Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.4.1 (myosin ATPase)
1,140 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Limited digestion of calmodulin (CaM)-dependent myosin light chain kinase from turkey gizzard with alpha-chymotrypsin in the presence of bound CaM generated an 80,000-dalton kinase fragment that was fully active in the absence of Ca2+. This kinase catalyzed specific Ca2+-independent phosphorylation of the 20,000-dalton light chain of myosin using isolated light chains, intact myosin, and actomyosin. Phosphorylation of myosin in the absence of Ca2+ allowed us to dissociate myosin phosphorylation from other potential Ca2+-dependent regulatory mechanisms, thus permitting an evaluation of the postulated central role of myosin phosphorylation in the regulation of smooth muscle contraction. Ca2+-independent myosin phosphorylation was found to cause loss of Ca2+ sensitivity of 1) actin-activated myosin ATPase activity in a crude actomyosin preparation, and 2) tension development in skinned smooth muscle fibers in the absence of Ca2+. Myosin phosphorylation is, therefore, the key event in actin activation of ATPase activity and initiation of contraction in skinned chicken gizzard fibers.
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PMID:Gizzard Ca2+-independent myosin light chain kinase: evidence in favor of the phosphorylation theory. 684 77

Protein phosphorylation may play a critical role in stimulus-coupled secretion of platelets. Some platelet proteins become phosphorylated on exposure to agents such as thrombin and collagen, and the smallest of these phosphoproteins (molecular weight 20,000), has been identified as a light chain of myosin. Phosphorylation of myosin light chain increases the activity of actin-activated myosin ATPase and the resultant contraction of the actomyosin presumably mediates the release reaction. Platelet myosin light chain kinase has been identified as a calcium-dependent enzyme requiring calmodulin for its activity. Calmodulin is a Ca2+-binding protein with a molecular weight of approximately 18,000 which seems to be involved in a wide variety of cellular processes. Although a growing body of evidence suggests that non-muscle actomyosin, such as that isolated from platelets, is regulated by Ca2+-calmodulin-dependent light chain phosphorylation, the precise relationship between the phosphorylation and the function of platelets is not clearly established. We now present pharmacological evidence that a calmodulin-mediated system, such as Ca2+-dependent myosin light chain phosphorylation, also plays an important role in the phenomenon of the release reaction. N-(6-aminohexyl)-5-chloro-1-napthalene-sulphonamide (W-7) (refs 13-15) is shown to bind selectively to calmodulin in vitro and inhibit its biological activity.
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PMID:Ca2+-calmodulin-dependent phosphorylation and platelet secretion. 743 2

Sheep aorta thin filaments were prepared by ultracentrifugation of an ATP-containing extract in the presence of different concentrations of ethanediol. Thin filaments prepared without ethanediol contained small quantities of tropomyosin (0.027 Tm:actin) and caldesmon (0.017 CD:actin) and activated the MgATPase of skeletal myosin independently of Ca2+. Ultracentrifugation in the presence of 10-20% ethanediol resulted in preparation of thin filaments with increased content of tropomyosin (0.17 Tm:actin) and caldesmon (0.04 CD:actin). These thin filaments possessed high Ca(2+)-sensitivity in activation of skeletal muscle myosin ATPase. Besides actin, tropomyosin and caldesmon, thin filaments contained gelsolin and filamin. Gelsolin content (0.007 gelsolin:actin) was independent of the presence of ethanediol. The filamin content decreased from 0.015 to 0.007 mol:mol actin when the ethanediol concentration was increased from 0 to 20%, and was negatively correlated with the Ca2+ sensitivity of thin filaments. In a reconstituted system, pure filamin or gelsolin affected caldesmon regulation of actomyosin ATPase. Gelsolin (0.01:actin) reduced the inhibition of actomyosin ATPase caused by caldesmon and increased the potency of Ca(2+)-calmodulin in reversing this inhibition. Filamin (0.007:actin) also decreased the inhibitory action of caldesmon on actin-activated myosin ATPase and also potentiated the reversal of this inhibition by calmodulin. We conclude that minor components of smooth muscle thin filaments (gelsolin and filamin) significantly modify caldesmon mediated regulation of actomyosin ATPase. We suggest a tropomyosin-mediated mechanism by which filamin or gelsolin could exert similar effects.
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PMID:Filamin and gelsolin influence Ca(2+)-sensitivity of smooth muscle thin filaments. 770 23

Ca(2+)-dependent myosin light chain (MLC) phosphorylation is an important step in the initiation of smooth muscle contraction. However, MLC phosphorylation alone cannot account for all aspects of contractile regulation, suggesting the involvement of other elements. In this article we present evidence obtained from Triton X-100 detergent skinned and intact tissue which demonstrates that vascular smooth muscle contraction can be initiated by a Ca(2+)-dependent mechanism that does not require prior MLC phosphorylation. We show that Ca2+ can initiate contractions supported by cytidine triphosphate (CTP) and that these contractions are inhibited by calmodulin antagonists, suggesting a Ca(2+)-calmodulin dependence of force distinct from that for MLC phosphorylation. Evidence is presented to demonstrate that carotid medial fibers contain a mitogen-activated protein (MAP) kinase which is activated by Ca2+ and may catalyze caldesmon phosphorylation. Based in part on our results and those of other investigators, we propose that direct Ca(2+)-calmodulin binding to caldesmon or phosphorylation of caldesmon by a Ca(2+)-dependent MAP kinase disinhibits caldesmon. Disinhibition of caldesmon allows an inherent basal level of actin-activated myosin ATPase activity to be expressed. The result is the slow development of force.
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PMID:Regulation of vascular smooth muscle contraction: myosin light chain phosphorylation dependent and independent pathways. 776 83

We have previously shown that caldesmon at low concentrations stimulates the interaction between actin, myosin, and ATP, while at high concentrations it inhibits the interaction [Ishikawa, R., Okagaki, T., Higashi-Fujime, S., & Kohama, K. (1991) J. Biol. Chem. 266, 21784-21790]. When the effect of caldesmon at low concentrations was monitored by measuring myosin ATPase activity in the absence of actin, the effect was slightly but significantly stimulatory; and at higher concentrations no inhibitory effect was observed. Therefore, we related the stimulatory effect with the myosin-binding property of caldesmon. In the presence of actin, a low concentration of caldesmon was not enough to evince the stimulatory effect: myosin concentration must also be low. This is because the stimulatory effect was obscured when myosin concentration was elevated. Ca(2+)-calmodulin abolished the stimulatory effect of caldesmon. However, the concentration of calmodulin required to abolish the stimulation was higher than that required to abolish the inhibition.
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PMID:Role of myosin in the stimulatory effect of caldesmon on the interaction between actin, myosin, and ATP. 826 10

Caldesmon is known to inhibit actin-activated myosin ATPase activity in solution, to inhibit force production when added to skeletal muscle fibers, and to alter actin movement in the in vitro cell motility assay. It is less clear that caldesmon can inhibit contraction in smooth muscle cells in which caldesmon is abundant. We now show that caldesmon and its 20-kDa actin-binding fragment are able to inhibit force in chemically skinned gizzard fiber bundles, which are activated by a constitutively active myosin light-chain kinase in the presence and absence of okadaic acid. This inhibitory effect is reversed by high concentrations of Ca2+ and calmodulin. Therefore, caldesmon may act by increasing the level of myosin phosphorylation required to obtain full activation. Our results also suggest that caldesmon does not act to maintain force in smooth muscle by cross-linking myosin with actin since competition of binding of caldesmon with myosin does not cause a reduction in tension.
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PMID:Caldesmon and a 20-kDa actin-binding fragment of caldesmon inhibit tension development in skinned gizzard muscle fiber bundles. 832 61

1. Using beta-escin and ionomycin-treated skinned smooth muscle strips of the rabbit mesenteric artery, the effects of calyculin A (CL-A, an inhibitor of type 1 and 2A phosphatases) on mechanical activities, phosphorylation of myosin light chain (MLC) and the relationship between the two were studied in Ca(2+)-free solution containing 4 mM EGTA and these effects were compared with those evoked by Ca2+. 2. The threshold concentration of Ca2+ required to increase either tension or MLC-phosphorylation was 0.1 microM and maximum effects were obtained at 10 microM. MLC was mainly monophosphorylated, rather than diphosphorylated, in the presence of Ca2+. ED50 value for Ca2+ was 0.54 microM for either tension or MLC-phosphorylation. The relationship between tension and MLC-phosphorylation is linear in the pCa range 7-5.5. 3. In Ca(2+)-free solution (containing either 20 mM EGTA or 4 mM EGTA with or without 4 mM BAPTA), 3 microM CL-A produced a contraction, the maximum amplitude of which was similar to that evoked by 10 microM Ca2+. CL-A (0.03-3 microM) concentration-dependently increased both tension and MLC-phosphorylation in Ca(2+)-free solution containing 4 mM EGTA. The threshold concentration of CL-A required for the increase in either tension or MLC-phosphorylation was 0.03 microM and maximum effects were obtained at 3 microM. In the presence of CL-A, MLC was not only monophosphorylated but also diphosphorylated. ED50 values for CL-A were 0.39 microM for tension, 0.44 microM for the monophosphorylated form of MLC and 0.54 microM for all phosphorylated (mono + di) forms. The relationship between tension and the monophosphorylated form of MLC was linear over the concentration range studied and was similar to that for Ca2+. 4. H-7 (3 microM, an inhibitor of protein kinase C) inhibited neither the tension nor phosphorylation of MLC induced by 10 microM Ca2+ or 3 microM CL-A. At a high concentration (30 microM), H-7 slightly inhibited both the tension and phosphorylation of MLC induced by either stimulant without a change in the tension-MLC-phosphorylation relationship. KN-62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinase II, did not modify either the tension or the phosphorylation of MLC induced by 10 microM Ca2+ or 3 microM CL-A. CK-II, another inhibitor of Ca(2+)-calmodulin-dependent protein kinase II, did not inhibit the contraction induced by 3 microM CL-A. 5. SM-1 (0.03-0.3 mM) and ML-9 (0.1 and 0.3 mM), inhibitors of MLC-kinase, each lowered the resting level of MLC-phosphorylation in Ca2+-free solution and also inhibited both the tension and MLC-phosphorylation induced by 10 microM Ca2+ or 3 microM CL-A, in a concentration-dependent manner.Neither SM-1 nor ML-9 modified the relationship between tension and either monophosphorylated or all phosphorylated (mono + di) forms of MLC in the presence of Ca2+ or CL-A.6. In a solution containing MgITP (the substrate for myosin ATPase but not for MLC-kinase) with no MgATP, 10 microM Ca2+ failed to produce contraction. Under these conditions, the amplitude of the contraction induced by 3 microM CL-A was greatly diminished in comparison with that induced in the presence of MgATP.7. The present results suggest that in smooth muscle cells of the rabbit mesenteric artery, CL-A in Ca2+-free solution, produces a maximum contraction through an indirect activation of Ca2+-calmodulin independent(constitutively active) MLC-kinase via its inhibitory action on MLC-phosphatases. Based on this evidence, it is hypothesized that, in these cells, a constitutively active MLC-kinase may be present, though its action may be concealed by that of endogenous MLC-phosphatase.
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PMID:Effects of calyculin A on tension and myosin phosphorylation in skinned smooth muscle of the rabbit mesenteric artery. 839 95

We expressed the following fragments of chicken gizzard caldesmon in the pMW 172/BL21 (DE3) system at 0.4-2.2 mg of pure protein/liter of culture: full-length smooth muscle caldesmon (CDh) (amino acids 1-756), nonmuscle caldesmon (CDl), amino acids 1-128 (N128), 1-578 (N578), 230-419, 606-756 (606C), and 658-756 (658C). CDh bound tropomyosin with a Kd of 1.5 microM; N578, 230-419, and 606C bound with affinities at least 2-5 fold lower; N128 bound weakly; and 658C did not bind. Only N128 and N578 bound to smooth muscle myosin, both about 10-fold weaker than CDh and CDl. Only 606C and 658C bound to actin-tropomyosin with affinities CDh = 606C > 658C. The binding to actin-tropomyosin was biphasic, whereas the binding to actin was monophasic, corresponding to the weak binding component found in the presence of tropomyosin. Calmodulin bound only to the C-terminal fragments with the same affinity as CDh. CDh, 606C, and 658C inhibited actin-tropomyosin-activated myosin ATPase, with maximal inhibition correlated with 1 caldesmon bound/14 actins, and inhibition was reversed by Ca(2+)-calmodulin. Thus, the actomyosin ATPase regulatory function, calmodulin binding, and most actin binding is located within the C-terminal 99 amino acids, whereas tropomyosin binding is distributed throughout the rest of the molecule.
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PMID:Binding and regulatory properties of expressed functional domains of chicken gizzard smooth muscle caldesmon. 849 61

A series of C-terminal deletion mutants of chicken gizzard smooth muscle caldesmon (CaD) were made using a polymerase chain reaction cloning strategy and a baculovirus expression system, and the precise locations of the functional domains of CaD involved in the regulation of actomyosin ATPase and the binding of actin, tropomyosin, and calmodulin were analyzed. Our results reveal a high affinity calmodulin-binding domain that consists of at least three calmodulin-binding determinants localized in residues 690-717, 658-689, and 628-657. The residues between positions 718 and 756 and positions 598 and 627 have no detectable calmodulin-binding site. A high affinity tropomyosin-binding domain is located between residues 718 and 756. The 159 residues at the C terminus of CaD contain multiple actin-binding determinants; the major ones are localized in the regions between residues 718 and 756 and residues 690 and 717. The amino acid residues between positions 718 and 756 contain the major determinant involved in the inhibition of the actin activation of smooth muscle myosin ATPase since CaD-(1-717) caused only 30% of the inhibition produced by the full-length CaD. Further deletion between residues 690 and 717 (CaD-(1-689) revealed a low level (10% of that seen for full-length CaD) of inhibition of the actomyosin ATPase. These data clearly demonstrate that the region of the last 66 amino acid residues at the CaD C terminus contains two or more major actin-binding motifs, one tropomyosin-binding domain, one high affinity calmodulin-binding determinant, and the domain that is responsible for the inhibition of the actin-activated ATPase of myosin.
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PMID:Characterization of the functional domains on the C-terminal region of caldesmon using full-length and mutant caldesmon molecules. 856 84

The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence was investigated with three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). Binding measurements in the presence of Ca2+ by separation on native gels and by carbodiimide-induced cross-linking showed a lower affinity for caldesmon in all the mutants. When Ca2+ was replaced by Ba2+ the affinity of calmodulin for caldesmon was further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase was virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retained their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflected their decreased affinity. Ca2+ -calmodulin produced a broadening in the signals of the NMR spectrum of the 10 kDa Ca(2+)-calmodulin-binding C-terminal fragment of caldesmon arising from tryptophans -749 and -779 and caused an enhancement of maximum tryptophan fluorescence of 49% and a 16 nm blue shift of the maximum. Ca(2+)-calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca(2+)-calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule.
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PMID:Multiple-sited interaction of caldesmon with Ca(2+)-calmodulin. 868 82


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