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 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 Electron microscopy of synthetic myosin filaments. Evidence for cross-bridge. Flexibility and copolymer formation.

1975 ◽  
Vol 67 (1) ◽  
pp. 93-104 ◽  
Author(s):  
T D Pollard
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Structural Features ◽  
Length Frequency ◽  
Single Population ◽  
Skeletal Muscle Myosin ◽  
Distinctive Features ◽  
Myosin Filaments ◽  
The Mean ◽  
Muscle Myosin

Electron micrographs of negatively stained synthetic myosin filaments reveal that surface projections, believed to be the heads of the constituent myosin molecules, can exist in two configurations. Some filaments have the projections disposed close to the filament backbone. Other filaments have all of their projections widely spread, tethered to the backbone by slender threads. Filaments formed from the myosins of skeletal muscle, smooth muscle, and platelets each have distinctive features, particularly their lengths. Soluble mixtures of skeletal muscle myosin with either smooth muscle myosin or platelet myosin were dialyzed against 0.1 M KC1 at pH 7 to determine whether the simultaneous presence of two types of myosin would influence the properties of the filaments formed. In every case, a single population of filaments formed from the mixtures. The resulting filaments are thought to be copolymers of the two types of myosin, for several reasons: (a) their length-frequency distribution is unimodal and differs from that predicted for a simple mixture of two types of myosin filaments; (b) their mean length is intermediate between the mean lengths of the filaments formed separately from the two myosins in the mixture; (c) each of the filaments has structural features characteristic of both of the myosins in the mixture; and (d) their size and shape are determined by the proportion of the two myosins in the mixture.

scholarly journals LOCALIZATION OF MYOSIN FILAMENTS IN SMOOTH MUSCLE

1968 ◽  
Vol 37 (1) ◽  
pp. 105-116 ◽  
Author(s):  
Robert E. Kelly ◽  
Robert V. Rice
Keyword(s):  
Smooth Muscle ◽  
Cross Section ◽  
Striated Muscle ◽  
Thick Filament ◽  
Longitudinal Axis ◽  
Thin Filaments ◽  
Chicken Gizzard ◽  
Thick Filaments ◽  
Myosin Filaments ◽  
Muscle Myosin

Thick myosin filaments, in addition to actin filaments, were found in sections of glycerinated chicken gizzard smooth muscle when fixed at a pH below 6.6. The thick filaments were often grouped into bundles and run in the longitudinal axis of the smooth muscle cell. Each thick filament was surrounded by a number of thin filaments, giving the filament arrangement a rosette appearance in cross-section. The exact ratio of thick filaments to thin filaments could not be determined since most arrays were not so regular as those commonly found in striated muscle. Some rosettes had seven or eight thin filaments surrounding a single thick filament. Homogenates of smooth muscle of chicken gizzard also showed both thick and thin filaments when the isolation was carried out at a pH below 6.6, but only thin filaments were found at pH 7.4. No Z or M lines were observed in chicken gizzard muscle containing both thick and thin filaments. The lack of these organizing structures may allow smooth muscle myosin to disaggregate readily at pH 7.4.

scholarly journals Assembly of smooth muscle myosin into side-polar filaments.

1977 ◽  
Vol 75 (3) ◽  
pp. 990-996 ◽  
Author(s):  
R Craig ◽  
J Megerman
Keyword(s):  
Smooth Muscle ◽  
Opposite Polarity ◽  
Skeletal Muscle Myosin ◽  
Myosin Filaments ◽  
Skeletal Myosin ◽  
Cross Bridges ◽  
Bipolar Filament ◽  
Muscle Myosin

The in vitro assembly of myosin purified from calf aorta muscle has been studied by electron microscopy. Two types of filament are formed: short bipolar filament similar to those formed from skeletal muscle myosin, and longer "side-polar" filaments having cross bridges with a single polarity along the entire length of one side and the opposite polarity along the other side. Unlike the case with skeletal myosin filaments, antiparallel interactions between myosin molecules occur along the whole length of side-polar filaments. The side-polar structure may be related to the in vivo form of myosin in vertebrate smooth muscle.

scholarly journals Gas6 Delays Senescence in Vascular Smooth Muscle Cells through the PI3K/ Akt/FoxO Signaling Pathway

2015 ◽  
Vol 35 (3) ◽  
pp. 1151-1166 ◽  
Author(s):  
Cheng-wei Jin ◽  
Hui Wang ◽  
Yan-qing Chen ◽  
Meng-xiong Tang ◽  
Guan-qi Fan ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Signaling Pathway ◽  
Vascular Smooth Muscle Cells ◽  
Cellular Senescence ◽  
Replicative Senescence ◽  
Muscle Cells ◽  
Specific Protein ◽  
Foxo Signaling Pathway

