Kinetics of interfacial catalysis by phospholipase A2 in intravesicle scooting mode, and heterofusion of anionic and zwitterionic vesicles.
In this and the following three papers we examine the kinetics of action of pig pancreatic phospholipase A2 on vesicles of anionic phospholipids without any additives. The results provide the first unequivocal demonstration of interfacial catalysis in intravesicle scooting mode. In this paper we describe the conditions in which the action of pig pancreatic phospholipase A2 on DMPMe (ester) vesicles in the absence of any additive commences without a latency. Under these conditions the free monomer substrate concentration is insignificant; the bilayer enclosed vesicle organization remains intact even when all the substrate in the outer monolayer has been hydrolyzed; the rate of intervesicle exchange and the rate of transbilayer movement (flip-flop) of molecules is negligibly slow; and the rate of fusion of vesicles is insignificant. Thus an enzyme molecule bound to one vesicle hydrolyzes all the DMPMe molecules in the outer monolayer of the vesicle by a first-order process with a rate constant of 0.6 per min at 30 degrees C; or viewed another way, one enzyme molecule in a DMPMe vesicle can hydrolyze all the available substrate molecules at the rate of 3000 per min. At low anion concentrations excess substrate vesicles are not hydrolyzed unless the rate of intervesicle exchange of the bound enzyme is stimulated by anions in the aqueous phase. Higher calcium concentrations promote not only homofusion of DMPMe vesicles but also heterofusion of DMPMe and DMPC vesicles. It is proposed that calcium-induced isothermal lateral phase separation in DMPMe vesicles induces defects in the bilayer organization, and such defects are the sites for phospholipase A2 binding and for heterofusion with DMPC (ester) vesicles which do not have such sites.
D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D020404:Glycerophospholipids; D006868:Hydrolysis; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008561:Membrane Fusion; D010712:Phosphatidic Acids / Q000378:metabolism*; D010740:Phospholipases / Q000494:pharmacology*; D010741:Phospholipases A / Q000494:pharmacology*; D054467:Phospholipases A2; D012235:Rhodamines / Q000378:metabolism
D020404:Glycerophospholipids; D008051:Lipid Bilayers; D010712:Phosphatidic Acids; D012235:Rhodamines; C029313:dimyristoylmethylphosphatidic acid; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; C044542:octadecyl Rhodamine B chloride; D002118:Calcium; D004134:Dimyristoylphosphatidylcholine