Inhibition of human cytosolic phospholipase A2 by human annexin V.
The ability of annexins, particularly annexin 1 (lipocortin 1), to inhibit phospholipase A2 (PLA2) is well known and a substrate depletion mechanism is now widely accepted as the explanation for most inhibitory studies. However, there are only a very limited number of reported studies involving annexins and the high-molecular-mass cytosolic PLA2 (cPLA2). In this study we have examined the effect of human recombinant annexin V, a potentially abundant cytosolic protein, on the ability of human recombinant cPLA2 to hydrolyse a variety of phospholipid substrates. The results show clearly that, under the conditions of our study, annexin V can inhibit cPLA2 activity by a mechanism of substrate depletion and that this inhibition is dependent on the nature of the phospholipids and the concentration of Ca2+ ions in the assay. The hydrolysis of 1-stearoyl 2-arachidonyl phosphatidylcholine by cPLA2 was not significantly affected by annexin V over a range of Ca2+ concentrations (1 microM-2.5 mM), a result that presumably reflects the zwitterionic nature of the phospholipid and the known inability of annexins to bind to such interfaces. In contrast, the hydrolysis of dioleoyl phosphatidylglycerol, which is an effective anionic phospholipid substrate for this enzyme, and more significantly that of 1-stearoyl 2-arachidonyl phosphatidic acid, were readily inhibited by annexin V, although these effects were Ca2+-dependent. The Ca2+ concentrations required for inhibition in the assay system in vitro are greater than those associated with Ca2+-stimulated events within the cell, suggesting that a role for annexin V in regulating cPLA2 activity might not involve a substrate depletion mechanism in vivo unless factors in addition to Ca2+ and phospholipids contribute to the binding of annexin V to cell membranes.
Buckland
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AG
Department of Biochemistry, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, U.K.
Wilton
D C
DC
D017304:Annexin A5 / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D003600:Cytosol / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D010712:Phosphatidic Acids / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000494:pharmacology
D017304:Annexin A5; D004791:Enzyme Inhibitors; D010712:Phosphatidic Acids; D010715:Phosphatidylglycerols; D011994:Recombinant Proteins; C060203:1-stearoyl-2-arachidonoylphosphatidic acid; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium