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S100C (S100A11, calgizzarin) inhibits the actin-activated myosin Mg(2+)-ATPase activity of smooth muscle in a dose-dependent manner: its half-maximal effect occurs at a S100C/actin molar ratio of 0.05 and its maximal effect occurs at a ratio of 0.20. Furthermore, S100C was found to bind to actin with a stoichiometry of 1:6-7 in the presence of Ca(2+), with an affinity of 1 x 10(-6) M determined by cosedimentation assays. Other Ca(2+)-binding proteins such as S100A1, S100A2, S100B, and calmodulin did not inhibit actin-activated myosin Mg(2+)-ATPase activity. Calmodulin, S100A1, and S100B reversed the inhibitory effect of calponin in a Ca(2+)-dependent manner, S100A2 had no effect, and S100C had additional inhibitory effects. The results suggest that S100C might be involved in the regulation of actin-activated myosin Mg(2+)-ATPase activity through its Ca(2+)-dependent interaction with actin filaments., (Copyright 2000 Academic Press.)

The present study was performed to investigate the effects of propiverine hydrochloride (1-methyl-4-piperidyl diphenylpropoxyacetate hydrochloride, P-4), a novel anti-pollakiuric agent, on the contractile proteins of smooth muscle. P-4 (30-300 microM) inhibited the activity of native actomyosin adenosine triphosphatase (ATPase) that had been freshly purified from canine urinary bladder, and calmodulin at 10 microM overcame this inhibition. P-4 also inhibited myosin light chain kinase from smooth muscle in a dose-dependent manner. However, at 300 microM, P-4 was unable to inhibit by 50% the activity of trypsin-treated myosin light chain kinase, which was independent of Ca2+/calmodulin. 1 mol of calmodulin bound 4 to 5 mol of [14C]P-4 in a Ca2+-dependent manner with a K(d) of 77.4 microM. These results indicate that calmodulin is one of the intracellular target molecules for P-4 and that inhibition of the action of calmodulin by P-4 might cause the inhibition of actomyosin ATPase activity, with subsequent relaxation of the smooth muscle of the urinary bladder.

In permeabilized smooth muscle, exogenously applied calponin binds to myofibrils and reduces Ca(2+)-activated tension (Itoh, T., Suzuki, S., Suzuki, A., Nakamura, F., Naka, M., and Tanaka, T. (1994) Pflügers Arch. Eur. J. Physiol. 427, 301-308). A calponin peptide (calponin Phe173-Arg185), which inhibits the binding of calponin to actin, blocks the action of calponin and enhances the contraction induced by submaximal Ca2+ in permeabilized vascular smooth muscle. Unlike calmodulin, this peptide enhances the Ca(2+)-induced contraction without a corresponding increase in the level of myosin light chain phosphorylation. These results suggest that calponin decreases the sensitivity of smooth muscle to Ca2+ at a given level of myosin light chain phosphorylation.

A myosin phosphatase was purified from chicken gizzard smooth muscle. The holoenzyme is a trimer and consists of 130,000-, 38,000-, and 20,000-Da subunits (in agreement with the results of Alessi et al.: Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M., and Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035). The catalytic subunit, 38,000 Da, is the type 1 delta isoform, and its derived amino acid sequence is identical to the rat isoform. The larger subunit bound to myosin and also interacted with the catalytic subunit. cDNA clones encoding the large subunit were isolated from chicken gizzard cDNA libraries. Overlapping clones indicated the presence of two isoforms, and open reading frames of 2889 and 3012 bases were obtained. These encoded proteins of 963 and 1004 amino acids, with masses of 106,700 and 111,600 Da, respectively. The insert in the larger isoform is in the center of the molecule, at residues 512-552. The N-terminal third of the molecule is composed of eight repeat sequences, similar to the cdc10/SWI6 or ankyrin repeat. Myosin binding and binding to the catalytic subunit are properties of a 58,000-Da fragment that represents the N-terminal part of the molecule.

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.

Phosphorylation of the 20,000 molecular weight (MW) light chain of platelet myosin is associated with the activation of platelets and subsequent release of platelet granules, and the protein kinase catalysing this phosphorylation has been identified as the Ca2+/calmodulin-dependent enzyme, myosin light chain kinase. Tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), which activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), can also cause platelet aggregation and phosphorylation of a 20,000-MW peptide in blood platelets. It was therefore of interest to ascertain whether the 20,000-MW peptide phosphorylated in platelets was the light chain of myosin and whether TPA-induced phosphorylation of the 20,000-MW peptide could be differentiated from thrombin-induced phosphorylation. We now report that TPA-induced activation of platelets is associated with the phosphorylation of the 20,000-MW light chain of myosin, that it appears to be mediated mainly through protein kinase C and that the site phosphorylated in the myosin light chain is distinct from that phosphorylated by myosin light chain kinase.

