KINETIC-ANALYSIS OF PHOSPHOLIPASE C-BETA ISOFORMS USING PHOSPHOLIPID-DETERGENT MIXED MICELLES - EVIDENCE FOR INTERFACIAL CATALYSIS INVOLVING DISTINCT MICELLE BINDING AND CATALYTIC STEPS

Phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P-2) hydrolysis by three different beta-isoforms of phospholipase C (PLC) was examined to investigate the catalytic action of these extracellular signal-regula ted enzymes. Depletion of phospholipase C from solution by incubation with sucrose-loaded vesicles of differing compositions followed by ult racentrifugation demonstrated stable attachment of PLC to the vesicles from which an equilibrium association constant of PLC with PtdIns (4, 5)P-2 could be determined. A mixed micellar system was established to assay PLC activity using dodecyl maltoside, which behaved as an essent ially inert diluent of PtdIns (4,5)P-2 with respect to PLC beta activi ty. Kinetic analyses were performed to test whether PLC beta activity was dependent on both bulk PtdIns (4,5)P-2 concentration and surface c oncentration in the micelles as has been shown for other lipid metabol ising enzymes. Each of the PLC beta isoforms behaved similarly in thes e analyses, which indicated the involvement of at least two binding ev ents. Interfacial Michaelis constants were calculated to be between 0. 1-0.2 mol fraction for all three enzymes, and K-s (the equilibrium dis sociation constant of PLC for lipid) ranged between 100-200 mu M. The apparent multiple interfacial binding events did not appear to result from lipid-induced PLC beta oligomerization implying that PLC beta mon omers possess more than one lipid-binding site, Surface dilution of PL C catalyzed PtdIns (4,5)P-2 hydrolysis was assessed in the presence of increasing concentrations of various nonsubstrate phospholipids, whic h profoundly reduced PLC activity, suggesting that these lipids may in hibit enzyme action. The data indicate that G protein-regulated isofor ms of PLC operate with separate lipid binding and catalytic steps and imply that under physiological conditions, PLC beta isoforms operate u nder first-order conditions. These findings may have implications for the mechanisms of regulation of PLC beta s by G protein subunits.