Background/Aims: Growth arrest-specific protein 6 (Gas6) is a cytokine that can be synthesized by a variety of cell types and secreted into the extracellular matrix. Previous studies have confirmed that Gas6 is involved in certain pathophysiological processes of the cardiovascular system through binding to its receptor, Axl. In the present study, we investigated the role of Gas6 in cellular senescence and explored the mechanisms underlying its activity. Methods: We used vascular smooth muscle cells (VSMCs) to create two cellular senescence models, one for replicative senescence (RS) and one for induced senescence (IS), to test the hypothesis that Gas6 delays senescence. Results: Gas6-treated cells appear relatively younger compared with non-Gas6-treated cells. In particular, Gas6-treated cells displayed decreased staining for SA-β-Gal, fewer G1 phase cells, and decreased levels of p16INK4a and p21Cip1 expression; conversely, Gas6-treated cells displayed more S phase cells and significantly increased proliferation indexes. Furthermore, in both the IS and RS models with Gas6 treatment, the levels of PI3K, p-Akt, and p-FoxO3a decreased following Axl inhibition by R428; similarly, the levels of p-Akt and p-FoxO3a also decreased following PI3K inhibition by LY294002. Conclusion: Gas6/Axl signaling is essential for delaying the cellular senescence process regulated by the PI3K/Akt/FoxO signaling pathway.

scholarly journals Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments.

1985 ◽  
Vol 101 (5) ◽  
pp. 1897-1902 ◽  
Author(s):  
J R Sellers ◽  
J A Spudich ◽  
M P Sheetz
Keyword(s):  
Smooth Muscle ◽  
Actin Filaments ◽  
Smooth Muscles ◽  
Smooth Muscle Myosin ◽  
Vitro Assay ◽  
Skeletal Muscle Myosin ◽  
Myosin Filaments ◽  
Muscle Myosin ◽  
Rate Of Movement

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.

Proteins of the contractile mechanism of mammalian smooth muscle and their possible location in the cell

1964 ◽  
Vol 160 (981) ◽  
pp. 517-524 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sedimentation Rate ◽  
Striated Muscle ◽  
Muscle Actin ◽  
Trypsin Treatment ◽  
Skeletal Muscle Myosin ◽  
Uterine Smooth Muscle ◽  
Muscle Myosin ◽  
Viscous Behaviour

Dorothy M. Needham speaking. Since the pioneer work of Csapo and his colleagues, beginning about fifteen years ago, it has been realized that from uterine smooth muscle can be extracted a protein closely resembling skeletal-muscle actomyosin in its viscous behaviour, sedimentation rate and electrophoretic mobility. (See, for example, Csapo 1948, 1949, 1950, 1959; Csapo, Erdos, Naeslund & Snellman 1950; Naeslund & Snellman 1951). Later work, in which the properties of purified preparations of myosin, actin and actomyosin have been studied, bears out these earlier conclusions. Thus, for example, we have shown (Needham & Williams 1963 b ) that skeletal-muscle myosin will react normally with uterus actin to give the highly viscous actomyosin; and similarly uterus myosin with skeletal-muscle actin. In both types of experiment the results indicated that the two proteins associated together in about the same proportions as when both are derived from skeletal muscle. Uterus actomyosin may be fragmented by carefully controlled trypsin treatment giving light and heavy meromyosins which, so far as they have been studied, show similar properties to the meromyosins from skeletal-muscle actomyosin (Needham & Williams 1959; Cohen, Lowey & Kucera 1961). Smooth muscle, however, does contain very strikingly less actomyosin than striated muscle, only 6 to 10 mg/g wet wt as compared with about 70 mg/g wet wt in skeletal muscle (Needham & Williams 1963 a ).

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

scholarly journals The Heavy-chain Stoichiometry of Smooth Muscle Myosin is a Characteristic of Smooth Muscle Tissues

1988 ◽  
Vol 41 (4) ◽  
pp. 409 ◽  
Author(s):  
Mukhallad A Mohammad ◽  
Malcolm P Sparrow
Keyword(s):  
Smooth Muscle ◽  
Heavy Chain ◽  
Relative Proportion ◽  
Sodium Dodecyl ◽  
Smooth Muscle Myosin ◽  
Human Bronchus ◽  
Heavy Chains ◽  
Tissue Extracts ◽  
Muscle Tissues ◽  
Muscle Myosin

The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3�6 times more abundant in toad stomach, 2�3 in rabbit myometrium, 2�0 in rat femoral artery, 1�3 in guinea pig ileum, 0�93 in pig trachea and 0�69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and nonmuscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts were electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.

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