| Popis: |
Phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2) hydrolysis by three different β-isoforms of phospholipase C (PLC) was examined to investigate the catalytic action of these extracellular signal-regulated enzymes. Depletion of phospholipase C from solution by incubation with sucrose-loaded vesicles of differing compositions followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles from which an equilibrium association constant of PLC with PtdIns (4, 5) P2 could be determined. A mixed micellar system was established to assay PLC activity using dodecyl maltoside, which behaved as an essentially inert diluent of PtdIns (4, 5) P2 with respect to PLCβ activity. Kinetic analyses were performed to test whether PLCβ activity was dependent on both bulk PtdIns (4, 5) P2 concentration and surface concentration in the micelles as has been shown for other lipid metabolising enzymes. Each of the PLCβ isoforms behaved similarly in these analyses, which indicated the involvement of at least two binding events. Interfacial Michaelis constants were calculated to be between 0.1-0.2 mol fraction for all three enzymes, and K(the equilibrium dissociation constant of PLC for lipid) ranged between 100-200 μM. The apparent multiple interfacial binding events did not appear to result from lipid-induced PLCβ oligomerization implying that PLCβ monomers possess more than one lipid-binding site. Surface dilution of PLC-catalyzed PtdIns (4, 5) P2 hydrolysis was assessed in the presence of increasing concentrations of various nonsubstrate phospholipids, which profoundly reduced PLC activity, suggesting that these lipids may inhibit enzyme action. The data indicate that G protein-regulated isoforms of PLC operate with separate lipid binding and catalytic steps and imply that under physiological conditions, PLCβ isoforms operate under first-order conditions. These findings may have implications for the mechanisms of regulation of PLCβs by G protein subunits. |
