Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The inhibitory effect of calmodulin antagonists, synthetic peptide analogs of the pseudosubstrate domain of smooth muscle MLC kinase, and an inhibitor based on the sequence of MLC were examined using bovine aortic actomyosin and isolated chicken gizzard MLC. Much lower concentrations of the peptides were necessary to inhibit actomyosin ATPase activity than to inhibit superprecipitation. In contrast, calmodulin antagonists inhibited both ATPase activity and superprecipitation at similar concentrations. The peptide analogs were competitive with isolated MLC, but not calmodulin, for inhibition of MLC kinase. These results suggest that in addition to the calmodulin dependence of MLC phosphorylation, a second calmodulin-like protein may be important in actin-myosin interactions. The data also suggest that the pseudosubstrate hypothesis may not completely account for regulation of MLC kinase activity.
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PMID:Peptide analogs of the pseudosubstrate domain of smooth muscle myosin light chain kinase inhibit actomyosin ATPase activity at concentrations that do not inhibit superprecipitation. 141 4

Previous studies have identified changes of mechanical properties of airway smooth muscle (ASM) from a canine model of atopic airway hyperreactivity. These changes, including increased maximum shortening capacity (delta Lmax) and early shortening velocity (Vo), may be responsible for the airway hyperresponsiveness in asthma. We have suggested that these changes may be due to increased actomyosin ATPase activity, controlled via phosphorylation of the 20 kD myosin light chain (MLC20) by MLC kinase (MLCK). Therefore, ATPase activity, MLC20 phosphorylation, and MLCK content and activity were assessed in tracheal and bronchial smooth muscles (TSM and BSM) of ragweed pollen-sensitized dogs (S) and their littermate controls (C). Specific ATPase activities from STSM and SBSM were significantly higher than their control counterparts (CTSM, CBSM). Phosphorylation of MLC20 in STSM was greater both at rest and during electrical stimulation due to the increased amount of MLCK in STSM and SBSM by 30 and 25%, respectively. MLCK activity was also increased significantly in STSM and SBSM (from 46.99 +/- 8.33 and 42.85 +/- 5.92 to 91.9 +/- 6.43 and 64.12 +/- 7.88 32P mmol/mg fresh tissue weight/min respectively [mean +/- SEM]). When normalized to the amount of MLCK in the tissue, however, specific MLCK activity in STSM and SBSM was similar to that in controls. It is unlikely that myosin phosphatase plays any role in the changes of MLC20 phosphorylation in sensitized animals. Peptide mapping showed no visible change in primary structure of MLCK in STSM and SBSM compared with those of controls. We report that ASM actomyosin ATPase activity is increased in STSM and SBSM. The increased ATPase activity is the result of increased MLC20 phosphorylation, the latter likely resulting from the increased MLCK content, which may account for the hyperresponsiveness found in ASM from these animals.
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PMID:Ragweed sensitization-induced increase of myosin light chain kinase content in canine airway smooth muscle. 144 4

It seems clear that a simple Ca2+ dependent switch (MLC phosphorylation) cannot completely explain all of the disparate mechanical and energetic results obtained under numerous experimental conditions in numerous laboratories. Some of the problems of the simple switch model are that: 1. Force can be developed in the complete absence of increases in MLC phosphorylation; 2. Crossbridge cycling rate, as measured by either shortening velocity or directly by ATPase activity, can be regulated independent of changes in MLC phosphorylation; and 3. Ca2+ can directly influence both force and crossbridge cycling rate. Thus, we believe that there are two distinct Ca2+ dependent regulatory systems which normally act in parallel to contract smooth muscle. One of these is the Ca2+ dependent MLC phosphorylation-dephosphorylation. system which is likely to be responsible for the rapid development of force. The other is the hypothesized Ca2+ dependent system which is probably responsible for the slow development of force as well as the maintenance of previously developed force, represented in Figure 5 as K8. This second system involves a calmodulin-like protein with a higher Ca2+ sensitivity than that for the Ca(2+)-calmodulin-MLC kinase system. Under most conditions, the total force attained by smooth muscle in response to stimulation is the result of the concerted activation of both of these regulatory systems. The available information is consistent with this hypothesis of two regulatory systems functioning in parallel. In addition to the information presented in this chapter, work from a number of laboratories (Moreland and Ford, 1982; Fujiwara et al., 1989; Kitazawa et al., 1989; Somlyo et al., 1989; Kubota et al., 1990; Kitazawa and Somlyo, this volume) have suggested the possibility that a regulated MLC phosphatase may functionally alter the Ca2+ sensitivity of the contractile filaments. There is evidence suggesting that the sensitivity of MLC kinase to activation by Ca2+ and calmodulin may be regulated (Stull et al., this volume). Protein kinase C has been postulated to play an important role in the regulation of myofilament Ca2+ sensitivity (Nishimura et al., this volume). MgADP has been suggested to affect the kinetics of latchbridge attachment and detachment (Kerrick and Hoar, 1987; Nishimura and van Breemen, 1989). Cooperativity between crossbridges as described by Somlyo et al. (1988) and Siegman et al. (this volume) might also be an important component in the regulation of smooth muscle contraction.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of a smooth muscle contraction: a hypothesis based on skinned fiber studies. 180 23

Ca(2+)-calmodulin-dependent phosphorylation of the 20-kDa smooth muscle myosin light chain (MLC) results in high shortening velocities and rapid stress development. The stress maintained after a reduction in Ca2+ is associated with a decrease in MLC phosphorylation and velocity of shortening. This Ca(2+)-dependent stress without proportional MLC phosphorylation has been termed "latch" and has been postulated to reflect a population of dephosphorylated noncycling cross bridges or "latch bridges." Mg2+ is necessary for contraction of smooth muscle, and in high concentrations, Mg2+ elicits contractions that are MLC phosphorylation independent. The purpose of this study was to test the hypothesis that high concentrations of Mg2+ directly induce latch-bridge formation. This was accomplished by comparing the characteristics of Mg(2+)-induced contractions of Triton X-100-skinned swine carotid media with the known characteristics of the Ca(2+)-dependent latch state. In the absence of Ca2+, free Mg2+ (3-20 mM) caused an increase in the velocity of shortening and a concentration-dependent increase in stress, with no detectable increase in MLC phosphorylation. Mg(2+)-induced contractions could be supported by CTP, which is a substrate for the actin-activated myosin adenosinetriphosphatase but not the MLC kinase. Stress development in response to Mg2+ was abolished at long tissue lengths, which also inhibit the expression of latch bridges. The calmodulin antagonist, trifluoperazine (TFP), inhibited the MLC phosphorylation-independent contractions elicited by Mg2+. TFP also inhibited the latch state. The results of this study support the existence of a regulatory system in vascular smooth muscle that is independent of the MLC phosphorylation system and can be directly activated by pharmacological levels of Mg2+.
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PMID:Characterization of magnesium-induced contractions in detergent-skinned swine carotid media. 182 25

Human platelet myosin forms 10S and 6S conformations, and its Ca(2+)- and Mg(2+)-ATPase activities are parallel with the transition between 10S and 6S conformation, as judged by the gel filtration, intrinsic fluorescence, and viscosity methods. The 20,000-dalton myosin light chain (LC20) is phosphorylated by both myosin light chain kinase (MLC kinase) and Ca2+, phospholipid-dependent protein kinase (protein kinase C [PKC]). The phosphorylation (1 mol of phosphate/mol of LC20) by MLC kinase shifts the equilibrium toward the 6S conformation, but that by PKC does not. The prephosphorylation of myosin by PKC prevents the effect of phosphorylation by MLC kinase on actin-activated Mg(2+)-ATPase activity, but not the effect on conformational change. Inhibition of actin-activated ATPase activity by PKC is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. These results suggest that sequential phosphorylation of myosin by both kinases plays an important role in the ATPase activities of human platelet myosin.
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PMID:Effect of phosphorylation of myosin light chain by myosin light chain kinase and protein kinase C on conformational change and ATPase activities of human platelet myosin. 183 91

Vasopressin (VP) and other hormones that elevate intracellular Ca2+ concentration also increase tight junctional permeability in the liver cell. Data derived from study of other tissues suggest that microfilaments are instrumental in regulating tight junctional permeability. By analogy to microfilament contraction in smooth muscles, it is likely that the transduction pathway for these hormones involves Ca(2+)-stimulated complex formation between calmodulin and myosin light chain (MLC) kinase with activation of this latter enzyme. MLC kinase then phosphorylates MLC, which, in the presence of actin, exerts adenosinetriphosphatase activity and produces microfilament contraction. This transduction pathway in the hepatocyte remains speculative. To demonstrate the likelihood of this pathway, we stimulated isolated hepatocytes with 10(-8) M VP and assayed MLC phosphorylation. We did this by immunoprecipitation of myosin from homogenates of liver cells prelabeled with [32P]-orthophosphate. We used a polyclonal antibody raised in rabbits against rat liver cell myosin. Our data demonstrate that VP is a potent stimulator of MLC phosphorylation. Maximal rises in intracellular Ca2+ and maximal MLC phosphorylation occur within 40 s of VP administration. The dose-response curve for MLC phosphorylation by VP is similar to that for tight junctional permeabilization in perfused liver with maximal effect at about 10(-8) M VP. The calcium ionophore A23187 also stimulated MLC phosphorylation. MLC phosphorylation, therefore, is at least coincident with, and probably responsible for, tight junctional permeabilization caused by elevation of intracellular Ca2+ in the liver cell.
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PMID:Vasopressin and A23187 stimulate phosphorylation of myosin light chain-1 in isolated rat hepatocytes. 187

The mechanism of the inhibitory effect of caffeine on smooth muscle contraction was examined using chicken gizzard. Caffeine (0.1-5 mmol/l) inhibited the KCl-induced contraction of the muscle with an IC50 of 1.1 mmol/l. Forskolin (0.01-10 mumol/l) also inhibited KCl-induced contraction. The inhibitory effect of caffeine was potentiated by a low concentration of forskolin (0.3 mumol/l) and the inhibitory effect of forskolin was potentiated by a low concentration of caffeine (0.1 mmol/l). Although caffeine and forskolin increased tissue cyclic AMP levels, caffeine inhibited the KCl-induced contraction more strongly than forskolin at a given cyclic AMP level. Caffeine (1-40 mmol/l) inhibited the contractions induced by 3 mumol/l Ca2+ in Triton X-100-permeabilized muscle. Caffeine (1-40 mmol/l) inhibited the phosphorylation of 20 kDa myosin light chain (MLC) in native actomyosin preparation and the inhibition was enhanced by decreasing the ATP concentration in the reaction medium. Calmodulin (CaM) activity, as monitored by Ca2+/CaM-dependent erythrocyte membrane (Ca2+ + Mg2+)-ATPase, was not affected by 20 mmol/l caffeine. Time-dependent dephosphorylation of MLC upon removal of Ca2+, an indicator of phosphate activity, was not affected by caffeine. Caffeine also inhibited the Ca2(+)-independent contraction in thiophosphorylated permeabilized muscle. These results indicate that caffeine inhibits smooth muscle contraction by a direct inhibition of MLC kinase and actin-myosin interaction. A part of the inhibitory effect may be mediated by cyclic AMP-dependent mechanism(s).
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PMID:Direct inhibition of chicken gizzard smooth muscle contractile apparatus by caffeine. 216 Jun 18

Previously, we have shown that okadaic acid (OA), isolated from black sponge (Halichondria okadai) causes contraction even in the absence of Ca++ in the saponin-permealized taenia isolated from guinea pig cecum. In the present study, mechanism of action of OA was examined using native actomyosin extracted from chicken gizzard smooth muscle. In the absence of Ca++, OA (0.1-1 microM) induced superprecipitation and increased the Mg++-adenosine triphosphatase activity. The OA-induced superprecipitation was enhanced by Ca++ at a concentration (greater than 0.1 microM) which did not activate the calmodulin-dependent myosin light chain (MLC) kinase. The effect of OA was not affected by the calmodulin inhibitor, trifluoperazine, at a concentration (100 microM) needed to inhibit the Ca++-induced response, but was inhibited markedly by the nonselective kinase inhibitors, amiloride (1 mM) and K-252a (5 microM). The OA-induced superprecipitation in the absence of Ca++ was accompanied by phosphorylation of the 20 K dalton MLC, which also was enhanced by low concentration of Ca++ (greater than 0.1 microM). OA did not change the phosphatase activity which dephosphorylates the phosphorylated MLC. An activator of Ca++- and phospholipid-dependent protein kinase, 12-O-tetradecanoylphorbol 13-acetate (1 microM), did not modulate superprecipitation or phosphorylation of MLC in the presence and absence of OA. Furthermore, inhibitors of Ca++ and phospholipid-dependent protein kinase, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (400 microM) and polymyxin B (100 micrograms/ml), affected neither superprecipitation nor phosphorylation of MLC induced by OA. With a reconstituted system containing purified myosin and MLC kinase, OA induced only slight phosphorylation of MLC.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Calcium-independent phosphorylation of smooth muscle myosin light chain by okadaic acid isolated from black sponge (Halichondria okadai). 282 58

The relaxant effects of amiloride and its analogues, benzamil, 5-(N,N-diethyl)-amiloride (DEAM) and 5-(N-ethyl-N-isopropyl)-amiloride (EIAM), were investigated using smooth muscle of guinea-pig taenia caeci and chicken gizzard. High K+-induced contractions of intact taenia and gizzard were inhibited by these compounds (1-100 microM) with the order of potency; benzamil greater than or equal to EIAM greater than DEAM greater than amiloride. Contractions of permealized taenia and gizzard were also inhibited by these compounds at concentrations 8-35 times higher than those needed to inhibit the contractions of intact tissues. These compounds inhibited 20 K myosin light chain (MLC) phosphorylation at the concentrations needed to inhibit the contraction in the permealized muscles. Calmodulin (CaM) activity, as monitored by erythrocyte membrane (Ca2+ + Mg2+)-ATPase and phosphodiesterase activities, was inhibited by DEAM and EIAM at similar concentrations as those to inhibit the MLC phosphorylation. Benzamil also inhibited CaM activity at concentrations 4-8 times higher than those required to inhibit MLC phosphorylation. However, amiloride failed to inhibit CaM activity. Among these compounds, amiloride and benzamil inhibited Ca2+/CaM-independent MLC phosphorylation due to trypsin-treated MLC kinase. Taenia tissue gradually accumulated these compounds and the tissue/medium ratio exceeded 3.5-17 after a 3-hr incubation period. These results indicate that amiloride and its analogues inhibit smooth muscle contraction mainly by the direct inhibition of MLC phosphorylation. The inhibitory effect of amiloride may be attributable to the inhibition of MLC kinase, whereas the inhibitory effect of DEAM and EIAM may largely be attributable to the inhibition of CaM. Benzamil may inhibit contraction by the inhibition of both MLC kinase and CaM. Differences in the drug-sensitivity between intact and permealized tissues may be attributable to the difference in drug accumulation by the cell.
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PMID:Direct inhibition of contractile apparatus by analogues of amiloride in the smooth muscle of guinea-pig taenia caecum and chicken gizzard. 293 May 91

The mechanism of the inhibitory effect of Li+ on contraction was examined in guinea pig ileal longitudinal smooth muscle. Li(+)-substitution (68.4 mM) reversed contractions induced by high K+ (45.4 mM), carbachol (1 microM) and histamine (1 microM) without changing the cytosolic Ca2+ level. Li+ also had no effect on the increase in 45Ca2+ uptake stimulated by high K+. High K+ transiently increased myosin light chain (MLC) phosphorylation, reaching a peak at 6-9 sec. Li(+)-substitution inhibited the high K(+)-induced MLC phosphorylation. In permeabilized ileal strips, contraction induced by 1 microM Ca2+ was inhibited by 10 mM Li+. The inhibitory effect was antagonized by increasing the concentration of Ca2+ or calmodulin. In the permeabilized muscle in which MLC was previously thiophosphorylated with 1 mM ATP gamma S and 3 microM Ca2+, ATP induced contraction in Ca2+ free buffer. Li+ added during this contraction did not show an inhibitory effect. In contrast, when 30 mM Li+ was added during the thiophosphorylation, the contraction induced by the subsequent addition of ATP was inhibited. Li+ (30 mM) changed neither the rate of relaxation induced by removing external Ca2+ in permeabilized muscle nor the rate of dephosphorylation of MLC induced by crude phosphatase extracted from the ileum. Li+ (15 mM), on the other hand, inhibited the rate of phosphorylation of MLC caused by crude MLC kinase extracted from the ileum. Li+ did not inhibit the calmodulin activity as measured with the (Ca2+ +Mg2+)-ATPase activity of the erythrocyte membrane. These results suggest that the inhibitory effect of Li+ on contractions is attributable to the inhibition of MLC kinase in guinea pig ileum.
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PMID:The inhibitory effect of Li+ on contractile elements of intestinal smooth muscle. 749 73


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