Characterization of myosin light-chain kinase from bovine adrenal medulla.
Partially purified bovine adrenal medullary myosin light-chain kinase (MLCK) possesses a Stoke's radius of 79 A and a sedimentation coefficient of 3.95 +/- 0.45 S, yielding a native molecular weight of 150,000 +/- 17,000 g/mol and a frictional ratio of 2.24. It catalyzes the phosphorylation of the isolated light chain of skeletal muscle myosin and the light chain of intact adrenal medullary myosin, but not phosphorylase b or histone. The activation of MLCK by calmodulin is specific and dose dependent, yielding a K0.5 value of 9.0 nM; the dose response curve with respect to free Ca2+ is biphasic, exhibiting a stimulatory phase at low free Ca2+ concentrations (K0.5 = 0.17 microM) and an inhibitory phase at higher free Ca2+ concentrations (400-3000 microM). Michaelis-Menten kinetics are observed for ATP, yielding a Km for ATP of 25 microM and a Vmax of 23.2 nmol/min/mg. However, positive cooperative kinetics are observed for the skeletal muscle myosin light chain, yielding a Hill coefficient of 3.57, a K0.5 for light chain of 27 microM and a Vmax of 16.6 nmol/min/mg. A stoichiometry of phosphorylation of approximately 1 mol of phosphate/mol of skeletal muscle myosin light chain was observed. Therefore, adrenal medullary MLCK is similar in most, but not all, of its physical and kinetics properties to MLCKs isolated from other sources and may serve to regulate actin-myosin contractile activity in the adrenal medulla.
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004589:Electrophoresis, Disc; D004789:Enzyme Activation; D007700:Kinetics; D009219:Myosin-Light-Chain Kinase; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D002147:Calmodulin; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D002118:Calcium