A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. II. P2Y-purinergic receptor and G-protein-mediated regulation of the purified enzyme reconstituted with turkey erythrocyte ghosts.
The preceding paper describes purification and properties of a 150-kDa polyphosphoinositide-specific phospholipase C from a cytosolic fraction of turkey erythrocytes (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507). Turkey erythrocytes express a P2Y-purinergic receptor that employs an unidentified G-protein to activate phospholipase C (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890; Cooper, C. L., Morris, A. J., and Harden, T. K. (1989) J. Biol. Chem. 264, 6202-6206). This paper describes receptor and G-protein regulation of the purified turkey erythrocyte phospholipase C after reconstitution of the enzyme using [3H]inositol pre-labeled turkey erythrocyte ghosts as acceptor membranes. These membranes contain polyphosphoinositides labeled to high specific radioactivity and display reduced responsiveness of their endogenous phospholipase C to P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of purified enzyme had no effect on basal inositol phosphate production, but markedly increased P2Y-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of purified phospholipase C with 10 micrograms of acceptor membrane protein produced half-maximal effects, and maximal activity was observed with reconstitution of 100 ng of purified enzyme. Agonist and guanine nucleotide-regulated phospholipase C activity measured using a reconstitution assay co-purified with phospholipase C activity detected using exogenously provided phosphatidylinositol 4,5-bisphosphate during purification of the 150-kDa protein. Only the maximal rate of inositol phosphate formation attained upon activation was increased in the presence of the purified phospholipase C. K0.5 values for adenosine 5'-O-(2-thiodiphosphate), guanosine 5'-3-O-(thio)triphosphate, and A1F4- activation of the purified enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes.
Morris
A J
AJ
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599.
Waldo
G L
GL
Downes
C P
CP
Harden
T K
TK
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000097:blood* / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007294:Inositol / Q000097:blood; D007295:Inositol Phosphates / Q000097:blood; D007700:Kinetics; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000097:blood* / Q000302:isolation & purification; D011983:Receptors, Purinergic / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology; D014422:Turkeys
D007295:Inositol Phosphates; D011983:Receptors, Purinergic; D013873:Thionucleotides; C030812:adenosine 5'-O-(2-thiodiphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D019204:GTP-Binding Proteins; D002118:Calcium