Purification and characterization of protein kinase C from rabbit iris smooth muscle. Myosin light-chain phosphorylation in vitro and in intact muscle.
Protein kinase C of rabbit iris smooth muscle was purified by the sequential use of three chromatographic steps, i.e. anion-exchange (DEAE-cellulose), gel filtration (Sephadex G-150) and substrate affinity (protamine-agarose), and its properties were investigated by using as substrate myosin light-chain protein (MLC) isolated from the same tissue. The enzyme appeared as a single band on SDS/polyacrylamide-gel electrophoresis, with a molecular mass of approx. 80 kDa. Histone H-1 and iris muscle MLC, but not rabbit skeletal-muscle MLC, were effective substrates for the enzyme, with apparent Km values of 3.0 and 16.6 microM respectively. The enzyme, with MLC as substrate, had the following characteristics. (a) Its activity was dependent on Ca2+ and phosphatidylserine (PS). In the presence of Ca2+ and PS, diolein and phorbol dibutyrate (PDBu) increased its activity by 61 and 65% respectively. Half-maximal activation of the enzyme (Ka) occurred at 10 microM free Ca2+, and in the presence of diolein and PDBu the apparent Ka for Ca2+ was decreased to 3 microM and 2 microM respectively. (b) Studies on the relative potency of various cofactors in activating the enzyme revealed that PS, phorbol myristate acetate and 1-stearoyl-2-arachidonylglycerol were the most potent of the phospholipids, phorbol esters and diacylglycerols respectively. (c) H-7, a protein kinase C inhibitor, inhibited MLC phosphorylation in a dose-dependent manner, with 50% inhibition at 10 microM. (d) Addition of carbamoylcholine (for 1 min) or PDBu (for 25 min) to iris sphincter muscle prelabelled with [32P]Pi specifically increased MLC phosphorylation, and only the stimulatory effect of the muscarinic agonist was blocked by atropine. The data provide additional support for a role for protein kinase C in the contractile response of the iris smooth muscle.
Howe
P H
PH
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-3331.
Abdel-Latif
A A
AA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D007498:Iris / Q000201:enzymology; D009130:Muscle, Smooth / Q000201:enzymology* / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity
D004075:Diglycerides; D010703:Phorbol Esters; D010743:Phospholipids; D011493:Protein Kinase C; D009218:Myosins; D002118:Calcium