title: Receptor- and G-protein-regulated 150-kDa avian phospholipase C: inhibition of enzyme activity by isoenzyme-specific antisera and nonidentity with mammalian phospholipase C isoenzymes established by immunoreactivity and peptide sequence.
abstract: A 150-kDa phospholipase C previously was purified from turkey erythrocytes and shown to be a P2Y-purinergic receptor- and guanine nucleotide-binding protein-regulated enzyme [J. Biol. Chem. 265:13508-13514 (1990)]. The relationship of this enzyme to the 150-kDa mammalian phospholipase C isoenzymes, termed phospholipase C-beta and -gamma, has been examined. Four antisera to the turkey erythrocyte phospholipase C recognized the avian enzyme in immunoblots but failed to recognize phospholipase C-gamma; one of the these weakly recognized phospholipase C-beta. Antibodies to phospholipase C-beta and -gamma failed to recognize the turkey erythrocyte phospholipase C. However, two antibodies raised against peptide sequence in regions of conserved sequence common to mammalian phospholipase C isoenzymes recognized the 150-kDa turkey erythrocyte phospholipase C. Antisera against the native form of the turkey erythrocyte phospholipase C inhibited the activity of this enzyme against phosphatidylinositol 4,5-bisphosphate presented as a component of mixed phospholipid vesicles or of mixed phospholipid and sodium cholate micelles; inhibition occurred as a decrease in Vmax, with no apparent change in Km for substrate or in the Ca2+ dependence of phospholipase C activity. Catalytic activity of phospholipase C-beta or -gamma against exogenous substrate was unaffected by antisera to the turkey erythrocyte enzyme. Antisera against the native form of the turkey erythrocyte phospholipase C also partially inhibited (50-60% inhibition) the capacity of AIF4- or adenosine 5'-O-(beta-thio) diphosphate plus guanosine 5'-O-(gamma-thio) triphosphate to stimulate phosphoinositide hydrolysis in ghosts prepared from [3H]inositol-prelabeled turkey erythrocytes. Moreover, the capacity of the purified 150-kDa enzyme to reconstitute receptor and G-protein-regulated phospholipase C activity in purified turkey erythrocyte plasma membranes devoid of this activity was completely inhibited by antisera to the turkey erythrocyte enzyme. Five peptides that were purified by high performance liquid chromatography from a tryptic digest of the turkey erythrocyte 150-kDa phospholipase C had no recognizable sequence homology with any deduced sequence of the mammalian phospholipase C isoenzymes. One turkey erythrocyte phospholipase C-derived peptide had clear homology with sequence in the first (X-domain) conserved region common to at least three of the mammalian phospholipase C isoenzymes, and another 16-amino acid peptide had partial sequence homology with the second (Y-domain) conserved region common to the mammalian enzymes. An 8-amino acid peptide from the tryptic digest had 75% homology with a sequence near the carboxyl terminus of mammalian phospholipase C-beta.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599.
authors.forename: G L
authors.initials: GL
authors.lastname: Waldo
authors.forename: A J
authors.initials: AJ
authors.lastname: Morris
authors.forename: D G
authors.initials: DG
authors.lastname: Klapper
authors.forename: T K
authors.initials: TK
authors.lastname: Harden
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D004912:Erythrocytes / Q000201:enzymology; D019204:GTP-Binding Proteins / Q000502:physiology*; D006868:Hydrolysis; D007106:Immune Sera; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011489:Protein Denaturation; D011817:Rabbits; D011983:Receptors, Purinergic / Q000502:physiology*; D012689:Sequence Homology, Nucleic Acid; D014422:Turkeys / Q000097:blood*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000276:immunology
chemical_list: D000906:Antibodies; D007106:Immune Sera; D007527:Isoenzymes; D010716:Phosphatidylinositols; D010743:Phospholipids; D011983:Receptors, Purinergic; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins