scholarly journals A tight-binding interaction between smooth-muscle native thin filaments and heavy meromyosin in the presence of MgATP

1989 ◽  
Vol 259 (1) ◽  
pp. 303-306 ◽  
Author(s):  
S B Marston
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Thin Filament ◽  
Tight Binding ◽  
Muscle Proteins ◽  
Actin Monomer ◽  
Heavy Meromyosin ◽  
Binding Interaction ◽  
Thin Filaments ◽  
Myosin Phosphorylation

The binding of the Ca2+-regulated native thin filaments from vascular smooth muscle to vascular smooth-muscle heavy meromyosin was measured in the presence of 3 mM-MgATP. At 25 degrees C and I 0.25 binding had an affinity of 1 X 10(-6)-0.3 X 10(-6) M-1 with a stoichiometry of one molecule bound to one actin monomer. The Km for the activation of heavy-meromyosin ATPase was 20-50 microM. Thin filament-heavy meromyosin binding was not altered by Ca2+ (pCa 9-4) or the extent of myosin phosphorylation. With skeletal-muscle heavy meromyosin affinity was 0.023 X 10(6) M-1 in parallel with activation of the ATPase (Km 54 microM). It is concluded that tight binding is specific to smooth-muscle proteins and that it is not related to the ATPase activation site.

scholarly journals Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle

1990 ◽  
Vol 272 (2) ◽  
pp. 305-310 ◽  
Author(s):  
S Marston
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Gel Electrophoresis ◽  
Thin Filament ◽  
Critical Concentration ◽  
Actin Monomer ◽  
Heavy Meromyosin ◽  
Thin Filaments

Ca2(+)-regulated native thin filaments were extracted from sheep aorta smooth muscle. The caldesmon content determined by quantitative gel electrophoresis was 0.06 caldesmon molecule/actin monomer (1 caldesmon molecule per 16.3 actin monomers). Dissociation of caldesmon and tropomyosin from the thin filament and the depolymerization of actin was measured by sedimenting diluted thin filaments. Actin critical concentration was 0.05 microM at 10.1 and 0.13 at 10.05 compared with 0.5 microM for pure F-actin. Tropomyosin was tightly bound, with half-maximal dissociation at less than 0.3 microM thin filaments (actin monomer) under all conditions. Caldesmon dissociation was independent of tropomyosin and not co-operative. The concentration of thin filaments where 50% of the caldesmon was dissociated (CD50) ranged from 0.2 microM (actin monomer) at 10.03 to 8 microM at 10.16 in a 5 mM-MgCl2, pH 7.1, buffer. Mg2+, 25 mM at constant I, increased CD50 4-fold. CD50 was 4-fold greater at 10(-4) M-Ca2+ than at 10(-9) M-Ca2+. Aorta heavy meromyosin (HMM).ADP.Pi complex (2.5 microM excess over thin filaments) strongly antagonized caldesmon dissociation, but skeletal-muscle HMM.ADP.Pi did not. The behaviour of caldesmon in native thin filaments was indistinguishable from caldesmon in reconstituted synthetic thin filaments. The variability of Ca2(+)-sensitivity with conditions observed in thin filament preparations was shown to be related to dissociation of regulatory caldesmon from the thin filament.

scholarly journals Phosphorylation of the calcium ion-regulated thin filaments from vascular smooth muscle A new regulatory mechanism?

1981 ◽  
Vol 197 (1) ◽  
pp. 127-139 ◽  
Author(s):  
M Walters ◽  
S B Marston
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Thin Filament ◽  
Sodium Dodecyl ◽  
Regulatory System ◽  
Specific Protein ◽  
Maximum Activity ◽  
Thin Filaments ◽  
Skeletal Muscle Myosin ◽  
Muscle Myosin

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.

scholarly journals Ca2+-dependent regulation of vascular smooth-muscle caldesmon by S.100 and related smooth-muscle proteins

1991 ◽  
Vol 277 (3) ◽  
pp. 819-824 ◽  
Author(s):  
K Pritchard ◽  
S B Marston
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Actin ◽  
Bovine Brain ◽  
Protein Yield ◽  
Muscle Actin ◽  
Muscle Proteins ◽  
Related Protein ◽  
Thin Filaments ◽  
S 100 ◽  
Related Proteins

