
AN
D016718:Arachidonic Acid / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D007938:Leukemia; D008563:Membrane Lipids / Q000378:metabolism*; D009000:Monocytes; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D007527:Isoenzymes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D010743:Phospholipids; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2