Calcium-independent myosin light chain kinase of smooth muscle. Preparation by limited chymotryptic digestion of the calcium ion dependent enzyme, purification, and characterization.
Limited alpha-chymotryptic digestion of Ca2+-, calmodulin-dependent myosin light chain kinase partially purified from smooth muscle (turkey gizzard) yielded a Ca2+-independent form of the enzyme. Digestion to yield the Ca2+-independent kinase required the enzyme complexed with Ca2+-calmodulin; when digestion was performed on the apoenzyme, i.e., in the absence of Ca2+, the dependence of kinase activity on Ca2+ was retained. The Ca2+-independent kinase was purified by ion-exchange chromatography and shown to have an apparent molecular weight of approximately 80000. The specific activity of the freshly prepared enzyme was 6.5 +/- 0.2 mumol of Pi incorporated min-1 mg-1 in the presence of Ca2+ and 8.3 +/- 0.3 mumol min-1 mg-1 in the absence of Ca2+, using the isolated light chains of gizzard myosin as the substrate. The Ca2+-independent enzyme also phosphorylated the 20000-dalton light chains of purified myosin and crude actomyosin from turkey gizzard. The Km of the Ca2+-independent kinase for Mg2+-ATP (54 muM) was not significantly different from that of the native, CA2+-dependent enzyme (68 muM). These observations indicate maintenance of the integrity of the active site after digestion with alpha-chymotrypsin. It is suggested that the loss of Ca2+ sensitivity of the kinase after limited proteolysis is due to loss of the calmodulin-binding site from the 80000-dalton fragment. The two sites of phosphorylation by the cyclic AMP dependent protein kinase were also removed by the chymotryptic hydrolysis.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000378:metabolism*; D005895:Gizzard, Avian / Q000201:enzymology; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase; D009994:Osmolar Concentration; D011494:Protein Kinases / Q000378:metabolism*; D014422:Turkeys
D002147:Calmodulin; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D002918:Chymotrypsin; D002118:Calcium