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: KEGG:D03291 (CaCl2)
6,342 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The thin filaments of vascular smooth muscle (pig aorta) contain a Ca2+-sensitive regulatory system that resembles troponin-tropomyosin [Marston, Trevett & Walters (1980) Biochem. J. 185, 355-365]. Our thin-filament preparations also contain enzymes that phosphorylate and dephosphorylate a specific protein. Initial rate of phosphorylation was 0.42 +/- 0.10 (95% confidence limits) mumol of Pi/min per g of thin filaments; half-maximal incorporation was obtained in 4 1/2 min, and a maximum of 1.8 +/- 0.1 mumol of Pi/g of thin filaments was incorporated after 40 min (conditions: 1 mM-MgATP, 60 mM-MgATP, 60 mM-KCl, 10 mM-imidazole, pH 7.0, 5 mM-MgCl2, 10 mM-NaN3, 0.5 mM-dithiothreitol, 0.1 mM-CaCl2, 25 degrees C). On gel electrophoresis in polyacrylamide (4-30% gradient)/0.25% sodium dodecyl sulphate gel over 75% of protein-bound phosphate was in a single protein of mol.wt. 21000. On electrophoresis in polyacrylamide (8%)/6 M-urea (pH 8.6) gel the phosphoprotein remained at the origin. Phosphorylation was associated with an increase in the concentration of high-affinity (K congruent to 10(6) M-1) Ca2+-binding sites from 0.8-1.5 to 6.3 mumol of Ca2+/g of thin filaments. Phosphorylation also changed the regulatory properties of the skeletal-muscle myosin-aorta thin-filament MgATPase; maximum activity was unaltered, but the phosphorylated thin filaments required only 0.36 microM-Ca2+ for half-activation compared with 2.7 microM-Ca2+ for unphosphorylated thin filaments. The possible regulatory role of thin-filament phosphorylation is discussed.
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PMID:Phosphorylation of the calcium ion-regulated thin filaments from vascular smooth muscle. A new regulatory mechanism? 645 87

Relaxation of tissues prepared from the swine carotid media following agonist (110 mM K+) washout was analyzed as a dual-exponential decay. The time course of the initial rapid phase (about 2 min) corresponded to myosin dephosphorylation and to the decay of the capacity to shorten isotonically. Because myosin was dephosphorylated to basal levels within 2 min, we hypothesize that the later, slow phase of relaxation (lasting up to 45 min) was due to a slow inactivation of nonphosphorylated cross bridges. Removing extracellular Ca2+ (0 mM CaCl2, 0.1 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid) greatly enhanced the rate of the slow phase of relaxation, and raising extracellular CaCl2 to 5 mM slowed relaxation significantly. A slow rate of Ca2+ removal to a final concentration that maintains resting tone appears to produce the slow phase of relaxation. These results support hypotheses based on other studies of contracting muscles. There appear to be two populations of cross bridges interacting with the thin filament: 1) phosphorylated and capable of rapid cycling, and 2) dephosphorylated cross bridges that can maintain stress. The latter reflect an unidentified regulatory mechanism, which appears to have a high sensitivity for Ca2+.
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PMID:Ca2+, myosin phosphorylation, and relaxation of arterial smooth muscle. 661 59

Myosin light chain kinase has been purified approximately 1700-fold from bovine thyroid gland, using Affigel blue column chromatography, ammonium sulfate fractionation (0-60% saturation), Sepharose 6B gel filtration, and calmodulin-Sepharose affinity column chromatography. This partially purified kinase was Ca2+ and calmodulin dependent for its activity and specifically phosphorylated the 20,000-dalton light chain of chicken gizzard myosin. At higher CaCl2 concentration, thyroid myosin light chain kinase demonstrated a novel inhibition in the presence of calmodulin. The mechanism of this Ca2+-dependent inhibition is not clear at present. The existence of myosin light chain kinase in thyroid gland provides additional support for the role of the contractile system in the secretion of thyroid hormones.
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PMID:Partial purification and characterization of myosin light chain kinase from bovine thyroid gland. 668 57

The role of myosin phosphorylation in regulating smooth muscle contraction has been investigated by quantitating the myosin phosphate content of tracheal smooth muscle frozen during contraction or relaxation. Myosin was purified from quick-frozen muscle samples with the aid of antibodies prepared against tracheal smooth muscle myosin, and the phosphate content was determined after separation of nonphosphorylated and phosphorylated myosin subunits by isoelectric focusing. The myosin phosphate content increased from an initial value of 0.50 to 1.1 mol of phosphate/mol of myosin within 3 min after the addition of 100 microM methacholine to resting tracheal smooth muscle. Myosin phosphorylation coincided temporally with the increase in isometric tension. Tracheal smooth muscles relaxed and the phosphate content decreased from 1.2 to 0.50 mol of phosphate/mol of myosin upon the addition of 10 microM atropine to muscles which had been previously contracted with 100 microM methacholine. Incubating methacholine-contracted muscles in a calcium-free Krebs buffer relaxed tracheal smooth muscles and reduced the phosphate content to 0.2 mol of phosphate/mol of myosin. Addition of 3 mM CaCl2 to these muscles elicited an increase in isometric tension concomitant with phosphorylation of myosin. These results support the hypothesis that myosin phosphorylation is important in regulating smooth muscle contraction.
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PMID:Myosin phosphorylation during contraction and relaxation of tracheal smooth muscle. 677 5

The binding of myosin subfragment-1 (S-1) to F-actin in the absence of nucleotide was examined by the sedimentation method using 1,5-IAEDANS-labeled S-1. We found that the binding affinity of F-actin to S-1 was dependent on the concentration of F-actin, and the binding was weaker at higher concentrations of F-actin. The apparent association constant determined from a linearly extrapolated Scatchard plot was 6.5 x 10(6) M-1 at 8.1 microns F-actin, and 1.7 x 10(7) M-1 at 2.0 microns F-actin in 120 mM KC1, 2 mM MgCl2, 0.1 mM CaCl2, and 20 mM Tris-acetate (pH 7.6) at 20 degrees C. Furthermore, the Scatchard plot revealed the existence of cooperativity in the binding of S-1 to F-actin. In order to obtain higher precision we developed a new method for the chromatographic determination of free S-1 in acto-S-1 solution. By this method we could determine free S-1 concentrations of the order of 10(-9) M easily and accurately. The above conclusion obtained by the sedimentation method was confirmed by this chromatographic method, and these effects can be well explained by considering the length distribution of F-actin. We propose an allosteric model in which both the length distribution and the polarity of F-actin are taken into consideration.
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PMID:The binding of myosin subfragment-1 to F-actin in the absence of nucleotide. Evidence for dependence on F-actin concentration. 713 Jan 52

The hypothesis that Ca2+ initiates contraction in smooth muscle by activating an endogenous myosin light chain kinase (MLCK) that phosphorylates the 20,000 dalton light chain (LC 20) of myosin was tested in tissues prepared from the media of swine carotid arteries. Unstimulated tissues with low levels of tone exhibited low levels of phosphorylated LC 20. On stimulation with a high-K+ physiological salt solution containing 1.6 mM CaCl2, LC 20 phosphorylation increased to 0.6 mol P/mol LC 20 within 30 s. This increase preceded force development, which required 2-4 min to attain a maximum steady-state value of 3.34 +/- 0.15 (SE) X 10(5) N/m2. These results support the hypothesis, as the stimulus was submaximal for the preparation. However, LC 20 phosphorylation declined significantly from its peak value before steady-state force was attained, reaching near control levels after 10 min of stimulation. The results suggest that Ca2+-stimulated LC 20 phosphorylation is an important physiological control mechanism but that additional factors are involved in the maintenance of tonic isometric force.
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PMID:Myosin light chain phosphorylation associated with contraction in arterial smooth muscle. 723 3

1. A method was developed for the isolation of essentially pure myosin light chains from perfused rat heart. The phosphorylation of the P-light chains was estimated by hydrolysis and measurement of phosphate released, by electrophoresis in 8 M-urea and by 32P incorporation in perfusion with [32P]Pi. 2. In control perfusions there was 0.5-0.6 mol of phosphate/mol of P-light chain. This was not changed by perfusion with 5 microM-adrenaline for 10-40s. Perfusion for 1 min with medium containing 7.5 mM-CaCl2, or for 30s with medium containing 118 mM-KCl, also did not change the phosphorylation of P-light chains. 3. It is concluded that phosphorylation of P-light chains is not important in mediating the action of inotropic agents in the heart.
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PMID:Phosphorylation of myosin light chains in perfused rat heart. Effect of adrenaline and increased cytoplasmic calcium ions. 747 33

