Concentration of enzyme-dependent activation of PLC-beta 1 and PLC-beta 2 by G alpha 11 and beta gamma-subunits.
Differential regulation of PLC-beta 1 and -beta 2 by the G-protein alpha-subunit, G alpha 11, and by G-protein beta gamma-subunits was studied utilizing recombinant PLC-beta 1 and -beta 2. Rat PLC-beta 1 and human PLC-beta 2 were purified after recombinant baculovirus-mediated expression in Sf9 cells. The catalytic properties of the purified recombinant isoenzymes were directly compared to PLC-beta 1 purified from bovine brain and PLC-beta 2 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 AlF(4-)-dependent activation was observed upon reconstitution of rPLC-beta 1 with the G-protein alpha-subunit, G alpha 11. Activation occurred with a concentration dependence on G alpha 11 for activation and elevation in reaction velocity that was similar to that of native PLC-beta 1. In contrast, G alpha 11 promoted only a small elevation in the catalytic rate of recombinant PLC-beta 2, which was also typical of the native isoenzyme. Maximal reaction rates with respect to PLC-beta isoenzyme concentration were achieved and indicated that rPLC-beta 2 required 10-fold greater concentrations of both G alpha 11 and of rPLC-beta 2 for activation than did rPLC-beta 1. rPLC-beta 1 and rPLC-beta 2 were also differentially regulated by beta gamma-subunits. This differential activation was not the result of different concentration dependencies on beta gamma-subunit for activation, but rather, the result of the greater degree to which the catalytic rate of PLC-beta 2 was elevated by beta gamma-subunits when compared to PLC-beta 1.
Paterson
A
A
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599-7365, USA.
Boyer
J L
JL
Watts
V J
VJ
Morris
A J
AJ
Price
E M
EM
Harden
T K
TK
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002417:Cattle; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics; D014422:Turkeys; D010738:Type C Phospholipases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D007527:Isoenzymes; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins