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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)

In this paper the correlation between phosphate incorporation into the regulatory light chain of myosin by a Ca2+-dependent myosin light chain kinase, and the Ca2+-sensitive ATPase activity and superprecipitation behaviour of arterial actomyosin, is described.
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PMID:A Ca2+-sensitive myosin light chain kinase, regulating pig carotid smooth muscle actomyosin ATPase. 624 12

Sepharose 4B conjugated with phosphorylated myosin light chains was used in affinity chromatography of a partially purified preparation of gizzard myosin light-chain phosphatase (MLCP) (Onishi et al. (1979) J. Biochem. 86, 1283-1290). The MLCP preparation thus purified contained, according to SDS gel electrophoresis, three components of 67,000 (67 K), 54,000 (54 K), 34,000 (34 K) daltons. In an accompanying report, Uchiwa et al. (J. Biochem. 91, 273-282 (1982)) described the purification of gizzard myosin light-chain kinase, which consisted of two subunits; 130 K and 17 K daltons. Using the purified preparations of MLCP and MLCK, it was demonstrated a) that reversible changes in the ATPase and superprecipitation activities occur as myosin light chains are enzymatically phosphorylated and dephosphorylated, and b) that addition of a very low concentration of Ca2+ and its removal cause reversible changes in the turbidity of actomyosin suspensions as well as in the state of phosphorylation of myosin light chains only when MLCK and MLCP are both present. These results provide strong support for the proposal (see Ikebe et al. (1977) J. Biochem. 80, 299-302) that MLCK and MLCP play a key role in the Ca2+ regulation in gizzard.
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PMID:Purification of gizzard myosin light-chain phosphatase, and reversible changes in the ATPase and superprecipitation activities of actomyosin in the presence of purified preparation of myosin light-chain phosphatase and kinase. 627 83

The various protein components of a reversible phosphorylating system regulating smooth muscle actomyosin Mg-ATPase activity have been purified. The enzyme catalyzing phosphorylation of smooth muscle myosin, myosin-kinase, requires Ca2+ and the Ca2+-binding protein calmodulin for activity and binds calmodulin in a ratio of 1 mol calmodulin to 1 mol of myosin kinase. Myosin kinase can be phosphorylated by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, and phosphorylation of myosin kinase that does not have calmodulin bound results in a marked decrease in the affinity of this enzyme for Ca2+-calmodulin. This effect is reversed when myosin kinase is dephosphorylated by a phosphatase purified from smooth muscle. When the various components of the smooth muscle myosin phosphorylating-dephosphorylating system are reconstituted, a positive correlation is found between the state of myosin phosphorylation and the actin-activated Mg-ATPase activity of myosin. Unphosphorylated and dephosphorylated myosin cannot be activated by actin, but the phosphorylated and rephosphorylated myosin can be activated by actin. The same relationship between phosphorylation and enzymatic activity was found for a chymotryptic peptide of myosin, smooth muscle heavy meromyosin. The findings reported here suggest one mechanism by which Ca2+ and calmodulin may act to regulate smooth muscle contraction and how cAMP may modulate smooth muscle contractile activity.
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PMID:Regulation of smooth muscle contractile proteins by calmodulin and cyclic AMP. 629 Feb 74

Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+-ATPase was purified 33-fold to a specific activity of 53 +/- 0.5 (12) mumol Pi X mg-1 X h-1, binding sites for strophantin 20-fold to a density of 56.3 +/- 5.3 (14) pmol/mg and that for the calcium antagonist nitrendipine 5.5-fold to a density of 0.72 +/- 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 +/- 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 +/- 4 (4) microliter/mg and 56.9 +/- 6 (4)% of the vesicles were right-side-out oriented. Several peptides of the purified membranes were phosphorylated in the presence of Mg . ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100 degrees C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP-dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22-kDa peptide and a 44-kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55-kDa. Ca2+/calmodulin-dependent phosphorylation of a 55-kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase. 'Depolarization' induced Ca2+ uptake which was measured according to Bartschat, D.K., Cyr, D.L. and Lindenmayer, G.E. [(1980) J. Biol. Chem. 255, 10044-10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg . ATP or Mg . ATP and cAMP-dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassium-stimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg . ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium-stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22-kDa peptide.
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PMID:Phosphorylation of purified bovine cardiac sarcolemma and potassium-stimulated calcium uptake. 630 17

One of the major proteins of the chicken intestinal microvillus is a calmodulin-binding protein of 105-110 kdaltons which has been tentatively identified as the bridge linking the microvillar filament bundle laterally to the membrane. We have treated isolated, membrane-intact brush borders with ATP and obtained solubilization of the 110-kdalton protein, calmodulin (CM), myosin, and lesser amounts of several other cytoskeletal proteins. Electron micrographs of ATP-extracted brush borders showed loss of the linkers between the actin filament bundle and the microvillar membrane, with "ballooning" of the membrane away from the filament bundle, particularly at the tip end. In brush borders treated with calcium and trifluoperazine to solubilize CM, precise arrangement and morphology of lateral bridges was unperturbed, but ATP treatment would no longer solubilize the 110-kdalton protein. This result suggests that associated CM is necessary for the ATP-induced solubilization of the 110-kdalton protein. A 110-kdalton protein-CM complex, with 110-kdalton protein: CM ratios of 1:1-2, was partially purified from ATP-extracts of brush borders by a combination of gel filtration and hydroxylapatite chromatography. The 110-kdalton protein-CM complex is an irregular, elongated molecule that ranged in size from 5 X 8 nm to 8 X 14 nm, with a Stokes' radius of 6.1 nm. This 110-kdalton protein-CM complex exhibited no Mg++-ATPase activity and no detectable myosin light chain kinase activity. In co-sedimentation assays, the 110-kdalton protein-CM bound to F-actin in the absence but not the presence of ATP. Both the interaction of the complex with actin and the binding of CM to the 110-kdalton protein were calcium-independent. Negative stains of F-actin and 110-kdalton protein-CM in the absence of ATP showed loosely organized aggregates of actin with the 110-kdalton protein-CM complex coating the surface of the filaments. On the basis of our data, and in agreement with previous calculations (Matsudaira, P.T., and D.R. Burgess, 1979, J. Cell Biol. 83:667-673), we suggest that the lateral bridge of the microvillus is composed of a dimer of the 110-kdalton protein with four associated calmodulins.
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PMID:Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells. 631 43

Trifluoperazine (TFP) inhibits superprecipitation and ATPase activity of smooth muscle actomyosin. This effect appears not to be due to the inhibitory effect of TFP on the Ca++-dependent modulator and the myosin light chain kinase, which are known to be cofactors required for activation of smooth muscle actomyosin.
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PMID:Effects of trifluoperazine on smooth muscle actomyosin. Short communication. 644 96

Three different reactions are known to occur in a combined system of skeletal actin, gizzard myosin, and gizzard native tropomyosin. They were studied as functions of ATP concentration. (a) At around 1 microM ATP in the presence of an ATP-regenerating system, two of the three reactions, superprecipitation and ATPase reaction, occurred independently of calcium and were not accompanied by the third reaction, phosphorylation of myosin light chains. (b) Relatively high concentrations of ATP were required for calcium-dependent phosphorylation and for calcium-activated ATPase reaction. It is suggested that the apparent Km value for myosin light-chain kinase and that for acto-phosphorylated myosin-ATPase are approximately 10(-4.0) M and 10(-5.5) M, respectively. (c) The calcium-dependent phosphorylation and the calcium- activated ATPase reaction were closely coupled, but they were only indirectly coupled with superprecipitation. (d) In some of the responses to change in ATP concentration, (a)-(c), skeletal acto-gizzard myosin was similar to skeletal acto-skeletal myosin. It was also similar in that sulfhydryl groups of myosin are involved in the calcium regulation of actomyosin-ATPase and its superprecipitation in both cases.
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PMID:Phosphorylation of gizzard myosin light chain and the ATPase reaction and superprecipitation of skeletal acto-gizzard myosin as functions of ATP concentration. 644 51

1. A purified preparation of myosin light-chain kinase (MLCK) was obtained from chicken gizzard, and it was shown to consist of two subunits; 130,000 (130 K)-dalton subunit and 17,000 (17 K)-dalton subunit. In amino acid composition the 130 K and 17 K subunits were identical with the 105 K and 17 K subunits of Dabrowska et al. (1977 and 1978), respectively. In disc gel electrophoresis, the 17 K subunit of our MLCK preparation responded to Ca2+ ions in the same way as bovine calmodulin, and differently from skeletal troponin C. There appeared to be one minor difference between 17 K subunit and calmodulin in the primary structure of the C-terminal region. 2. The Ca2+ and Sr2+ concentrations required for the three activities (ATPase and superprecipitation activities and MLCK activity) were measured. Two types of "reconstituted" myosin B were used; one contained 17 K subunit of gizzard MLCK and the other contained bovine brain calmodulin. The two types of "reconstituted" myosin B were practically identical with "natural" myosin B in the Ca2+ and Sr2+ requirements for the three activities measured above. 3. Both the extent and the activity of superprecipitation, and both the limited and steady activities of ATPase were measured. The MLCK activity was estimated in two ways; by urea gel electrophoresis and by measuring 32 P incorporation from [gamma-32P]ATP into myosin. The results thus obtained favor the kinase-phosphatase mechanism of calcium regulation of gizzard muscle contraction.
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PMID:Purification of chicken gizzard myosin light-chain kinase, and its calcium and strontium sensitivities as compared with those of superprecipitation and ATPase activities of actomyosin. 646 41

Glycerinated single fibres from the dorsal longitudinal muscle of Lethocerus maximus were isometrically contracted in MgATP-salines (10 microM Ca2+; 1.5 mM Mg2+; pH 6.7; 22 degrees C and 20 mM PEP; 100 U/ml pyruvate kinase). The ratio of ATPase activity to tension decreased by a factor of 2 after reducing the ATP-concentration from 15 to 0.5 mM. At all ATP-concentrations (0.5-15 mM), the fibres showed tension adjustments in response to small step changes in length characteristic to an actively contracting muscle: i) an elastic phase which did not depend on ATP-concentration ii) a quick phase of stress relaxation with at least two exponential components; iii) a phase of delayed tension generation. An increase in size of the length step and/or a decrease of ATP-concentration slowed the quick phase and the delayed phase. Similar results have been obtained with skinned cardiac muscle (pig right ventricle). To see, how the isolated contractile system is affected by an increase in the light chain phosphorylation, tension transients were studied in skinned right ventricular muscle fibres before and after incubation with ATP gamma S (2 mM), pure myosin light chain kinase (9 micrograms/ml), Calmodulin (1 microM) and Ca2+ (0.8 microM). While isometric tension development elicited by 20 microM Ca2+ in the ATP salt solution was barely affected in presence of the enzyme, the ATPase activity was decreased by about 25% of the control. There was also a marked decrease (about 50%) in the contraction velocity as determined by the recovery of tension following a quick release. Quick stretches cause an immediate increase in tension followed by a rapid fall and a subsequent rise in tension. The velocity of this tension rise decreased by approximately 30% after incubation with myosin light chain kinase.
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PMID:Tension transients in skinned muscle fibres of insect flight muscle and mammalian cardiac muscle: effect of substrate concentration and treatment with myosin light chain kinase. 661 Oct 37

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


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