Actin-myosin interaction kinetics of the intact rat portal vein were studied by analyzing force recovery after cessation of force-inhibiting length vibration. The time constant of postvibration force recovery averaged 0.86 +/- 0.04 s during short-term activation (< 12 s), and increased up to 1.59 +/- 0.02 s (cross-bridge downregulation) during sustained activation of more than 10 min. After the depletion of intracellular calcium stores, the depolarized preparation developed maximum force at an extracellular calcium concentration in excess of 50 mM CaCl2. The time constant of postvibration force recovery rose to 12.31 +/- 1.35 s after an activation period of 30 min. These retarded contraction kinetics may be caused either by low activation of the 20-kDa myosin light chain kinase or by high activity of the 20-kDa myosin light chain phosphatase. Addition of the phosphatase inhibitor okadaic acid (10 microM) during high calcium activation decreases the time constant to 8.04 +/- 0.86 s and appears to prevent the distinct retardation of the contraction kinetics. During submaximum activation (2.5 mM CaCl2), the time constant of postvibration force recovery stabilizes at 1.56 +/- 0.07 s, indicating downregulated cross-bridge kinetics, and is unaffected by phosphatase inhibition. For maximum barium activation, instead of calcium, 19.5 mM BaCl2 is required, which produced time constants of postvibration force recovery of 8.38 +/- 0.32 s. The addition of okadaic did not affect contraction kinetics during barium activation. The pronounced retardation of contraction kinetics that was observed after the maximum calcium activation of the previously calcium depleted, depolarized preparation is probably due to high phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Smooth muscle contraction kinetics at different calcium concentrations. 776 76

We have characterized the binding of rhodamine phalloidin to actin filaments and actin filaments saturated with either myosin subfragment-1 or tropomyosin in 50 mM KCl, 1 mM MgCl2 buffer at pH 7.0. Direct transient kinetic measurements of rhodamine phalloidin binding to actin filaments indicate an association rate constant of 2.8 x 10(4) M-1 s-1 and a dissociation rate constant of 4.8 x 10(-4) s-1. The ratio of the rate constants yields a dissociation equilibrium constant of 17 nM. From equilibrium measurements, the apparent affinity of rhodamine phalloidin for actin filaments is 116 nM. The difference between the affinities determined by equilibrium and kinetic experiments is attributed to the depolymerization of filaments at low actin concentrations in the equilibrium samples. The binding stoichiometry is one rhodamine phalloidin molecule per actin subunit. When myosin subfragment-1 and tropomyosin are bound to actin filaments, the rate constants for rhodamine phalloidin binding are the same as for actin alone and in agreement with the binding affinities measured in equilibrium experiments. Presumably these proteins stabilize the filaments. Neither substitution of CaCl2 for MgCl2 nor the inclusion of 20 mM phosphate altered the rate or equilibrium constants.
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PMID:Transient kinetic analysis of rhodamine phalloidin binding to actin filaments. 798 Nov 98

Thymosin beta 4 is able to form 1:1 complexes with monomeric (G) actin, thereby stabilizing the intracellular pool of unpolymerized actin. We have searched for factors that are able to induce the polymerization of actin from the actin:thymosin beta 4 complex. Phalloidin, subfragment 1 isolated from rabbit skeletal muscle myosin and chicken intestinal myosin I are demonstrated to be able to polymerize the actin from this complex in the presence of 1 mM MgCl2. Polymerization of actin was verified by the DNase I inhibition assay, by cosedimentation and from the fluorescence increase of pyrene-labelled actin. Actin filaments formed under the influence of subfragment 1 or phalloidin were visualized under the electron microscope after negative staining. Polymerization of skeletal muscle actin from the complex with thymosin beta 4 by phalloidin is accompanied by the hydrolysis of the actin-bound ATP to ADP. Polymerization was also induced by sonicated F-actin which possessed a high concentration of free filament ends. F-actin was severed by 0.01 M human cytoplasmic gelsolin, which is known to possess blocked+ends. Free, slowly growing-ends were unable to induce polymerization of actin from the thymosin beta 4 complex. However, when gelsolin on its own or in complex with two actin molecules was added to actin:thymosin beta 4 under nucleating conditions, it was found to be able to promote actin repolymerization provided that its concentration was close to the dissociation constant (Kd) of actin:thymosin beta 4. This Kd was found to be 0.4 microM in the presence of 1 mM MgCl2 and the absence of KCl and, thus, close to the critical concentration of actin polymerization under these conditions. The source of actin did not influence its polymerization from the thymosin beta 4 complex; rabbit skeletal muscle actin and porcine brain actin were polymerized with equal efficiency from their complexes with thymosin beta 4 by both phalloidin and myosin subfragment 1. Skeletal muscle, but not cytoplasmic actin, was found to be also polymerized in the presence of increased CaCl2 concentrations to values above 1 mM.
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PMID:Induction of the polymerization of actin from the actin:thymosin beta 4 complex by phalloidin, skeletal myosin subfragment 1, chicken intestinal myosin I and free ends of filamentous actin. 805 11


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