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 AlF4t--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.
|Number of pages||12|
|State||Published - Sep 1995|
Bibliographical noteFunding Information:
in producing the pVLI392/PLC-\[32 plasmid construct, and Drs. Rob Nicholas and Theresa M. Filtz for much valuable advice and assistance. We are indebted to Drs S. G. Rhee and J. L. Knopf for the gifts of the pIBI-PLC-~ 1 and pMT-PLC-\[~2 recombinant plasmids, respectively. We also thank John W. O'Tuel for his help in preparing this manuscript. A. P. was the recipient of a Wellcome Trust Travel Fellowship, and A. J. M. was the recipient of an American Heart Association Grant-In-Aid, #911524.
ASJC Scopus subject areas
- Cell Biology