Enhanced interfacial catalysis and hydrolytic specificity of phospholipase A2 toward peroxidized phosphatidylcholine vesicles.
The hydrolytic action of phospholipase A2 was examined with unilamellar vesicles composed of soybean phosphatidylcholine in terms of the calcium dependency of the enzyme and substrate specificity following lipid peroxidation. Experiments were performed under conditions where enzyme:substrate ratios were low, specifically in the range of one to five enzyme molecules for every 10 vesicle particles. Accordingly, low hydrolytic activities were found where less than 15% of the phospholipids were hydrolyzed under the various conditions of time, enzyme:substrate ratios, calcium concentrations, and extent of peroxidation utilized. Vesicle peroxidation increased the Ca2+ binding potential to a degree comparable to addition of the anionic phospholipid, dioleoylphosphatidic acid (DOPA). A remarkable similarity was found between the binding profiles for Ca2+ and phospholipase A2 activity; however, enzyme activity toward oxidized vesicles was beyond the increases observed for Ca2+ binding. Under conditions where approximately 5% of the phospholipids were peroxidized the effective Ca2+ concentration required for half-maximal activity was less than one-half that required for unoxidized vesicles. Peroxidation of vesicle phospholipids markedly increased the rate and extent of hydrolysis, even in the presence of DOPA or deoxycholate. Deoxycholate is known to induce vesicle fusion such that a larger proportion of enzyme is associated with a fewer number of enlarged vesicles. Using a dual isotope technique to measure hydrolysis of oxidized vs unoxidized phospholipids and covesicle preparations to study enzyme binding and activity, a significantly greater apparent intervesicle exchange of enzyme was found after peroxidation of vesicles with more than a twofold hydrolytic specificity toward the oxidized phospholipids. We postulate that a combination of structural and Ca2+ binding affinity changes are produced in membranes following lipid peroxidation which evoke an additive effect on PLA2 activity. Although oxidized phospholipids may serve as activators of phospholipase A2 by presenting the interface in a form where Ca2+ and enzyme binding and/or specific activity are increased, an additional and important factor appears to involve membrane fusion or vesicle-vesicle interactions. This process facilitates enzyme activity through the replenishment of substrates wherein the otherwise limited interaction of enzyme and substrate is overcome by more rapid or extensive vesicle fusion which increases access to the phospholipids available in the preparation.
Salgo
M G
MG
Department of Pathology, University of Southern California, Los Angeles 90033.
Corongiu
F P
FP
Sevanian
A
A
D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D006868:Hydrolysis; D007700:Kinetics; D008054:Lipid Peroxides / Q000378:metabolism; D008081:Liposomes; D010713:Phosphatidylcholines / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity
D008054:Lipid Peroxides; D008081:Liposomes; D010713:Phosphatidylcholines; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium