scholarly journals Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle

2008 ◽  
Vol 295 (3) ◽  
pp. C653-C660 ◽  
Author(s):  
Renaud Léguillette ◽  
Nedjma B. Zitouni ◽  
Karuthapillai Govindaraju ◽  
Laura M. Fong ◽  
Anne-Marie Lauzon
Keyword(s):  
Smooth Muscle ◽  
Alternative Hypothesis ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Tonic Muscle ◽  
Latch State ◽  
Muscle Myosin ◽  
Phasic Smooth Muscle

Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (νmax) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of νmax versus [MgADP] was significantly greater for tonic (−0.51 ± 0.04) than phasic muscle myosin (−0.15 ± 0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092 ± 0.022 pN for phasic muscle and 0.084 ± 0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state.

Phosphorylation of calponin in airway smooth muscle

1997 ◽  
Vol 272 (1) ◽  
pp. L115-L123 ◽  
Author(s):  
J. Pohl ◽  
S. J. Winder ◽  
B. G. Allen ◽  
M. P. Walsh ◽  
J. R. Sellers ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Inhibitory Effect ◽  
Tracheal Smooth Muscle ◽  
Actin Binding ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Kinase C ◽  
Gradient Gel Electrophoresis

Calponin is an actin-binding protein known to be a substrate in vitro for several protein kinases and phosphoprotein phosphatases. We tested the hypothesis that calponin is phosphorylated in vivo using canine tracheal smooth muscle strips metabolically labeled with 32Pi. Calponin was gel purified from muscles stimulated with 1 microM carbachol. Phosphorylation increased to 2.0 times the basal level of 178 +/- 26 counts per minute (cpm)/microgram calponin within 30 s to 350 +/- 64 cpm/micrograms. Two-dimensional nonequilibrium pH gradient gel electrophoresis resolved four charge isoforms of calponin in unstimulated muscle. Stimulation with carbachol induced an additional more acidic isoform. Phosphorylation of calponin in vitro with protein kinase C (PKC) also induced formation of additional acidic isoforms. The functional effect of phosphorylation was demonstrated using an in vitro motility assay in which unphosphorylated calponin (2 microM) caused a profound inhibition of actin sliding. Calponin phosphorylated by PKC did not inhibit actin sliding. The results show that phosphorylation of calponin occurs in intact tracheal smooth muscle and that phosphorylation of calponin in vitro alleviates the inhibitory effect of calponin on actomyosin function.

scholarly journals Smooth muscle myosin cross-bridge interactions modulate actin filament sliding velocity in vitro.

1990 ◽  
Vol 111 (2) ◽  
pp. 453-463 ◽  
Author(s):  
D M Warshaw ◽  
J M Desrosiers ◽  
S S Work ◽  
K M Trybus
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Myosin ◽  
Copolymer Composition ◽  
Weakly Bound ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Chemically Modified ◽  
Cross Bridges ◽  
Muscle Myosin

Although it is generally believed that phosphorylation of the regulatory light chain of myosin is required before smooth muscle can develop force, it is not known if the overall degree of phosphorylation can also modulate the rate at which cross-bridges cycle. To address this question, an in vitro motility assay was used to observe the motion of single actin filaments interacting with smooth muscle myosin copolymers composed of varying ratios of phosphorylated and unphosphorylated myosin. The results suggest that unphosphorylated myosin acts as a load to slow down the rate at which actin is moved by the faster cycling phosphorylated cross-bridges. Myosin that was chemically modified to generate a noncycling analogue of the "weakly" bound conformation was similarly able to slow down phosphorylated myosin. The observed modulation of actin velocity as a function of copolymer composition can be accounted for by a model based on mechanical interactions between cross-bridges.

Characterization of isoform diversity in smooth muscle myosin heavy chains

1994 ◽  
Vol 72 (11) ◽  
pp. 1351-1360 ◽  
Author(s):  
Christine A. Kelley ◽  
Robert S. Adelstein
Keyword(s):  
Smooth Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Smooth Muscle Myosin ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Amino Terminal ◽  
Muscle Myosin

In this paper we review some of our recent work on the structural and biochemical characterization of isoforms of the heavy chain of vertebrate smooth muscle myosin II. There exist both amino-terminal and carboxyl-terminal alternatively spliced isoforms of the smooth muscle myosin heavy chain (MHC). mRNA splicing at the 3′ end generates two MHCs, which differ in length and amino acid sequence in the carboxyl terminus. We will refer to the longer, 204-kDa isoform as MHC204 and the shorter, 200-kDa isoform as MHC200. We found that MHC204, but not MHC200, can be phosphorylated by casein kinase II on a serine near the carboxyl terminus, suggesting that these isoforms may be differentially regulated. The physiological significance of this phosphorylation is not known. However, as demonstrated in this paper, phosphorylation does not appear to affect filament formation, velocity of movement of actin filaments by myosin in an in vitro motility assay, actin-activated Mg2+ ATPase activity, or myosin conformation. Our results also show that MHC204 and MHC200 form homodimers, but not heterodimers. Purified MHC204 and MHC200 homodimers are not enzymatically different, at least as measured using an in vitro motility assay. The amino-terminal spliced MHC204 and MHC200 isoforms are the result of the specific insertion or deletion of seven amino acids near the ATP-binding region in the myosin head. We refer to these isoforms as inserted (MHC204-I; MHC200-I) or noninserted (MHC204; MHC200), respectively. In contrast to the carboxyl-terminal spliced isoforms, the amino-terminal spliced inserted and noninserted myosin heavy chain isoforms are enzymatically different. The inserted isoform, which is expressed in intestinal, phasic-type smooth muscle, has a higher actin-activated Mg ATPase activity and moves actin filaments at a greater velocity in an in vitro motility assay than the noninserted MHC isoform, which is expressed in tonic-type vascular smooth muscle. The results presented in this review suggest that the alternative splicing of smooth muscle mRNA results in at least four different isoforms of the myosin heavy chain molecule. The potential relevance of these molecular isoforms to smooth muscle function is discussed.Key words: myosin, heavy chain isoforms.

Thiophosphorylation independently activates each head of smooth muscle myosin in vitro

1995 ◽  
Vol 269 (5) ◽  
pp. C1160-C1166 ◽  
Author(s):  
D. E. Harris ◽  
C. J. Stromski ◽  
E. Hayes ◽  
D. M. Warshaw
Keyword(s):  
Smooth Muscle ◽  
Myosin Light Chain ◽  
Smooth Muscle Myosin ◽  
Mechanical Activity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Measured Activity ◽  
Coated Surface ◽  
Muscle Myosin

To determine whether thiophosphorylation of the 20-kDa myosin light chain activates each head of smooth muscle myosin independently of the head with which it is paired, chicken gizzard smooth muscle myosin was randomly thiophosphorylated, producing a mixture of unphosphorylated and singly and doubly thiophosphorylated myosin. Thiophosphorylation levels were measured by glycerol-urea gels, and the activity of this myosin was determined by actin-activated adenosinetriphosphatase measurements and in an in vitro motility assay, where the velocity of actin filaments moving over a myosin-coated surface is measured. Activity at each thiophosphorylation level was similar to that previously observed for mixtures of unphosphorylated and doubly thiophosphorylated myosin (D. E. Harris, S. S. Work, R. K. Wright, N. R. Alpert, and D. M. Warshaw. J. Muscle Res. Cell Motil. 15: 11-19, 1994). All doubly thiophosphorylated myosin was then formed into filaments and removed from randomly thiophosphorylated myosin by centrifugation. The remaining myosin (mixture of unphosphorylated and singly phosphorylated myosin), which could not polymerize because of their conformation, retained approximately 70% activity compared with mixtures of unphosphorylated and doubly thiophosphorylated myosin. Thus a thiophosphorylated smooth muscle myosin head can produce substantial biochemical and mechanical activity, even when it is paired with an unphosphorylated partner.

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.

Distribution of phenotypically disparate myocyte subpopulations in airway smooth muscle

2005 ◽  
Vol 83 (1) ◽  
pp. 104-116 ◽  
Author(s):  
Andrew J Halayko ◽  
Gerald L Stelmack ◽  
Akira Yamasaki ◽  
Karol McNeill ◽  
Helmut Unruh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Lung Development ◽  
Similar Phenotype ◽  
Low Levels ◽  
Phenotype Heterogeneity ◽  
Muscle Myosin

Phenotype and functional heterogeneity of airway smooth muscle (ASM) cells in vitro is well known, but there is limited understanding of these features in vivo. We tested whether ASM is composed of myocyte subsets differing in contractile phenotype marker expression. We used flow cytometry to compare smooth muscle myosin heavy chain (smMHC) and smooth muscle-α-actin (sm-α-actin) abundance in myocytes dispersed from canine trachealis. Based on immunofluorescent intensity and light scatter characteristics (forward and 90° side scatter), 2 subgroups were identified and isolated. Immunoblotting confirmed smMHC and sm-α-actin were 10- and 5-fold greater, respectively, in large, elongate myocytes that comprised ~60% of total cells. Immunohistochemistry revealed similar phenotype heterogeneity in human bronchial smooth muscle. Canine tracheal myocyte subpopulations isolated by flow cytometry were used to seed primary subcultures. Proliferation of subcultures established with myocytes exhibiting low levels of smMHC and sm-α-actin was ~2× faster than subcultures established with ASM cells with a high marker protein content. These studies demonstrate broad phenotypic heterogeneity of myocytes in normal ASM tissue that is maintained in cell culture, as demonstrated by divergent proliferative capacity. The distinct roles of these subgroups could be a key determinant of normal and pathological lung development and biology.Key words: flow cytometry, phenotype, heterogeneity, asthma, differentiation.

The in vitro motility assay parameters of actin filaments from Mytilus edulis exposed in vivo to copper ions

2009 ◽  
Vol 491 (1-2) ◽  
pp. 32-38 ◽  
Author(s):  
Natalia N. Vikhoreva ◽  
Petr G. Vikhorev ◽  
Maria A. Fedorova ◽  
Ralf Hoffmann ◽  
Alf Månsson ◽  
...  
Keyword(s):  
Mytilus Edulis ◽  
Actin Filaments ◽  
Copper Ions ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility
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