Isolation and Characterization of Myosin and Two Myosin Fragments from Human Blood Platelets

Adelstein, Robert S.; Pollard, Thomas D.; Kuehl, W. Michael

doi:10.1073/pnas.68.11.2703

Cited by 121 publications

(47 citation statements)

References 14 publications

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“…In contrast to platelet myosin, which elutes from Sepharose 4B with two peaks of ATPase activity, fibroblast myosin eluted only as a single peak. This single peak, which has the same RF as muscle myosin, indicates that unlike platelet myosin, fibroblast myosin has not undergone a partial proteolytic cleavage (10).…”

Section: Discussionmentioning

confidence: 89%

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Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Adelstein

Conti

Johnson

et al. 1972

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

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Myosin has been isolated from cloned mouse fibroblasts, line L-929. Fibroblast myosin: (i) binds to rabbit muscle actin and is dissociated from it by ATP, (ii) has an ATPase activity that is suppressed by Mg2+ in 0.6 M KCI and is activated by rabbit muscle actin in the presence of Mgg+ in 14 mM KCI, (iii) forms thin bi. polar aggregates in 0.1 M KCI when viewed in the electron microscope, (iv) possesses a heavy chain with the same mobility as muscle myosin (molecular weight 200,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these respects, fibroblast myosin appears to be similar to muscle myosin in structure and function.Since the work of Ishikawa et al. (1) on the presence of actinlike filaments in fibroblasts, the possible existence of a myosin counterpart in these cells has been raised. Recently, Yang and Perdue (2) succeeded in isolating and purifying actin from tertiary cultures of chick embryo cells and found it similar to muscle actin in its molecular properties. Furthermore, the the work of Bray (3) has indicated that cytoplasmic actin can be isolated from several different embryonic sources.cAMP has a marked effect on the motility, adhesion, morphology, and growth of fibroblasts grown in vitro (4-9). It is possible that some of the effects of cAMP could be mediated through one or more of the "contractile" proteins (actin, myosin, and troponin-tropomyosin). To explore this hypothesis, we began by attempting to isolate a myosin-like protein from cloned fibroblasts. Using a modification of a technique developed for the isolation of platelet myosin (10), we isolated myosin from cloned mouse fibroblasts grown in vitro. This protein has been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, ATPase assay with and without muscle actin, actin binding, and electron microscopy. MATERIALS AND METHODSCloned mouse (L-929) fibroblasts were grown either as monolayers or in spinner bottles. The cells were scraped off the sides of the bottles or sedimented from the suspended culture. They were washed three times in ten volumes of 0.9% NaCl-0.3% sodium citrate-5 mM dithiothreitol (S2threitol)-1 mM EDTA, and sedimented each time at 13,000 X g for 15 min.

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Section: Discussionmentioning

confidence: 89%

“…Polyacrylamide gel electrophoresis was performed as described (10). Instead of reduction and alkylation of sulfhydryl groups, the samples were dissolved in 1% SDS-1 mM sodium phosphate (pH 7.0)-50 mM S2threitol and boiled for 5 min just before electrophoresis.…”

Section: Purification Proceduresmentioning

confidence: 99%

Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Adelstein

Conti

Johnson

et al. 1972

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

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“…Although the amounts, and degree of organization of these proteins, and their association with regulatory components is of a much higher order in cells specialized for contraction, it can be assumed that basic mechanisms of contractility are similar in all cells (4,52,69,112) . The finding of tropomyosin-like protein in nonmuscle cells (19,84,112) makes credible the applicability of a scheme similar to that accepted for muscle to cytoplasmic contraction in general (4,112) . However, other more primitive modalities of regulation of the interaction of actin and myosin than the troponin-tropomyosin system (e .g .…”

