A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. I. Purification and properties.
Eighty-three percent of polyphosphoinositide-specific phospholipase C activity was recovered in a cytosolic fraction after nitrogen cavitation of turkey erythrocytes. This activity has been purified approximately 50,000-fold when compared to the starting cytosol with a yield of 1.7-5.0%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phospholipase C preparation revealed a major polypeptide of 150 kDa. The specific activity of the purified enzyme was 6.7-14.0 mumol/min/mg of protein with phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 4-phosphate as substrate. Phospholipase C activity was markedly dependent on the presence of Ca2+. The phospholipase C showed an acidic pH optimum (pH 4.0). At neutral pH, noncyclic inositol phosphates were the major products formed by the phospholipase C, while at pH 4.0, substantial formation of inositol 1:2-cyclic phosphate derivatives occurred. Properties of the purified 150-kDa turkey erythrocyte phospholipase C were compared with the approximately 150-kDa phospholipase C-beta and -gamma isoenzymes previously purified from bovine brain (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1987) J. Biol. Chem. 262, 12511-12518). The turkey erythrocyte phospholipase C differed from the two mammalian phospholipases with respect to the effect of sodium cholate on the rate of polyphosphoinositide hydrolysis observed. Moreover, when presented with dispersions of pure inositol lipids, phospholipases C-beta and -gamma displayed comparable maximal rates of polyphosphoinositide and phosphatidylinositol hydrolysis. By contrast, the turkey erythrocyte phospholipase C displays a marked preference for polyphosphoinositide substrates.
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
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D004912:Erythrocytes / Q000201:enzymology* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000097:blood*; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000097:blood* / Q000302:isolation & purification; D011956:Receptors, Cell Surface / Q000502:physiology; D015398:Signal Transduction; D013379:Substrate Specificity; D014422:Turkeys
D002793:Cholic Acids; D006882:Hydroxyapatites; D011956:Receptors, Cell Surface; D017886:Durapatite; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D019204:GTP-Binding Proteins; D019826:Cholic Acid; D002118:Calcium