Calcium-independent myosin light chain kinase of smooth muscle. Preparation by limited chymotryptic digestion of the calcium ion dependent enzyme, purification and characterization

Publisher Website
Google Scholar
Abstract
Limited .alpha.-chymotryptic digestion of Ca2+-, calmodulin-dependent myosin L 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 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 MW of .apprx. 80,000. The specific activity of the freshly prepared enzyme was 6.5 .+-. 0.2 .mu.mol of Pi incorporated min-1 mg-1 in the presence of Ca2+ and 8.3 .+-. 0.3 .mu.mol min-1 mg-1 in the absence of Ca2+, using the isolated L chains of gizzard myosin as the substrate. The Ca2+-independent enzyme also phosphorylated the 20,000-dalton L chains of purified myosin and crude actomyosin from turkey gizzard. The Km of the Ca2+-independent kinase for Mg2+-ATP (54 .mu.M) was not significantly different from that of the native, Ca2+-dependent enzyme (68 .mu.M). These observations indicate maintenance of the integrity of the active site after digestion with .alpha.-chymotrypsin. The loss of Ca2+ sensitivity of the kinase after limited proteolysis is apparently due to loss of the calmodulin-binding site from the 80,000-dalton fragment. The 2 sites of phosphorylation by the cAMP dependent protein kinase were also removed by the chymotryptic hydrolysis.