Smooth muscle myosin from chicken gizzard is phosphorylated by Ca2+-activated phospholipid-dependent protein kinase, protein kinase C, as well as by Ca2+/calmodulin-dependent kinase, myosin light chain kinase (Endo, T., Naka, M., and Hidaka, H. (1982) Biochem. Biophys. Res. Commun. 105, 942-948). We have now demonstrated the effect of phosphorylation by protein kinase C on the smooth muscle myosin molecule. In glycerol/urea polyacrylamide gel electrophoresis the 20,000-dalton light chain phosphorylated by protein kinase C co-migrated with that phosphorylated by myosin light chain kinase. Moreover, the light chain phosphorylated by both kinases migrated more rapidly than did the light chain phosphorylated by either myosin light chain kinase or protein kinase C alone. Myosin phosphorylated by protein kinase C formed a bent 10 S monomer while that phosphorylated by myosin light chain kinase was an unfolded and extended 6 S monomer in the presence of 0.2 M KCl. In addition, myosin phosphorylated by kinases had a sedimentation velocity of 7.3 S, thereby suggesting that the myosin was partially unfolded. The unfolded myosin was visualized electron microscopically. The fraction in the looped form was higher when for myosin phosphorylated by both kinases higher than for that phosphorylated by light chain kinase alone. Therefore, phosphorylation by protein kinase C does not lead to the change in myosin conformation seen with myosin light chain kinase.

The rate of phosphorylation and dephosphorylation of smooth muscle myosin by myosin light chain kinase and by two myosin light chain phosphatases (gizzard phosphatase IV and aorta phosphatase) are measured in various conditions; the relationship between the rate of phosphorylation and dephosphorylation of myosin and the myosin conformation is also studied. The rate of dephosphorylation of myosin was completely inhibited in the presence of 1 mM MgCl2 and ATP at low ionic strength where phosphorylated myosin forms a folded conformation. The inhibition was released when myosin formed either an extended monomer or filaments. The rate of phosphorylation of myosin was also affected by the conformation of myosin. The rate for a folded myosin was slower than those for an extended monomer and filamentous myosin. The phosphorylation and dephosphorylation of heavy meromyosin, subfragment-1, and the isolated 20,000-dalton light chain are not inhibited at low ionic strength, and the rate of phosphorylation and dephosphorylation was decreased with increasing ionic strength. KCl dependence of the rate of phosphorylation and dephosphorylation of myosin was normalized by using KCl dependence of subfragment-1, and it was found that the marked inhibition of the rate of phosphorylation and dephosphorylation of myosin is closely related to the change from an extended to a folded conformation of myosin.

Three forms of 20-kDa myosin light chain (MLC), unphosphorylated, monophosphorylated, and diphosphorylated MLC (designated 20K, 20K-P, and 20K-PP) were demonstrated in thrombin-stimulated human platelets by two different gel electrophoretic methods: in the presence of glycerol urea or in two dimensions (isoelectric and sodium dodecyl sulfate). The diphosphorylation of platelet 20-kDa MLC increased, dose dependently, up to 0.4 U/ml thrombin and reached 25% of platelet 20-kDa MLC. After mono- or diphosphorylated 20-kDa MLC from thrombin-stimulated platelets was digested with trypsin, the analysis using two-dimensional peptide mapping demonstrated that two different sites were phosphorylated by MLC kinase and protein kinase C, as noted in the case of 12-O-tetradecanoylphorbol-13-acetate-stimulated platelets (M. Naka, et al. (1983) Nature (London) 306, 490-492). The more rapid monophosphorylation was catalyzed preferentially by MLC kinase while the slower and additional phosphorylation was catalyzed mainly by protein kinase C. These results suggest the importance of distinguishing multiple site phosphorylation of 20-kDa MLC in thrombin-activated human platelets.

Myosin 20 K-Da light chain phosphorylation in human platelets was found to be catalyzed by MLCK in the early phase during collagen activation. The effect of newly synthesized selective inhibitor of MLCK, ML-9, on collagen induced platelet activation was investigated. ML-9 delayed the time course of the myosin 20 K-Da light chain phosphorylation, sequentially led to a delay in aggregation, secretion and phosphorylation of the 40K-Da peptide, in a dose-dependent fashion. It is proposed that the MLCK catalyzed phosphorylation of myosin 20 K-Da light chain may be an initial response and if so may influence the sequent reactions in the activation of platelets with collagen.