Section: Contractionmentioning

confidence: 99%

Action of Cytochalasin D on Cells of Established Lines

Miranda

Godman

Deitch

et al. 1974

The Journal of Cell Biology

216

119

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HeLa, Vero, L, HEp2, and MDBK cells respond immediately to 0.2–0.5 µg/ml cytochalasin D (CD) with sustained contraction (contracture), loss of microvilli, expression of endoplasmic contents (zeiosis), nuclear protrusion, and extension of cytoplasmic processes. The development of these changes is depicted, and the dose-response patterns in these cell lines are described. MDBK is generally most resistant and HeLa most sensitive to these effects of CD. Cells in G1 are most sensitive to CD; responsiveness decreases progressively during early S and is least in mid S through G2. CD inhibits transport of [14C]deoxyglucose in HeLa by about 45% but has no significant effect on hexose uptake in Vero and MDBK; sugar transport is thus apparently unrelated to any morphologic effect of CD. Although spreading and attachment are impeded, CD does not decrease and may even enhance the adhesiveness of established monolayers. Contraction appears to be a primary early effect of CD, upon which other visible changes follow. It is prevented by some inhibitors of energy metabolism (deoxyglucose and dinitrophenol) and does not occur in glycerinated models without ATP. The possible bases of the contractile response to CD are discussed. Although direct or indirect action of CD on some microfilaments may occur, a generalized structural disruption of contractile filaments by CD is considered unlikely.

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“…Moreover platelet myosin can be digested by proteolytic enzymes to yield two fragments, rod and head (1,4). These fragments have similar properties to the rod and subfragment-1 fragments produced by proteolytic cleavage of skeletal-muscle myosin (5).…”

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Phosphorylation of Human Platelet Myosin

Adelstein

Conti

Anderson

1973

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

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A preparation extracted from human blood platelets, which incorporates 32p from -y-labeled AT32P into one of the two light chains of platelet myosin and platelet myosin head is described. This phosphorylation, which appears to be due to an endogenous kinase, is specific for the myosin light chain in that no other protein extracted in 0.6 M KCI-15 mM Tris HCI (pH 7.5) is phosphorylated. The phosphorylated light chain, which has been purified by gel filtration, releases the covalently bound phosphate after incubation in alkali and not after incubation in acid.Human blood platelets contain three proteins which are similar to muscle contractile proteins. These proteins, platelet myosin, platelet actin, and platelet tropomyosin, have been purified and characterized so that their structure and function can be compared to muscle contractile proteins (1)(2)(3).Platelet myosin is similar to muscle myosin in being composed of two heavy chains (molecular weight 200,000) and two different light chains (molecular weight 20,000 and 15,000). Moreover platelet myosin can be digested by proteolytic enzymes to yield two fragments, rod and head (1, 4). These fragments have similar properties to the rod and subfragment-1 fragments produced by proteolytic cleavage of skeletal-muscle myosin (5).Functionally, platelet myosin and platelet myosin head bind to platelet and skeletal muscle actin and are dissociated from it by ATP. At low ionic strength, in the presence of Mg2+, the ATPase activity of platelet myosin head is activated by rabbit muscle actin. This activation is dependent on Ca2+ in the presence of muscle troponin-tropomyosin (1).Platelet actin, which resembles skeletal muscle actin in molecular weight and ability to undergo a G F (globular fibrous) transformation, activates the ATPase activity of of skeletal-muscle heavy meromyosin. This activation becomes Ca2+-dependent after the addition of rabbit skeletalmuscle troponin-tropomyosin (1). The third contractile protein, platelet tropomyosin, which resembles muscle tropomyosin in several physical and chemical properties, has a molecular weight lower than muscle tropomyosin. This protein has been isolated and characterized by Cohen and Cohen (2).The manner by which the interaction of platelet actin and myosin (as well as actin and myosin from cells other than platelets) is regulated is of current interest. One manner of control of this interaction might be through a troponintropomyosin complex, similar to that present in skeletalmuscle proteins. Only indirect evidence points to the existence of such a system in platelets (1, 2). Reversible phosphorylation of one of the contractile proteins offers another possible control mechanism for platelet contraction and/or aggrega-3115 tion. This is of particular interest, since a protein kinase has been isolated from platelets but the substrate for this kinase has yet to be identified (6).In this paper we describe a preparation extracted from washed, human platelets which incorporates 82p from -ylabeled AT32P into the light cha...

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Isolation and Characterization of Myosin and Two Myosin Fragments from Human Blood Platelets

Cited by 121 publications

References 14 publications

Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Action of Cytochalasin D on Cells of Established Lines

Phosphorylation of Human Platelet Myosin

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