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 small guanosine triphosphatase Rho is implicated in myosin light chain (MLC) phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in nonmuscle cells. The guanosine triphosphate (GTP)-bound, active form of RhoA (GTP.RhoA) specifically interacted with the myosin-binding subunit (MBS) of myosin phosphatase, which regulates the extent of phosphorylation of MLC. Rho-associated kinase (Rho-kinase), which is activated by GTP.RhoA, phosphorylated MBS and consequently inactivated myosin phosphatase. Overexpression of RhoA or activated RhoA in NIH 3T3 cells increased phosphorylation of MBS and MLC. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase.
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PMID:Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase) 866 97

The N-terminal region of skeletal myosin light chain-1 (MLC-1) binds to the C terminus of actin, yet the functional significance of this interaction is unclear. We studied a fragment (MLC-pep; residues 5-14) of the ventricular MLC-1. When added to rat cardiac myofibrils, 10 nM MLC-pep induced a supramaximal increase in the MgATPase activity at submaximal Ca2+ levels with no effect at low and maximal Ca2+ levels. A nonsense, scrambled sequence peptide had no effect at any pCa value. MLC-pep did not affect myosin KEDTA and CaATPase activities or actin-activated MgATPase activities in the absence or presence of tropomyosin. The MLC-pep did not alter the ability of troponin I to inhibit MgATPase activity. Moreover, when troponin I and troponin C were extracted from the myofibrils, the MLC-pep lost its ability to stimulate the ATPase rate. This effect was fully restored upon reconstitution of the extracted myofibrils with troponin I-troponin C complex. Thus, activation of MgATPase activity by the peptide required a full complement of thin filament regulatory proteins. Interestingly, the stimulatory effect occurred at a ratio of 4 peptides to 1 thin filament, suggesting that the peptide engages in a highly cooperative process that may involve activation of the entire thin filament.
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PMID:An essential myosin light chain peptide induces supramaximal stimulation of cardiac myofibrillar ATPase activity. 890 Jan 93

Although the actin-binding and actomyosin adenosinetriphosphatase (ATPase) inhibitory properties of calponin are well documented in vitro, its function in the smooth muscle cell has not been elucidated. To address this question, we utilized the ferret aortic smooth muscle cell, which shows a protein kinase C-dependent contraction even at pCa (-log [Ca2+]) 9.0 in the absence of a change in myosin light chain phosphorylation. Force was recorded from single, briefly permeabilized cells stimulated via a Ca(2+)-independent pathway by either phenylephrine or the epsilon isoenzyme of protein kinase C. Treatment of stimulated cells with wild-type recombinant calponin reduced steady-state contractile force by 45-60%. When calponin application preceded protein kinase C epsilon treatment, contraction was completely suppressed. On the other hand, calponin phosphorylated at Ser175 or mutant calponin with a Ser175 --> Ala replacement had no effect on contractile force. A peptide corresponding to Leu166-Gly194 of calponin, which included an actin-binding domain but excluded the actomyosin ATPase inhibitory region, was synthesized. Treatment of aortic smooth muscle cells with this peptide triggered a concentration-dependent contraction, presumably by alleviating the inhibitory effect of endogenous calponin. A control peptide with a scrambled sequence of the same residues produced no detectable contractile response. Although other interpretations are possible, these results are consistent with the view that calponin participates in thin filament-mediated regulation of smooth muscle contraction and that it may be part of a Ca(2+)-independent pathway downstream of protein kinase C epsilon.
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PMID:Effects of calponin on force generation by single smooth muscle cells. 892 96

Myosin is a highly conserved, ubiquitous protein found in all eukaryotic cells, where it provides the motor function for diverse movements such as cytokinesis, phagocytosis, and muscle contraction. All myosins contain an amino-terminal motor/head domain and a carboxy-terminal tail domain. Due to the extensive number of different molecules identified to date, myosins have been divided into seven distinct classes based on the properties of the head domain. One such class, class II myosins, consists of the conventional two-headed myosins that form filaments and are composed of two myosin heavy chain (MYH) subunits and four myosin light chain subunits. The MYH subunit contains the ATPase activity providing energy that is the driving force for contractile processes mentioned above, and numerous MYH isoforms exist in vertebrates to carry out this function. The MYHs involved in striated muscle contraction in mammals are the focus of the current review. The genetics, molecular biology, and biochemical properties of mammalian MYHs are discussed below. MYH gene expression patterns in developing and adult striated muscles are described in detail, as are studies of regulation of MYH genes in the heart. The discovery that mutant MYH isoforms have a causal role in the human disease familial hypertrophic cardiomyopathy (FHC) has implemented structure/function investigations of MYHs. The regulation of MYH genes expressed in skeletal muscle and the potential functional implications that distinct MYH isoforms may have on muscle physiology are addressed.
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PMID:The mammalian myosin heavy chain gene family. 897 Jul 33

The regulatory myosin light chain (MLC) is phosphorylated in cardiac muscle by Ca2+/calmodulin-dependent MLC kinase (MLCK) and is considered to play a modulatory role in the activation of myofibrillar adenosine triphosphatase (ATPase) and the process of force generation. Since the depression in cardiac contractile function in chronic diabetes is associated with a decrease in myofibrillar ATPase activity, we investigated changes in MLC phosphorylation in diabetic heart. Rats were made diabetic by injecting streptozotocin (65 mg/kg intravenously), and the hearts were removed 8 weeks later; some 6-week diabetic animals were injected with insulin (3 U/d) for 2 weeks. Changes in the relative MLC and MLCK protein contents were measured by electrophoresis and immunoblot assay, whereas phosphorylated and unphosphorylated MLCs were separated on 10% acrylamide/urea gel and identified by Western blot. MLC and MLCK contents were decreased markedly (40% to 45%) and MLC phosphorylation was decreased significantly (30% to 45%) in the diabetic rat heart homogenate in comparison to control values. The changes in MLC and MLCK content in diabetic heart were partially reversible, whereas changes in MLC phosphorylation were normalized upon treatment with insulin. These results suggest that decreased protein contents of MLC and MLCK and phosphorylation of MLC may contribute to the depression of cardiac myofibriliar ATPase activity and heart dysfunction in diabetic cardiomyopathy.
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PMID:Myosin light-chain phosphorylation in diabetic cardiomyopathy in rats. 900 73

A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-type, rat aorta 20-kDa myosin light chain (MLC20) or a mutant form of MLC20 in which Thr18 and Ser19 were mutated into alanines. These mutations result in a MLC20 that cannot be phosphorylated by myosin light chain kinase. An 11-amino acid epitope from c-myc was added to both MLC20 sequences to facilitate identification of these proteins. Madin-Darby canine kidney cells were stably transduced, and MLC20 expression was demonstrated by Western blot analysis using a myc-specific antibody. MLC20 exchange was demonstrated by purifying myosin from the transduced cells and repeating the Western blot analysis. Actin-activated adenosinetriphosphatase assays on the purified myosins demonstrated approximately 50% decrease in the rate of ATP hydrolysis by the myosin containing the mutant MLC20. Transepithelial electrical resistance was decreased and mannitol flux was increased across monolayers of cells expressing mutant MLC20. These data demonstrate that MLC20 phosphorylation is involved in regulating paracellular permeability and epithelial barrier function.
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PMID:Expression of a mutant myosin light chain that cannot be phosphorylated increases paracellular permeability. 912 98

Increased maximum velocity of shortening (Vmax), increased shortening ability (delta Lmax) and decreased relaxation rate have been reported for arterial smooth muscle from 16- to 18-week-old spontaneously, hypertensive rats (SHR) compared with age-matched normotensive Wistar-Kyoto rats (WKY). Vmax is dependent on actomyosin ATPase activity, and this activity is in turn dependent on the level of phosphorylation of the 20-kDa myosin light chain (MLC20) normally a function of calcium concentration. In this article, methods are described and data are presented from studies addressing possible intracellular regulatory mechanisms that might lead to the altered contractility of the SHR arterial muscle. In one study, myofibrillar protein was extracted from 16- to 18-week-old SHR and WKY caudal arterial muscle. The Mg(2+)-activated ATPase activity was measured under conditions where the Ca2+ concentration was controlled. In another study, the amount of myosin present and relative proportions of the myosin heavy chain (MHC) isoforms were determined by quantitative SDS-PAGE using heavy molecular weight standards and bovine serum albumin as the standard for concentration. In a third study, MLC20 phosphorylation levels in electrically stimulated arterial muscle were determined by urea glycerol gel electrophoresis and Western blot analyses. The SHR (n = 6) myofibrillar ATPase liberated 0.011 +/- 0.003 mumol Pi/mg myosin/min, which was significantly more than the 0.006 +/- 0.001 mumol Pi/mg myosin/min liberated by the WKY (n = 4) myofibrillar ATPase (P < 0.05). Consistent with the increased ATPase activity, phosphorylation of MLC20 was increased by 2.8 times as much in the SHR compared with the WKY electrically stimulated arterial muscle. However, there was no difference in MHC isoform pattern in the SHR compared with the WKY arterial muscle in contrast to the findings of at least one other laboratory. This discrepancy is discussed. The data reviewed in this article lead to the conclusions that an increased actin-activated myosin ATPase activity and MLC20 phosphorylation are likely responsible for the increased velocity of shortening previously reported in SHR arterial muscle and the increased ATPase activity is not a function of an increased myosin content or of altered MHC isoform pattern in the SHR muscle.
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PMID:Arterial muscle myosin heavy chains and light chains in spontaneous hypertension. 918 11

1. Stellettamide A (ST-A), a novel marine toxin isolated from a marine sponge, inhibited high K+(72.7 mM)-induced contraction in the smooth muscle of guinea-pig taenia coli with an IC50 of 88 microM. 2. In the taenia permeabilized with Triton X-100, ST-A inhibited Ca2+ (3 and 10 microM)-induced contractions with an IC50 of 46 microM for 3 microM Ca2+ and 105 microM for 10 microM Ca2+. In the permeabilized taenia, calyculin-A (300 nM), a potent inhibitor of type-1 and type-2A phosphatases, induced sustained contraction in the absence of Ca2+. ST-A had no effect on this contraction. 3. ST-A inhibited Mg2+-ATPase activity in native actomyosin prepared from chicken gizzard with an IC50 of 25 microM. 4. In a reconstituted smooth muscle contractile system containing calmodulin, myosin light chain (MLC) and MLC kinase, ST-A inhibited MLC phosphorylation with an IC50 of 152 microM. The inhibitory effect of ST-A was antagonized by increasing the concentration of calmodulin. 5. ST-A inhibited calmodulin activity, assessed by Ca2+/calmodulin-dependent enzymes, (Ca2+-Mg2+)-ATPase of erythrocyte membrane, with an IC50 of 100 microM and phosphodiesterase prepared from bovine cardiac muscle with an IC50 of 52 microM. The inhibitory effect on phosphodiesterase activity was antagonized by increasing the calmodulin concentration. 6. Interaction between ST-A and calmodulin was demonstrated by instantaneous quenching of the intrinsic tyrosine fluorescence of calmodulin by ST-A (3-300 microM). Similar results were obtained in the presence or absence of Ca2+ suggesting that ST-A binds to calmodulin and that Ca2+ is not essential for the binding of ST-A to calmodulin. 7. These results suggest that ST-A, isolated from marine metabolites, is a novel inhibitor of calmodulin.
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PMID:Stellettamide-A, a novel inhibitor of calmodulin, isolated from a marine sponge. 925 8

The p21-activated kinase (PAK) family includes protein phosphotransferases regulated by the GTPases rho, rac, and cdc42. Sequence homology, activation mechanism, and substrate specificity suggest that the well-characterized human placenta S6/H4 kinase is a member of this family. In these studies, S6/H4 kinase purified to homogeneity from human placenta was activated in vitro by cdc42-GTP, or protease incubation and MgATP-dependent autophosphorylation. The cdc42-activated enzyme demonstrated an Mr 60,000, and shares sequence homology with the gammaPAK family. Antipeptide antibodies against one of the autophosphorylation site sequences recognized a single p60 protein in the purified placenta preparation or Jurkat cell extracts. An autophosphorylated Mr 40,000 protein, previously identified as the catalytic domain of the enzyme, was also detected by the antibody after protease activation. Crude PAK60 obtained from Mono Q chromatography of Jurkat cell extracts and purified placenta enzyme catalyzed phosphorylation of histone H4 and myelin basic protein as well as a variety of synthetic peptides previously identified as S6/H4 kinase substrates. In addition, Jurkat myosin II and the regulatory myosin light chain were phosphorylated by the Jurkat and placenta gammaPAK. Synthetic peptides were used to demonstrate that the site of light chain phosphorylation occurs at the serine which results in ATPase activation. The data suggest that human gammaPAK may regulate cell motility by a GTP-dependent and calcium-independent mechanism.
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PMID:Myosin phosphorylation by human cdc42-dependent S6/H4 kinase/gammaPAK from placenta and lymphoid cells. 939 38

Chronic asthma is characterized by hypertrophy and hyperplasia of airway smooth muscle cells (SMC) that limit airflow by a geometric effect. Whether contractility of airway SMC is altered is not clear. Cultured cells were used as a model of hyperplasia. Phenotypic changes seen indicated conversion to a synthetic, weakly contractile type. At confluence, although limited reversal of protein changes was seen, no restoration in contractility occurred. Phenotypic modulation of postconfluent cultured airway SMC under prolonged serum deprivation (arrested cells) is reported here. Two phenotypically distinct groups of cells were identified in primary airway SMC cultures: 1) elongated spindle-shaped cells, which expressed large amounts of smooth muscle contractile and regulatory proteins, and 2) flat and stellate cells, which expressed very little. The first group showed a surprising shortening capacity and a velocity that was even greater than that of the freshly isolated cells, whereas the second group became spherical and noncontractile. Even more surprising was that the myosin heavy chain (MHC) isoform (SM-B) generally said to be associated with the higher shortening velocity disappeared from the cell, while the content of the key rate-limiting regulating enzyme, myosin light chain kinase (MLCK), increased 30-fold. We conclude that a functional, contractile phenotype of airway SMC can be obtained by prolonged serum deprivation. We speculate that the increased contractility could be the result of increased phosphorylation of the 20-kDa myosin light chain resulting from increased content of smooth muscle MLCK rather than any increase in endogenous MHC ATPase activity. This model may be useful for study of SMC differentiation and contraction.
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PMID:Serum deprivation induces a unique hypercontractile phenotype of cultured smooth muscle cells. 961 7


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