Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2.
Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A(2) (sPLA(2)s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA(2)s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 microm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA(2)s, with Me-Indoxam being the most generally potent sPLA(2) inhibitor. A dramatic correlation exists between the ability of the sPLA(2)s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA(2)s are uniquely efficient in this regard.
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D051379:Mice; D015394:Molecular Structure; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010741:Phospholipases A; D004229:Dithiothreitol