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: (1) 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, (2) 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 k_a = 0.5 μM); synergistic activation by 1,2-sn-diolein, phospholipid and calcium ions; and rank order of specific phospholipid activational potency: phosphatidylserine > phosphatidic acid > phosphatidylinositol > phosphatidylethanolamine > 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 (k_d {reversed tilde equals}19 nM), saturable, low-capacity ({reversed tilde equals}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. In addition, catalytic activity is associated with high-affinity, specific phorbol ester-binding properties that correspond closely to the activational characteristics of this enzyme in this target organ.