Cyclic adenosine 3',5'-monophosphate-dependent regulation of purified bovine aortic calcium/calmodulin-dependent myosin light chain kinase.
Myosin light chain kinase was extracted from bovine aortic muscularis by a low ionic strength buffer containing 50% glycerol. It was purified 130-fold with a 10% yield by anion-exchange chromatography followed by affinity chromatography on calmodulin-Sepharose. The enzyme was 95% calcium/calmodulin-dependent and exhibited a specific activity of 2-6 mumol/min per mg. It phosphorylated the myosin regulatory light chain exclusively. The apparent Kd for calmodulin was 6.3 nM. Upon phosphorylation of the enzyme by the catalytic subunit of cyclic AMP-dependent protein kinase, its affinity for calmodulin decreased 4-fold, without alteration of the V. When examined by SDS-polyacrylamide gel electrophoresis, the purified enzyme was made up of two major peptides (Mr 142 000 and 131 000, respectively), with a minor 80 000 dalton peptide. All these peptides were 32P-labeled after incubation with [gamma-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. Also, after non-denaturing polyacrylamide gel electrophoresis, they all exhibited myosin light chain kinase activity, suggesting that the 131 000 and 80 000 dalton species are proteolytic products of the native enzyme of Mr 142 000. Vascular smooth muscle myosin light chain kinase is therefore soluble, calcium/calmodulin dependent and phosphorylatable by cyclic AMP-dependent protein kinase with concomitant decrease in its affinity for calmodulin. These features account for the beta-adrenergic relaxation of vascular smooth muscle.
D000818:Animals; D001011:Aorta / Q000201:enzymology; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase