Calmodulin-linked equilibria in smooth muscle myosin light chain kinase

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Abstract
Competition experiments using 9-anthroylcholine, a fluorescent dye that undergoes calmodulin-dependent binding by smooth muscle myosin light chain kinase [Malencik, D. A., Anderson, S. R., Bohnert, J. L., and Shalitin, Y, S. (1982) Biochemistry 21, 4031], demonstrate a strongly stabilizing interaction between the adenosine 5''-triphosphate and myosin light chain binding sites operating within the enzyme-calmodulin complex but probably not in the free enzyme. The interactions in the latter case may be even slightly destabilizing. The fluorescence enhancement in solutions containing 5.0 .mu.M each of the enzyme and calmodulin is directly proportional to the maximum possible concentration of bound calcium on the basis of four calcium binding sites. Evidently, all four calcium binding sites of calmodulin contribute about equally to the enhanced binding of 9-anthroylcholine by the enzyme. Fluorescence titrations on solutions containing 1.0 .mu.M enzyme plus calmodulin yield a Hill coefficient of 1.2 and K = 0.35 .+-. 0.08 .mu.M calcium. Three proteolytic fragments of smooth myosin ligh chain kinase, apparent products of endogenous proteolysis, were isolated and characterized. All three possess calmodulin-dependent catalytic activity. Their interactions with 9-anthroylcholine, in both the presence and absence of calmodulin, are similar to those of the native enzyme. However, the stabilities of their complexes with calmodulin vary. The corresponding dissociation constants range from 2.8 nM for the native enzyme and 8.5 nM for the 96 K fragment to .apprx. 15 nM for the 68K and 90K fragments [0.20 N KCl, 50 mM 3-(N-morpholino)propanesulfonic acid, and 1 mM CaCl2, pH 7.3, 25.degree. C]. A coupled fluorometric assay, modified from a spectrophotometric assay for adenosine cyclic 3'',5''-phosphate dependent protein kinase [Cook, P. F., Neville, M. E., Vrana, K. E., Hartl, F. T., and Roskoski, R. (1982) Biochemistry 21, 5794], has provided the first continuous recordings of myosin light chain kinase is a responsive enzyme, whose activity adjusts rapidly to changes in solution conditions.