The effect of phosphorylation of gizzard myosin on actin activation

Gorecka, Alicja; Aksoy, Mark O.; Hartshorne, D.J.

doi:10.1016/0006-291x(76)90286-2

Cited by 175 publications

(82 citation statements)

References 15 publications

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“…Following his report, Aksoy et al (21) and G6recka et al (10) demonstrated that addition of a crude kinase from chicken gizzard to chicken gizzard actomyosin resulted in an increase in myosin phosphorylation and in actin-activated myosin ATPase activity. Neither report mentioned the effect of phosphorylation using purified smooth muscle myosin free of kinase and phosphatase.…”

Section: Discussionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

“…The 35-70% ammonium sulfate fraction (10-15 mg/ml), which contained myosin, actin, and tropomyosin as well as other proteins, was used for phosphorylation, dephosphorylation, and purification of myosin. Prior to use, it was dialyzed overnight against [10][11][12][13][14][15][16][17][18][19][20] …”

Section: Introductionmentioning

confidence: 99%

“…Dephosphorylation of platelet myosin results in a decrease in the actinactivated ATPase activity (6). Although these experiments indicated a role for phosphorylation in controlling actin-activation of myosin in nonmuscle cells, there was no evidence for such control in muscle.Recent preliminary reports from a number of laboratories have suggested that phosphorylation of smooth muscle myosin may play a role in the actin-activation and Ca2+ regulation of smooth muscle myosin ATPase activity (8)(9)(10). In this paper we report the isolation and purification of phosphorylated and METHODS All procedures were carried out at 40 unless noted as otherwise.…”

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confidence: 99%

“…Recent preliminary reports from a number of laboratories have suggested that phosphorylation of smooth muscle myosin may play a role in the actin-activation and Ca2+ regulation of smooth muscle myosin ATPase activity (8)(9)(10). In this paper we report the isolation and purification of phosphorylated and Abbreviations: NaDodSO4, sodium dodecyl sulfate; EDTA, (ethylenedinitrilo)tetraacetic acid; DTT Phosphorylation was terminated by raising the KCI concentration to 1 M, making the solution 0.1 M with respect to phosphate buffer, placing the samples on ice, and applying the samples to a Sepharose 4B column after 2 min of chilling.…”

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confidence: 99%

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Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.

Chacko¹,

Conti²,

Adelstein³

1977

Proc. Natl. Acad. Sci. U.S.A.

299

154

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A 35-70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from Phosphorylation of a light chain of myosin has been shown to occur in myosins isolated from both muscle and cytoplasmic sources (1, 2). In each case, covalent phosphorylation occurs on a specific light chain of myosin in the size range of 18,000 to 20,000 daltons-e.g., white skeletal muscle, 18,000; red skeletal muscle, 20,000; cardiac muscle, 20,000; smooth muscle, 20,000; and platelet, 20,000 (1-3).Phosphorylation is catalyzed by a specific enzyme, myosin light chain kinase, which differs in the cases of cytoplasmic and muscle myosin in that the former enzyme is independent of Ca2+ for activity whereas the latter enzyme requires Ca2+ (1, 4, 5). Dephosphorylation of the myosin light chain is catalyzed by an exogenous (6) or endogenous phosphatase (7, 28).Phosphorylation of platelet myosin has been shown to control the interaction between platelet actin and myosin; phosphorylated myosin is activated by actin to a greater extent (5-to 7-fold) than is nonphosphorylated myosin (6). Dephosphorylation of platelet myosin results in a decrease in the actinactivated ATPase activity (6). Although these experiments indicated a role for phosphorylation in controlling actin-activation of myosin in nonmuscle cells, there was no evidence for such control in muscle.Recent preliminary reports from a number of laboratories have suggested that phosphorylation of smooth muscle myosin may play a role in the actin-activation and Ca2+ regulation of smooth muscle myosin ATPase activity (8)(9)(10). In this paper we report the isolation and purification of phosphorylated and METHODS All procedures were carried out at 40 unless noted as otherwise. Deionized water was used throughout. Preparation of smooth muscle actomyosin One-month-old male guinea pigs were anesthetized and their vasa deferentia were removed, collected in balanced salt solution (Hanks'), and carefully freed from connective tissue under a dissecting microscope. The cleaned specimens were pooled (usually 120 in number weighing approximately 5 g) and homogenized in 35 ml of the extraction buffer: 60 mM KCI, 40 mM imidazole-HCl (pH 7.1), 4 mM (ethylenedinitrilo)tetraacetic acid (EDTA), 10 mM ATP, and 10 mM dithiothreitol (DTT). A tissue homogenizer was used for a total of 2 min and the material being homogenized was chilled in ice. The homogenized material was sedimented at 48,000 X g for 20 min to yield a cloudy supernatant and a pellet. Immediately after addition of ATP and MgCl2 to 10 mM, the supernatant was fractionated into 0-35% and 35-70% fractions by addition of a saturated ammonium sulfat...

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Section: Discussionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.

Chacko¹,

Conti²,

Adelstein³

1977

Proc. Natl. Acad. Sci. U.S.A.

299

154

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Thin Filament Structure and the Steric Blocking Model

Lehman

2016

Comprehensive Physiology

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By interacting with the troponin-tropomyosin complex on myofibrillar thin filaments, Ca2+ and myosin govern the regulatory switching processes influencing contractile activity of mammalian cardiac and skeletal muscles. A possible explanation of the roles played by Ca2+ and myosin emerged in the early 1970s when a compelling "steric model" began to gain traction as a likely mechanism accounting for muscle regulation. In its most simple form, the model holds that, under the control of Ca2+ binding to troponin and myosin binding to actin, tropomyosin strands running along thin filaments either block myosin-binding sites on actin when muscles are relaxed or move away from them when muscles are activated. Evidence for the steric model was initially based on interpretation of subtle changes observed in X-ray fiber diffraction patterns of intact skeletal muscle preparations. Over the past 25 years, electron microscopy coupled with three-dimensional reconstruction directly resolved thin filament organization under many experimental conditions and at increasingly higher resolution. At low-Ca2+, tropomyosin was shown to occupy a "blocked-state" position on the filament, and switched-on in a two-step process, involving first a movement of tropomyosin away from the majority of the myosin-binding site as Ca2+ binds to troponin and then a further movement to fully expose the site when small numbers of myosin heads bind to actin. In this contribution, basic information on Ca2+-regulation of muscle contraction is provided. A description is then given relating the voyage of discovery taken to arrive at the present understanding of the steric regulatory model.

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