1. We have investigated the ability of bovine brain S.100, and of three related proteins from sheep aorta smooth muscle, to confer Ca(2+)-sensitivity on thin filaments reconstituted from smooth-muscle actin, tropomyosin and caldesmon. 2. At 37 degrees C in pH 7.0 buffer containing 120 mM-KCl, approximately stoichiometric amounts of S.100 reversed caldesmon's inhibition of the activation of myosin MgATPase by smooth-muscle actin-tropomyosin. The [S.100] which reversed by 50% the inhibition by caldesmon (the E.C.50) was 2.5 microM when [caldesmon] = 2-3 microM in the assay mixture. When [KCl] was decreased to 70 mM, E.C.50 = 11.5 microM; at 25 degrees C in 70 mM-KCl, up to 20 microM-S.100 had no effect. When skeletal-muscle actin rather than smooth-muscle actin was used to reconstitute thin filaments, 20 microM-S.100 did reverse inhibition by caldesmon, at 25 degrees C in buffer containing 70 mM-KCl. This dependence on conditions is also characteristic of the calmodulin-caldesmon interaction. 3. These results suggested that S.100 or a related protein might interact with caldesmon in smooth muscle. We therefore attempted to prepare such a protein from sheep aorta. Three proteins were purified: an Mr-17,000 protein (yield 16 mg/kg), an abundant Mr-11,000 protein (yield 48 mg/kg), and an Mr-9000 protein (yield 4 mg/kg). Neither of the last two low-Mr proteins had any effect on activation of myosin MgATPase by reconstituted thin filaments. The protein of Mr 17,000 had Ca(2+)-sensitizing activity, and behaved exactly like brain calmodulin in the assay system.

Regulation of vascular smooth muscle tone

1994 ◽  
Vol 72 (8) ◽  
pp. 919-936 ◽  
Author(s):  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Contractile Response ◽  
Muscle Tone ◽  
Myosin Phosphorylation ◽  
Smooth Muscle Tone ◽  
Vascular Smooth Muscle Tone

Vascular smooth muscle tone is regulated primarily by the sarcoplasmic free Ca2+ concentration, which determines the level of myosin phosphorylation. Stimulation of the muscle results in an increase in free [Ca2+], whereupon Ca2+ binds to calmodulin, inducing a conformational change enabling calmodulin to interact with and activate myosin light chain kinase. The active Ca2+∙calmodulin∙myosin light chain kinase complex catalyses the phosphorylation of serine-19 of the two 20-kDa light chains of myosin; this triggers cross-bridge cycling and the development of force. Relaxation follows restoration of free [Ca2+] to the resting level, whereupon calmodulin dissociates from myosin light chain kinase, which is thereby inactivated, and myosin is dephosphorylated by myosin light chain phosphatase and remains detached from actin. Overwhelming evidence now exists in favour of the central role of myosin phosphorylation–dephosphorylation in smooth muscle contraction–relaxation. However, considerable evidence supports the existence of additional, secondary mechanisms that can modulate the contractile state of smooth muscle either by altering the Ca2+ sensitivity of the contractile response or otherwise modulating one of the molecular events occurring downstream of the Ca2+ signal, e.g., the interaction of phosphorylated myosin heads with actin. The interplay of several regulatory elements confers on the contractile response of vascular smooth muscle the high degree of flexibility and adaptability required for the effective regulation of blood pressure.Key words: calcium, myosin, protein kinases, protein phosphatases, signal transduction, regulation of contraction, caldesmon, calponin.

scholarly journals Skeletal muscle contractions stimulate cGMP formation and attenuate vascular smooth muscle myosin phosphorylation via nitric oxide

FEBS Letters ◽  
1998 ◽  
Vol 431 (1) ◽  
pp. 71-74 ◽  
Author(s):  
Kim S. Lau ◽  
Robert W. Grange ◽  
Wen-Jinn Chang ◽  
Kristine E. Kamm ◽  
Ingrid Sarelius ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Muscle Contractions ◽  
Smooth Muscle Myosin ◽  
Myosin Phosphorylation ◽  
Muscle Myosin ◽  
Cgmp Formation

Ca2+- and Myosin Phosphorylation–independent Relaxation by Halothane in K+-depolarized Rat Mesenteric Arteries

2003 ◽  
Vol 99 (3) ◽  
pp. 656-666 ◽  
Author(s):  
Isao Tsuneyoshi ◽  
Dongya Zhang ◽  
Walter A. Boyle
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Mesenteric Arteries ◽  
Dependent Manner ◽  
Resistance Arteries ◽  
Concentration Dependent Manner ◽  
Myosin Phosphorylation ◽  
High K ◽  
Mlc Phosphorylation ◽  
Depolarized Smooth Muscle

