Catalytic and receptor-binding properties of the calcium-sensitive phospholipid-dependent protein kinase (protein kinase C) in swine luteal cytosol.
We have evaluated the catalytic and receptor-binding properties of protein kinase C in swine luteal cytosol using two complementary approaches: assay of catalytic activity assessed as the enzymatic transfer of radiolabeled phosphate to histone III-s acceptor protein in the presence of specific phospholipid, diacylglycerol, or phorbol ester and ionic calcium; and, the high-affinity binding of [3H]phorbol-12,13-dibutyrate ([3H]PDB) to the protein kinase C receptor. Catalytic properties of pig luteal protein kinase C included: absolute dependence on calcium ions for maximal activation (approximate ka = 0.5 microM); synergistic activation by 1,2-sn-diolein, phospholipid and calcium ions; and rank order of specific phospholipid activational potency: phosphatidylserine greater than phosphatidic acid greater than phosphatidylinositol greater than phosphatidylethanolamine greater than phosphatidylcholine. The enzyme was also activated by specific phorbol esters at the following half-maximally effective (ED50) concentrations: 12-O-tetradecanoylphorbol-13-acetate (TPA) 11 nM; phorbol-12,13-dibenzoate (PDBe) 26 nM; phorbol-12,13-dibutyrate (PDBu) 33 nM; mezerein 65 nM; and phorbol-12,13-diacetate (PDA) 130 nM. Phorbol-ester receptor properties of protein kinase C included specific, high-affinity (kd congruent to 19 nM), saturable, low-capacity (congruent to 44 pmol/mg protein) [3H]PDB binding sites. Moreover, the rank order of the equilibrium binding ID50s for various phorbol compounds was similar to that of catalytic ED50s: viz. 3 nM TPA; 8 nM PDBe; 16 nM PDBu; 19 nM mezerein; and 590 nM PDA. Thus, swine luteal cytosol contains catalytically active protein kinase C with specific phospholipid sensitivity, synergistic activation by diacylglycerol, phospholipid and calcium, and a strict dependence on ionic calcium concentrations that is influenced markedly by the presence of diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)
Wheeler
M B
MB
Veldhuis
J D
JD
D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D003338:Corpus Luteum / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D011112:Polymyxin B / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000378:metabolism*; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004075:Diglycerides; D010703:Phorbol Esters; D010718:Phosphatidylserines; D011955:Receptors, Drug; D011971:Receptors, Immunologic; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D011112:Polymyxin B; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium