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)

Conformational changes of the region involving 17K dalton light chain (G2) in gizzard myosin induced by ATP and its analogs were studied by a combination of light chain exchange and fluorescence spectroscopy. The cysteinyl residues of myosin light chain mixture, 20K dalton G1 and 17K dalton G2 chain, were labeled with a fluorescent thiol reagent, N-(1-anilionon-aphthyl-4ymaleimide (ANM). Chicken gizzard myosin was incubated with a large excess of the fluorescence labeled exogenous light chains in 2 M urea. After removal of urea and unbound light chains, a strong fluorescent band was observed at the position of the 17K dalton light chain (G2) of myosin on SDS PAGE. There was no difference in enzymatic activity and light chain composition between myosins before and after light chain exchange. The emission spectrum of ANM label in 17K dalton light chain in the bound state in myosin showed a fluorescence enhancement and a blue shift on adding ATP but not ADP. The spectral change disappeared after conversion of ATP to ADP. Various nucleoside triphosphates other than ATP had similar effects on the fluorescence spectrum. The relative effectiveness of most nucleotides on the fluorescence spectral change of ANM in the light chain bound in myosin agreed well with that on tryptophan residues in myosin, except in the case of ADP and CTP. These results suggest that 17K dalton light chain of gizzard myosin is closely related to the ATPase site of myosin.
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PMID:Involvement of 17K dalton light chain of smooth muscle myosin in substrate-induced conformational change. 735 26

To determine whether thiophosphorylation of the 20-kDa myosin light chain activates each head of smooth muscle myosin independently of the head with which it is paired, chicken gizzard smooth muscle myosin was randomly thiophosphorylated, producing a mixture of unphosphorylated and singly and doubly thiophosphorylated myosin. Thiophosphorylation levels were measured by glycerol-urea gels, and the activity of this myosin was determined by actin-activated adenosinetriphosphatase measurements and in an in vitro motility assay, where the velocity of actin filaments moving over a myosin-coated surface is measured. Activity at each thiophosphorylation level was similar to that previously observed for mixtures of unphosphorylated and doubly thiophosphorylated myosin (D. E. Harris, S. S. Work, R. K. Wright, N. R. Alpert, and D. M. Warshaw. J. Muscle Res. Cell Motil. 15: 11-19, 1994). All doubly thiophosphorylated myosin was then formed into filaments and removed from randomly thiophosphorylated myosin by centrifugation. The remaining myosin (mixture of unphosphorylated and singly phosphorylated myosin), which could not polymerize because of their conformation, retained approximately 70% activity compared with mixtures of unphosphorylated and doubly thiophosphorylated myosin. Thus a thiophosphorylated smooth muscle myosin head can produce substantial biochemical and mechanical activity, even when it is paired with an unphosphorylated partner.
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PMID:Thiophosphorylation independently activates each head of smooth muscle myosin in vitro. 749 5

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

1. The effects of inorganic phosphate (P(i)) on force, Ca(2+)-force relationship, ATPase activity, maximal shortening velocity (Vmax) and rate of tension development were investigated in chemically skinned preparations of smooth muscle from the guinea-pig taenia coli. 2. In maximally thiophosphorylated fibres, P(i) in the range 1-40 mM inhibited isometric force, with a reduction of 20% at 20 mM P(i). 3. The relative force was similar at all [Ca2+], i.e. the Ca(2+)-force relationship was not affected, when 20 mM P(i) was present. 4. After photolytic release of ATP from caged ATP in maximally thiophosphorylated fibres in the presence of 20 mM P(i), tension rose to a lower level but with a higher rate constant than in the absence of P(i). 5. Inorganic phosphate (20 mM) did not affect the ATP hydrolysis in fibres activated at intermediate [Ca2+] or by maximal thiophosphorylation. 6. Inorganic phosphate (20 mM) decreased force but did not influence Vmax in maximally activated fibres. At lower levels of activation by Ca2+, P(i) increased the Vmax and decreased force slightly without affecting the degree of myosin light chain phosphorylation. 7. We conclude that P(i) influences cross-bridge reactions associated with force generation in smooth muscle. These reactions are not rate limiting for cross-bridge turnover under isotonic or isometric conditions in maximally activated smooth muscle fibres, since P(i) did not influence Vmax or the rate of ATP turnover. 8. Since P(i) increased Vmax in submaximally activated muscles, we propose that, under these conditions, shortening velocity is rate limited by cross-bridge states, reached early after attachment, which impose a mechanical resistance to shortening.
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PMID:Effects of inorganic phosphate on cross-bridge kinetics at different activation levels in skinned guinea-pig smooth muscle. 760 32

Smooth muscle hypertrophy is often found in tissue subjected to abnormal physical stress. To determine if physical stress (strain) per se could increase the contractile potential of airway smooth muscle (ASM), we compared cultured ASM cells subjected to strain to control cells (no strain) for rates of 1) myosin light chain kinase (MLCK)-mediated myosin light chain (LC20) phosphorylation, 2) actin-activated myosin ATPase, and 3) myosin light chain phosphatase-mediated myosin dephosphorylation. Lysates from strained cells showed increases in both LC20 phosphorylation activity and actomyosin ATPase activity but decreased rates of phosphatase-dependent myosin dephosphorylation. The increased LC20 phosphorylation activity and ATPase activity of the strained cells were accompanied by increases in cellular content of MLCK and myosin, respectively, compared with control. Because the cultured ASM cells exposed to strain expressed higher MLCK activity and actomyosin ATPase activity but lower myosin light chain phosphatase activity, these data suggest that physical stress in part determines ASM potential for contractile state.
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PMID:Mechanical strain increases contractile enzyme activity in cultured airway smooth muscle cells. 761 41

The phosphorylation of an M(r) 20,000 myosin light chain (MLC20) promotes the generation of contractile force through actin-myosin adenosine triphosphatase in most agonist-mediated vascular smooth muscle cell contraction. However, the role of calcium-mediated contractile processes in sustained arterial narrowing after subarachnoid hemorrhage remains unknown. In a femoral artery model of vasospasm, whole blood was applied to arteries in 54 rats for periods of 2 to 10 days; the contralateral artery treated with platelet-rich plasma served as matched control. During the early stage of vasospasm (Days 2-5), in the media of arteries exposed to blood, MLC20 phosphorylation (including diphosphorylated forms) increased significantly (30-38%; P < 0.05); total medial MLC20 during this interval was comparable to that in controls. After 5 days, however, total MLC20 decreased markedly (> 90%; P < 0.01) compared with controls; phosphorylated MLC20 was undetectable during this interval. MLC20-mediated contractile processes may be prominent in the early stages of arterial narrowing after subarachnoid hemorrhage; later stages are associated with the loss of MLC20 and the possible persistence of arterial narrowing by other mechanisms.
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PMID:The time course of myosin light-chain phosphorylation in blood-induced vasospasm. 764

Two anti-17,000 Da myosin light chain (LC17) monoclonal antibodies (MM2 and MM10), which increase the actin-activated Mg(2+)-ATPase activity of dephosphorylated smooth muscle myosin, inhibited the exchange of the 20,000 Da regulatory light chain of myosin (LC20). MM2, which shows higher potency of activation of ATPase activity, inhibited the exchange more extensively than MM10, suggesting that there is a correlation between the activation of ATPase activity and the inhibition of the LC20 exchange. The inhibition of the exchange was observed for intact myosin and heavy meromyosin but not subfragment 1, suggesting that the heavy chain at the head-rod junction is involved in the inhibition of LC20 exchange by anti-LC17 antibodies. Alternatively, the interaction between the two heads of the myosin molecule may influence the inhibition of LC20 exchange. These results suggest that LC20 interacts with both LC17 and the heavy chain, and the interaction between LC20 and LC17 is involved in the activation of actin-activated ATPase activity of smooth muscle myosin.
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PMID:Inhibition of 20-kDa myosin light chain exchange by monoclonal antibodies against 17-kDa myosin light chain. 772 54

The controversial finding that the thick filaments of smooth muscle can be evanescent leads to the hypothesis that the large functional range of this muscle is accommodated by plastic rearrangements that place more thick filaments in series at longer lengths. Our preliminary finding that the shortening velocity and compliance of dog tracheal muscle were strongly dependent on adapted muscle length, while force was much less length dependent, supports this hypothesis (V.R. Pratusevich, C.Y. Seow, and L.E. Ford. Biophys. J. 66: A139, 1994). The hypothesis leads to two further corollaries. The first is that the lengthening of the thick filaments that must accompany their reformation will cause a series to parallel transition: fewer long filaments span the muscle length, but the longer filaments have more cross bridges acting in parallel. The second is that there is more than one activating mechanism in smooth muscle. It is known that myosin light chain phosphorylation activates the actomyosin ATPase, but this same phosphorylation also causes a structural change that facilitates filament formation. The consideration that the unaggregated, phosphorylated myosin must be prevented from competing with myosin in thick filaments and hydrolyzing ATP suggests that there must be a second mechanism that must allow the thin filaments to interact selectively with filamentous myosin. This need for a second activating mechanism may explain the presence of tropomyosin, calponin, and caldesmon on thin filaments. Although the two corollaries follow from the initial hypothesis, it should be emphasized that the three are not mutually dependent, and that the proof or disproof of any one of them would not prove or disprove the others.
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PMID:Plasticity in smooth muscle, a hypothesis. 776 73

Calponin has been implicated in the regulation of smooth muscle contraction as a result of its ability to inhibit the actin-activated Mg ATPase of smooth muscle myosin. This inhibitory effect is abolished by phosphorylation of calponin by Ca2+/calmodulin-dependent protein kinase II or protein kinase C, and restored following dephosphorylation by a type 2A protein phosphatase. Confocal immunofluorescent images of isolated smooth muscle cells colabeled with antibodies to calponin and actin or to calponin and tropomyosin indicate that calponin is present on thin filaments throughout the cell cytoplasm. Both calponin phosphorylation and myosin light chain phosphorylation increased in intact smooth muscle tissue strips when they contracted in response to carbachol or the phosphatase inhibitor okadaic acid. These results support the hypothesis that calponin phosphorylation-dephosphorylation plays a role in regulating smooth muscle contraction.
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PMID:Calponin and smooth muscle regulation. 776 87

To study asthmatic airway smooth muscle we developed a canine model of ragweed pollen sensitized, airway hyperresponsiveness because of the difficulties in obtaining human tissue. Tracheal and bronchial smooth muscles from sensitized dogs were shown to possess greater ability to shorten and higher maximum shortening velocity (Vo), both of which contribute to the excessive narrowing of airways typical of human asthma. However, maximum force production remained normal, demonstrating the dissociation between the behaviour of shortening and force. Because we found no evidence of inflammation, hypertrophy, or hyperplasia in the sensitized airway smooth muscles, we felt this is a model of early disease and should provide insight into early and perhaps primary pathogenetic mechanisms. Vo is known to be determined by actomyosin ATPase, which in smooth muscle is activated via phosphorylation of the 20-kDa myosin light chain (MLC20) by myosin light chain kinase (MLCK). Therefore, ATPase activity, MLC20 phosphorylation, and MLCK were investigated. Sensitized tracheal and bronchial smooth muscles showed significantly higher ATPase activity, and a higher level of MLC20 phosphorylation, resulting from increased MLCK activity, a consequence of the measured increase in total quantity of MLCK rather than in specific activity. Since MLCK is activated by binding with Ca(2+)-calmodulin complex, intracellular Ca2+ concentration and calmodulin activity were also assessed, but no difference was found between sensitized and control animals. Our study suggests that increased MLCK quantity may be the cause of airway hyperresponsiveness found in sensitized animals, and future investigation should be focused on depicting the reason for the elevated MLCK.
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PMID:Early changes in airway smooth muscle hyperresponsiveness. 776 91


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