Purification and characterization of bovine cardiac calmodulin-dependent myosin light chain kinase.

M.P Walsh,B Vallet,F Autric,J.G Demaille

doi:10.1016/s0021-9258(19)86440-2

Abstract

Myosin light chain kinase, which is located primarily in the soluble fraction of bovine myocardium, has been isolated and purified approximately 1200-fold with 16% yield by a three-step procedure. The approximate content of soluble myosin light chain kinase in heart is calculated to be 0.63 microM. The isolated kinase is active only as a ternary complex consisting of the kinase, calmodulin, and Ca2+; the apparent Kd for calmodulin is 1.3 nM. The enzyme also exhibits a requirement for Mg2+ ions. Myosin light chain kinase is a monomeric enzyme with Mr = 85,000. The enzyme exhibits a Km for ATP of 175 microM, and a K0.5 for the regulatory light chain of cardiac myosin of 21 microM. The optimum pH is 8.1. Kinase activity is specific for the regulatory light chain of myosin. The specific activity of the isolated enzyme (30 nmol 32P/min/mg of protein) is considerably less than and corresponding values reported for the skeletal and smooth muscle light chain kinases. This is probably due to proteolysis during extraction of the myocardium, a phenomenon which has, as yet, proven impossible to eliminate. In contrast to the smooth muscle enzyme (Adelstein, R.S., Conti, M.A., Hathaway, D.R., and Klee, C.B. (1978) J. Biol. Chem. 253, 8347-8350), the cardiac kinase is not phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.

Highlights

Myosin light chain kinase, which is located primarily in the soluble fraction of bovine myocardium, has been isolated and purified approximately
To verify that all the myosin light chain kinase activity was, located in the soluble fraction, myofibrillar pellets were extracted under the following conditions: (a) 1 volume of Buffer A containing Triton X-100
Proteolysis of Myosin Light Chain Kinase-We considered the possibility that the low specific activity of the isolated myosin light chain kinase may be due to phosphorylation of a serine or threonine residue close to either terminus of the molecule; such phosphorylation sites are known to exist in, for example, glycogen synthase [47, 48], smooth muscle myosin light chain kinase [8], cGMP-dependent protein kinase [49], and many other proteins

Summary

PROCEDURES

Materials-[y-32P]ATP (300 to 400 mCi/mmol) was prepared by the method of Glynn and Chappell [19] from 32Pi In certain experiments (indicated in the text) a partially purified, calmodulin-free regulatory light chain fraction was prepared from the appropriate light chain fraction by ion exchange chromatography performed essentially as described by Nairn and Perry [27] This fraction was devoid of contaminating calmodulin as evidenced by its lack of activation of calmodulin-deficient myosin light chain kinase; it contained approximately. The eluate from each slice was assayed for kinase activity as described above after addition of 0.4 mg of partially purified regulatory light chain fraction of bovine cardiac myosin. The reaction was terminated by adding 30 1.11of the reaction mixture (containing 5 pg of myosin light chain kinase) to an equal volume of 0.5 M Tris-HCl, pH 6.8, 1% sodium dodecyl sulfate, 30%. Myosin light chain kinase activity was assayed immediately as described above in the presence of 0.1 mM CaC12 or 5 mM EGTA

RESULTS

Regulatory

DISCUSSION

Methods