The kinetics of the phospholipase A2-catalyzed hydrolysis of Egg phosphatidylcholine in unilamellar vesicles. Product inhibition and its relief by serum albumin.
Only the lecithin in the outer leaflet (representing 70% of the total) of egg lecithin unilamellar vesicles is hydrolyzed by Crotalus atrox phospholipase A2. Hydrolyzed vesicles remain intact and impermeable to ionic solutes. The fatty acids produced in the hydrolysis remain on the vesicle and are only partially ionized at neutral pH due to electrostatic repulsions. About 40% of the lysolecithin product is desorbed from the vesicle. In the presence of a large excess of bovine serum albumin, the reaction is first order with respect to both the enzyme and the substrate. At 21 degrees C, pH 7.2, I = 0.16 M, and [Ca2+] = 7 mM, the second order rate constant is kex(2) = 1.5 X 10(6) M-1 s-1. In the absence of albumin, the reaction is inhibited competitively by both the monomeric (KIm = 4.5 X 10(-8) M) and micellar (nKIa = 3.7 X 10(-7) M) forms of lysolecithin ([critical micelle concentration] = 4.3 X 10(-6) M). Bovine serum albumin complexes two molecules of lysolecithin with a dissociation constant, Kb = 5 X 10(-8) M. With substoichiometric albumin, the reaction is biphasic, and, when the albumin is saturated with lysolecithin, the kinetics become similar to those observed in the absence of albumin. The action of phospholipase A2 shows that in unilamellar vesicles there is only one major lecithin conformation in the outer leaflet, or that all conformations are rapidly interconvertible.
D003435:Crotalid Venoms / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005227:Fatty Acids / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D012710:Serum Albumin, Bovine / Q000494:pharmacology*
D003435:Crotalid Venoms; D005227:Fatty Acids; D008081:Liposomes; D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; D012710:Serum Albumin, Bovine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2