Renata Dabrowska

[30] Smooth muscle myosin light chain kinase

Publisher Summary Myosin light chain kinase catalyzes the transfer of the γ-phosphate of adenosine triphosphate (ATP) to a serine residue on a specific class of myosin light chains. Myosin light chain kinases are specific for the phosphorylatable light chains of myosin. Myosin of the same tissue is the preferred substrate of a given kinase. In the case of smooth muscle myosin, the two 20,000-dalton light chains are phosphorylated and the site of phosphorylation is serine-19. The myosin light chain kinase from all muscle sources is dependent on Ca 2+ and calmodulin—a feature that is particularly useful in assay procedures. The assay usually involves the quantitation of acid-stable phosphate incorporated into the myosin light chain using Mg 2+ - [γ- 32 P]ATP as the phosphate donor in the presence and absence of Ca 2+ and calmodulin. Either the isolated 20,000-dalton light chain or the intact myosin molecule can be used as the substrate—that is, phosphate acceptor.

Composition of the myosin light chain kinase from chicken gizzard.

Abstract The Ca 2+ -dependent protein kinase (ATP:myosin light chain phosphotransferase) from chicken gizzard smooth muscle requires two proteins for enzymatic activity. These have approximate molecular weights of 105,000 and 17,000 daltons. The isolation procedure for each component is described. Neither component alone markedly alters either the actin-moderated ATPase activity or the phosphorylation of myosin. Activation of ATPase activity by a combination of the two components occurred only in the presence of Ca 2+ and was always accompanied by the phosphorylation of myosin. The simultaneous activation of ATPase activity and myosin phosphorylation establishes a direct correlation between the two events.

A Ca2+- and modulator-dependent myosin light chain kinase from non-muscle cells

Abstract A myosin light chain kinase has been obtained in a partially purified form from human blood platelets and bovine brain. The kinase from both sources required Ca2+ and the modulator protein for its activity. The subunit molecular weight is approximately 105,000 daltons. These kinases are therefore similar to the smooth muscle kinase ( Dabrowska, R., Aromatorio, D., Sherry, J. M. F., and Hartshorne, D. J. (1977) Biochem. Biophys. Res. Commun. 78, 1263–1272) . It is suggested that the role of the myosin light chain kinase is similar in both muscle and non-muscle cells and serves to activate the contractile apparatus, via the phosphorylation of myosin, in response to an increase in the intracellular free Ca2+ concentration.

The binding of smooth muscle myosin light chain kinase to actin

Summary Myosin light chain kinase isolated from turkey gizzards binds to skeletal muscle actin. The binding is not influenced significantly by the presence, or absence, of Ca 2+ , calmodulin, gizzard tropomyosin and Mg 2+ -ATP. The myosin light chain kinase is removed from the actin filaments as the ionic strength is increased. The possibility of non-specific binding to actin is considered unlikely since the interaction is not affected by the presence of excess bovine serum albumin and also by the finding that the Ca 2+ -independent form of the myosin light chain kinase does not bind to actin. The binding of myosin light chain kinase to gizzard myosin is not as marked, and under conditions similar to those used to demonstrate binding to actin only about 10% of the kinase is associated with the myosin aggregates.

Caldesmon-induced inhibition of ATPase activity of actomyosin and contraction of skinned fibres of chicken gizzard smooth muscle

Caldesmon induces inhibition of MG2+‐ATPase activity of actomyosin and relaxation of skinned fibers of chicken gizzard smooth muscle without influencing the level of myosin light chain‐1 phosphorylation. Both these effects are reversed by calmodulin at a high molar excess over caldesmon in the presence of Ca2+.

The effects of caldesmon extraction on mechanical properties of skinned smooth muscle fibre preparations

The role of caldesmon in the regulation of smooth muscle contraction was investigated in chemically skinned smooth muscle fibres from the guineapig taenia coli. A 19-kDa C-terminal fragment of caldesmon gave a minor (<5%) reduction of force in fully thiophosphorylated fibres, but reduced force by about 50% at intermediate activation levels without affecting the level of light chain phosphorylation. An extraction procedure was developed using incubation in solutions containing high Mg2+ concentrations. Protein analysis revealed a selective decrease in the amount of caldesmon in the fibres. Maximal active force per cross-sectional area was unaffected. The Ca2+ dependence of active force was shifted towards lower Ca2+ concentrations and became less steep. The effects of extraction of caldesmon could in part be reversed by incubation in a solution containing purified caldesmon. The results are consistent with the hypothesis that caldesmon in smooth muscle thin filaments inhibits force generation and plays a role in regulating cooperative attachment of cross-bridges at sub-maximal levels of activation in smooth muscle.

Separation and characterization of the constituents of troponin

Hartshorne and coworkers [l] have found recently that troponin, a protein which in the form of a complex with tropomyosin sensitizes actomyosin to changes in concentration of calcium ions (for review see [2]) can be separated at pH 1 .O into two fractions. One of them, troponin B, inhibited the Mg*‘-stimulated ATPase activity of DAM* [3] independently of the concentration of Ca2+ and of the presence of tropomyosin whereas a second fraction, troponin A, had no influence on DAM per se, but sensitized troponin B to Ca 2+ ions in the presence of tropomyosin. Two similar fractions were later obtained by Schaub and Perry [4] with the use of SE-Sephadex chromatography Our previous experiments also showed that preparations of troponin are heterogenous; moreover, disc electrophoresis and chromatography on DEAESephadex-A-50 column indicated the presence of more than two fractions [5]. By using the latter method the protein fractions were obtained and some of their properties are described in this paper.

Some functional properties of nonpolymerizable and polymerizable tropomyosin.

SummaryThe binding of125I-labelled nonpolymerizable (brain or carboxypeptidase A-treated skeletal muscle) and polymerizable (intact skeletal muscle) tropomyosin to muscle F-actin was studied by ultracentrifugation under various conditions. The amount of nonpolymerizable tropomyosin bound to F-actin both in 0.1m KCl and in 7mm MgCl2 was much lower than that of the polymerizable one. In the presence of MgCl2 the amount of nonpolymerizable tropomyosin bound to F-actin approached saturation level. Under these conditions, however, the amount of skeletal muscle tropomyosin bound exceeded saturation, suggesting formation of both head-to-tail polymers and side-to-side aggregates. The latter seems to be responsible for the inhibition of acto-heavy meromyosin ATPase activity which is caused by skeletal muscle tropomyosin but not by nonpolymerizable tropomyosin.Nonpolymerizable tropomyosin can substitute for the rabbit skeletal muscle tropomyosin in the regulatory system operating in skeletal muscle. Inhibition of ATPase activity of acto-heavy meromyosin by nonpolymerizable tropomyosin in the presence of troponin and the absence of calcium ions is less than that obtained with polymerizable tropomyosin. The inhibition of ATPase activity is directly correlated with the extent of binding of nonpolymerizable tropomyosin to F-actin under the conditions of the ATPase assay.

Comparative studies of chicken gizzard and rabbit skeletal tropomyosin

Abstract 1. 1. Physicochemical and functional properties of chicken gizzard tropomyosin were compared to those of rabbit skeletal tropomysin. 2. 2. In spite of the different role tropomyosin plays in smooth and skeletal muscle, the properties and structure of gizzard and skeletal tropomyosin are very similar. 3. 3. Chicken gizzard tropomyosin has the same ability to bind to actin and troponin as the skeletal one and can fully substitute the latter in its function in the regulatory system operating in skeletal muscle.

The effect of caldesmon on actin-myosin interaction in skeletal muscle fibers

The effects of caldesmon on structural and dynamic properties of phalloidin-rhodamine-labeled F-actin in single skeletal muscle fibers were investigated by polarized microphotometry. The binding of caldesmon to F-actin in glycerinated fibers reduced the alterations of thin filaments structure and dynamics that occur upon the transition of the fibers from rigor to relaxing conditions. In fibers devoid of myosin and regulatory proteins (ghost fibers) the binding of caldesmon to F-actin precluded structural changes in actin filaments induced by skeletal muscle myosin subfragment 1 and smooth muscle tropomyosin. These results suggest that the restraint for the alteration of actin structure and dynamics upon binding of myosin heads and/or tropomyosin evoked by caldesmon can be related to its inhibitory effect on actin-myosin interaction.