Background Volatile anesthetics inhibit vascular smooth muscle contraction, but the mechanisms responsible are uncertain. In this study, the effects of halothane on Ca2+ signaling and Ca2+ activation of contractile proteins were examined in high K+-depolarized smooth muscle from rat mesenteric resistance arteries. Methods Vessels were cannulated and held at a constant transmural pressure (40 mmHg). Image analysis and microfluorimetry were used to simultaneously measure vessel diameter and smooth muscle intracellular [Ca2+] concentration ([Ca2+]i). Myosin light chain (MLC) phosphorylation was measured using the Western blotting technique. Results Step increases in extracellular [Ca2+] concentration (0-10 mM) in high K+ (40 mM)-depolarized smooth muscle produced incremental increases in [Ca2+]i, MLC phosphorylation, and contraction. Halothane (0.5-4.5%) inhibited contraction in a concentration-dependent manner, but the decrease in [Ca2+]i was small, and there was a marked shift in the [Ca2+]i-contraction relationship to the right, indicating an important Ca2+ desensitizing effect. Halothane (0.5-4.5%) did not affect MLC phosphorylation or the [Ca2+]-MLC phosphorylation relationship, but the MLC phosphorylation-contraction relationship was also shifted rightward, indicating an "MLC phosphorylation" desensitizing effect. In contrast, control relaxations produced by the Ca2+ channel blocker nifedipine were accompanied by decreases in both [Ca2+]i and MLC phosphorylation, and nifedipine had no affect on the [Ca2+]i-contraction, [Ca2+]i-MLC phosphorylation, and MLC phosphorylation-contraction relationships. Conclusions In high K+-depolarized vascular smooth muscle, halothane relaxation is largely mediated by a Ca2+ and MLC phosphorylation desensitizing effect. These results suggest that the relaxing action of halothane is independent of the classic Ca2+-induced myosin phosphorylation contraction mechanism.

scholarly journals Ca2+ can control vascular smooth-muscle thin filaments without caldesmon phosphorylation

1986 ◽  
Vol 237 (2) ◽  
pp. 605-607 ◽  
Author(s):  
S B Marston
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Binding Protein ◽  
Direct Interaction ◽  
Half Time ◽  
Thin Filaments

The Ca2+-dependent regulation of the activation of myosin MgATPase by vascular-smooth-muscle thin filaments involves caldesmon. This effect may be due to the direct interaction of caldesmon with a Ca2+-binding protein such as calmodulin or phosphorylation of caldesmon by a Ca2+-dependent kinase. I have found that Ca2+ switches on aorta thin filaments in less than 10 s, whereas the caldesmon in the thin filaments is phosphorylated only slowly (half-time greater than 10 min) and the maximum phosphorylation is very low (1 molecule per 7 molecules of caldesmon). I conclude that the phosphorylation of caldesmon hypothesis is untenable.

scholarly journals Calcium ion-regulated thin filaments from vascular smooth muscle

1980 ◽  
Vol 185 (2) ◽  
pp. 355-365 ◽  
Author(s):  
S B Marston ◽  
R M Trevett ◽  
M Walters
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Atpase Activity ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin C ◽  
Myosin Atpase ◽  
Protein G ◽  
Thin Filaments ◽  
Myosin Atpase Activity

Myosin and actin competition tests indicated the presence of both thin-filament and myosin-linked Ca2+-regulatory systems in pig aorta and turkey gizzard smooth-muscle actomyosin. A thin-filament preparation was obtained from pig aortas. The thin filaments had no significant ATPase activity [1.1 +/- 2.6 nmol/mg per min (mean +/- S.D.)], but they activated skeletal-muscle myosin ATPase up to 25-fold [500 nmol/mg of myosin per min (mean +/- S.D.)] in the presence of 10(-4) M free Ca2+. At 10(-8) M-Ca2+ the thin filaments activated myosin ATPase activity only one-third as much. Thin-filament activation of myosin ATPase activity increased markedly in the range 10(-6)-10(-5) M-Ca2+ and was half maximal at 2.7 × 10(-6) M (pCa2+ 5.6). The skeletal myosin-aorta-thin-filament mixture gave a biphasic ATPase-rate-versus-ATP-concentration curve at 10(-8) M-Ca2+ similar to the curve obtained with skeletal-muscle thin filaments. Thin filaments bound up to 9.5 mumol of Ca2+/g in the presence of MgATP2-. In the range 0.06-27 microM-Ca2+ binding was hyperbolic with an estimated binding constant of (0.56 +/- 0.07) x 10(6) M-1 (mean +/- S.D.) and maximum binding of 8.0 +/- 0.8 mumol/g (mean +/- S.D.). Significantly less Ca2+ bound in the absence of ATP. The thin filaments contained actin, tropomyosin and several other unidentified proteins. 6 M-Urea/polyacrylamide-gel electrophoresis at pH 8.3 showed proteins that behaved like troponin I and troponin C. This was confirmed by forming interspecific complexes between radioactive skeletal-muscle troponin I and troponin C and the aorta thin-filament proteins. The thin filaments contained at least 1.4 mumol of a troponin C-like protein/g and at least 1.1 mumol of a troponin I-like protein/g.

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