Binding of phospholipase A2 to zwitterionic bilayers is promoted by lateral segregation of anionic amphiphiles.
Catalytic action of phospholipase A2 is appreciably influenced by the organization and dynamics of bilayers of glycerophosphocholines (Apitz-Castro et al. (1988) Biochim. Biophys. Acta 688, 341-348). However, such effects of the quality of the interface are not observed with bilayers of glycerophosphoryl methanol and other anionic phospholipids (Jain et al. (1986) Biochim. Biophys. Acta 860, 435-447). Such differences between the catalytic susceptibility of zwitterionic versus anionic bilayers are due to a large difference in the affinity of the enzyme for these interfaces. Binding to phospholipase A2 to zwitterionic interfaces can be promoted in the presence of certain anionic additives. For example in the pre-steady-state phase of hydrolysis, segregation of the nacently produced products of hydrolysis could promote binding of phospholipase A2 to regions of higher anionic charge density in the zwitterionic interface. In this paper we show that the dynamics of segregation of the nacently produced products of hydrolysis in zwitterionic bilayers can be readily followed by monitoring the fluorescence intensity of the cationic dye NK-529 (Yu and Jain (1989) Biochim. Biophys. Acta 980, 15-22). The fluorescence emission characteristics of NK-529 change appreciably due to self-quenching of the bound dye molecules as the fatty acid molecules segregate in the bilayer. The kinetics of segregation of fatty acids during the course of hydrolysis of bilayers of zwitterionic phospholipids by phospholipase A2 exhibits an unequivocal correlation with a variety of phenomena that are observed during the transition from the pre-steady-state phase to the steady-state phase of hydrolysis in the reaction progress curves as a function of temperature and in the presence of lipophilic additives.
Jain
M K
MK
Department of Chemistry, University of Delaware, Newark 19716.
Yu
B Z
BZ
Kozubek
A
A
D000838:Anions; D002232:Carbocyanines / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005456:Fluorescent Dyes; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology; D013010:Sonication; D013050:Spectrometry, Fluorescence
D000838:Anions; D002232:Carbocyanines; D005227:Fatty Acids; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010743:Phospholipids; D012492:Salts; C051385:1,1',3,3,3',3'-hexamethylindocarbocyanine; C047037:1,2-ditetradecylphosphatidylcholine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D004134:Dimyristoylphosphatidylcholine