Concentration of enzyme-dependent activation of PLC-β1 and PLC-β2 by Gα₁₁ and βγ-subunits

Differential regulation of PLC-β1 and -β2 by the G-protein α-subunit, Gα₁₁, and by G-protein βγ-subunits was studied utilizing recombinant PLC-β1 and -β2. Rat PLC-β1 and human PLC-β2 were purified after recombinant baculovirus-mediated expression in Sf9 cells. The catalytic properties of the purified recombinant isoenzymes were directly compared to PLC-β1 purified from bovine brain and PLC-β2 partially purified from HL60 polymorphonuclear neutrophils. The recombinant isoenzymes were indistinguishable from the native isoenzymes with respect to dependence of reaction velocity on bulk PtdIns(4,5)P₂ substrate concentration, pH, and free Ca²⁺ concentration. Marked AlF₄^t--dependent activation was observed upon reconstitution of rPLC-β1 with the G-protein α-subunit, Gα₁₁. Activation occurred with a concentration dependence on Gα₁₁ for activation and elevation in reaction velocity that was similar to that of native PLC-β1. In contrast, Gα₁₁ promoted only a small elevation in the catalytic rate of recombinant PLC-β2, which was also typical of the native isoenzyme. Maximal reaction rates with respect to PLC-β isoenzyme concentration were achieved and indicated that rPLC-β2 required 10-fold greater concentrations of both Gα₁₁ and of rPLC-β2 for activation than did rPLC-β1. rPLC-β1 and rPLC-β2 were also differentially regulated by βγ-subunits. This differential activation was not the result of different concentration dependencies on βγ-subunit for activation, but rather, the result of the greater degree to which the catalytic rate of PLC-β2 was elevated by βγ-subunits when compared to PLC-β1.