Isolation of two myosin light-chain kinases from bovine carotid artery and their regulation by phosphorylation mediated by cyclic AMP-dependent protein kinase.
Myosin light-chain kinase was purifed from bovine carotid artery. Approx. 90% of myosin kinase was extracted in the supernatant fraction with buffer containing EDTA during myofibril preparation. The soluble fraction yielded two distinct peaks on DEAE-Sephacel chromatography. Peak I was eluted at a conductance of 11-12mmho and was completely dependent on Ca(2+)-calmodulin for its activity. Peak II was eluted at a conductance of 13-14mmho and showed approx. 15% Ca(2+)-independent activity. The myosin kinases I and II were further purified by affinity chromatography by using calmodulin coupled to Sepharose 4B, which resulted in 960-and 650-fold purification of type I and type II kinases respectively. Myosin kinase II activity was completely Ca(2+)-dependent after affinity chromatography on the calmodulin-Sepharose column. Myosin kinases I and II were phosphorylated by cyclic AMP-dependent protein kinase. In the presence of bound calmodulin 0.5-0.7mol of phosphate was incorporated/mol of myosin kinases I and II. On the other hand, in the absence of bound calmodulin 1-1.4mol of phosphate was incorporated/mol of kinases I and II. Phosphorylation in the absence of calmodulin significantly decreased the myosin kinase activity of both enzymes, and the decrease in myosin kinase activity was due to a 3-5-fold increase in the amount of calmodulin required for half-maximal stimulation of both type I and type II kinases. The regulation of myosin kinase activity by cyclic AMP-dependent phosphorylation would suggest that beta-adrenergic-mediated relaxation of vascular smooth muscle may be partly due to the direct interaction of cyclic AMP at the site of contractile proteins.
D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002852:Chromatography, Ion Exchange; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*
D002147:Calmodulin; D007527:Isoenzymes; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase