Identification of two forms of myosin light chain kinase in turkey gizzard.

Two forms of myosin light chain kinase from turkey gizzard are separable by ion-exchange chromatography. One is the well-characterized 130,000 Mr enzyme. Purification of the second form by affinity chromatography on calmodulin--Sepharose showed it to consist of two polypeptide chains of Mr 136,000 and 141,000. This form of the enzyme required Ca2+ and calmodulin for activity, was specific for the Mr 20,000 light chain of myosin, and appeared to phosphorylate the same site on the light chain as the Mr 130,000 enzyme. The low-Mr gizzard kinase may be a proteolytic fragment of a higher-Mr species or these may represent different isoenzymes.

Correlation of intrinsic fluorescence and conformation of smooth muscle myosin.

The addition of ATP to turkey gizzard myosin causes an enhancement of the intrinsic tryptophan fluorescence. The level of fluorescence enhancement is determined by the myosin conformation. The transition of myosin from the folded (10 S) state to the extended (6 S) state is accompanied by a decrease in the fluorescence level. Phosphorylation-dephosphorylation of myosin does not directly influence fluorescence and will induce changes only if the myosin conformation is altered. Under the appropriate conditions, phosphorylation of myosin favors the transition of 10 S to 6 S. The phosphorylation dependence of the associated fluorescence decrease is not linear, and it is proposed that the phosphorylation of both light chains is required for the full transition. The tryptophan residues involved respond to the binding of ATP at the hydrolytic sites. Since the fluorescence properties of gizzard myosin are influenced by the myosin conformation, it is reasonable to assume that the active sites are also modified by the shape of the myosin molecule.

Correlation of enzymatic properties and conformation of smooth muscle myosin.

In the presence of adenosine 5'-triphosphate (ATP) and 1-10 mM MgCl2, the relative viscosity (eta rel) of dephosphorylated gizzard myosin is reduced markedly over a range of KCl from 0.35 to 0.15 M. Sedimentation patterns show that the decrease in eta rel is due to the conversion of the 6S to 10S forms of myosin. Under similar conditions, eta rel of phosphorylated myosin is not altered, and at 0.2 M KCl, the 10S form is not observed. In 1 and 2 mM MgCl2 and less than 0.2 M KCl, 10S can be formed from both phosphorylated myosin plus ATP and dephosphorylated myosin minus ATP. In the presence of ethylenediaminetetraacetic acid (EDTA), the decrease of eta rel and corresponding change in sedimentation pattern are independent of ATP and show only a dependence on KCl. Therefore, ATP and dephosphorylation are not obligatory for the 6S to 10S transition. In all instances, the 6S-10S transition of monomeric myosin is paralleled by an alteration of adenosine-5'-triphosphatase (ATPase) activity; i.e., the KCl dependence of the two processes is the same. Transition from 6S to 10S causes a decrease in Mg2+-and Ca2+-ATPase activity of myosin and an increase in K+-EDTA-ATPase activity. The relationship between myosin shape and the ATP dependence of Mg2+-ATPase activity also is consistent with this generalization. The phosphorylation dependence of the viscosity transition from 6S to 10S is not linear, and phosphorylation of both heads is required for the complete transition. In contrast, the ATP dependence of the transition is linear, and the binding of 2 mol of ATP/myosin is required for maximum effect.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Effects of magnesium chloride on smooth muscle actomyosin adenosine-5'-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers.

The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5'-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (less than or equal to 1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)