The assembly of ribbon-shaped structures in low ionic strength extracts obtained from vertebrate smooth muscle

1973 ◽  
Vol 265 (867) ◽  
pp. 203-212 ◽  
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Diffraction Analysis ◽  
Average Diameter ◽  
Smooth Muscle Myosin ◽  
Optical Diffraction ◽  
The Core ◽  
Regular Arrangement ◽  
Low Ionic Strength ◽  
Muscle Myosin

In actomyosin extracts from smooth muscle obtained at low ionic strength, an assembly of protein into long ribbon-shaped elements is observed to take place. These ribbons which range up to about 100 nm in width and up to many micrometres in length exhibit a strong repeat period of about 5.6 nm. Optical diffraction analysis shows that they possess a long repeat of 39.1 nm ± 0.4 nm. Tropomyosin purified from vertebrate smooth muscle can be induced to form the same ribbon-shaped elements. On removal of salt from solution the ribbons dissociate into fine filaments of average diameter about 8 nm which show subfilaments of about 2 to 3 nm diameter. In crude preparations the ribbons occur in solution together with myosin. If such preparations are left to stand for several days, ribbons may be found that show a visible 14 nm period which appears to arise from the presence of a regular arrangement of projections. Smooth muscle myosin alone assembles into cylindrical filaments which exhibit a regular arrangement of projections along their entire length, indicating an absence of polarity. These results indicate, as have those recently obtained from section material, that the myosin-containing component of vertebrate smooth muscle contains a protein that forms the core of the filament, which is responsible for its ribbon-like shape and which probably determines the polarity of the attached myosin molecules. It is proposed that this protein is tropomyosin.

scholarly journals Phosphorylation by casein kinase II affects the interaction of caldesmon with smooth muscle myosin and tropomyosin

1993 ◽  
Vol 290 (2) ◽  
pp. 437-442 ◽  
Author(s):  
N V Bogatcheva ◽  
A V Vorotnikov ◽  
K G Birukov ◽  
V P Shirinsky ◽  
N B Gusev
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Smooth Muscle Myosin ◽  
Myosin Phosphorylation ◽  
Chymotryptic Peptide ◽  
Molecular Masses ◽  
Low Ionic Strength ◽  
Muscle Myosin

Smooth muscle caldesmon was phosphorylated by casein kinase II, and the effects of phosphorylation on the interaction of caldesmon and its chymotryptic peptides with myosin and tropomyosin were investigated. The N-terminal chymotryptic peptide of caldesmon of molecular mass 27 kDa interacted with myosin. Phosphorylation of Ser-73 catalysed by casein kinase II resulted in a 2-fold decrease in the affinity of the native caldesmon (or its 27 kDa N-terminal peptide) for smooth muscle myosin. At low ionic strength, caldesmon and its N-terminal peptides of molecular masses 25 and 27 kDa were retarded on a column of immobilized tropomyosin. Phosphorylation of Ser-73 led to a 2-4-fold decrease in the affinity of caldesmon (or its N-terminal peptides) for tropomyosin. Thus phosphorylation of Ser-73 catalysed by casein kinase II affects the interaction of caldesmon with both smooth muscle myosin and tropomyosin.

scholarly journals Myosins of secretory tissues

1978 ◽  
Vol 77 (3) ◽  
pp. 827-836 ◽  
Author(s):  
RE Ostlund ◽  
JT Leung ◽  
DM Kipnis
Keyword(s):  
New York ◽  
Smooth Muscle ◽  
Salivary Gland ◽  
Ionic Strength ◽  
Smooth Muscle Myosin ◽  
Myosin Atpase ◽  
Cell Fractionation ◽  
Secretory Tissue ◽  
Low Ionic Strength ◽  
Muscle Myosin

Myosin has been purified from the principal pancreatic islet of catfish, hog salivary gland, and hog pituitary. Use of the protease inhibitor Trasylol (FBA Pharmaceuticals, New York) was essential in the isolation of pituitary myosin. Secretory tissue myosins were very similar to smooth muscle myosin, having a heavy chain of 200,000 daltons and light chains of 14,000 and 19,000 daltons. Salivary gland myosin cross-reacted with antibodies directed toward both smooth muscle myosin and fibroblast myosin, but not with antiskeletal muscel myosin serum. The specific myosin ATPase activity measured in 0.6 M KCl was present. Tissues associated with secretion of hormone granules contained substantial amounts of this ATPase, rat pancreatic islets having 4.5 times that of rat liver. Activation of low ionic strength myosin ATPase by actin could not be demonstrated despite adequate binding of the myosin to muscle actin and elution by MgATP. The myosins were located primarily in the cytoplasm as determined by cell fractionation and were quite soluble in buffers of low ionic strength.

scholarly journals Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding.

1987 ◽  
Vol 105 (6) ◽  
pp. 3007-3019 ◽  
Author(s):  
K M Trybus ◽  
S Lowey
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Conformational Changes ◽  
Myosin Head ◽  
Smooth Muscle Myosin ◽  
Detectable Effect ◽  
Skeletal Muscle Myosin ◽  
Polar Type ◽  
Low Ionic Strength ◽  
Muscle Myosin

Small bipolar filaments, or "minifilaments," are formed when smooth muscle myosin is dialyzed against low ionic strength pyrophosphate or citrate/Tris buffers. Unlike synthetic filaments formed at approximately physiological ionic conditions, minifilaments are homogeneous as indicated by their hypersharp boundary during sedimentation velocity. Electron microscopy and hydrodynamic techniques were used to show that 20-22S smooth muscle myosin minifilaments are 380 nm long and composed of 12-14 molecules. By varying solvents, a continuum of different size polymers in the range of 15-30S could be obtained. Skeletal muscle myosin, in contrast, preferentially forms a stable 32S minifilament (Reisler, E., P. Cheung, and N. Borochov. 1986. Biophys. J. 49:335-342), suggesting underlying differences in the assembly properties of the two myosins. Addition of salt to the smooth muscle myosin minifilaments caused unidirectional growth into a longer "side-polar" type of filament, whereas bipolar filaments were consistently formed by skeletal muscle myosin. As with synthetic filaments, addition of 1 mM MgATP caused dephosphorylated minifilaments to dissociate to a mixture of folded monomers and dimers. Phosphorylation of the regulatory light chain prevented disassembly by nucleotide, even though it had no detectable effect on the structure of the minifilament. These results suggest that differences in filament stability as a result of phosphorylation are due largely to conformational changes occurring in the myosin head, and are not due to differences in filament packing.

scholarly journals Smitin, a novel smooth muscle titin–like protein, interacts with myosin filaments in vivo and in vitro

2002 ◽  
Vol 156 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Kyoungtae Kim ◽  
Thomas C.S. Keller
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Smooth Muscle Myosin ◽  
Globular Domain ◽  
Contractile Apparatus ◽  
Myosin Filaments ◽  
Muscle Myosin

Smooth muscle cells use an actin–myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

Extractability and properties of the contractile proteins of vertebrate smooth muscle

1973 ◽  
Vol 265 (867) ◽  
pp. 169-181 ◽  
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Striated Muscle ◽  
Smooth Muscles ◽  
Contractile Proteins ◽  
Biosynthetic Activity ◽  
Salting Out ◽  
Low Ionic Strength ◽  
The Comparative Study ◽  
Muscle Myosin

The vertebrate smooth muscles differ from the striated ones by their larger extracellular space, the smaller size of their cells and their high content in extracellular components. Furthermore, the smooth muscle cell is a bifunctional biological unit able to carry on also an important biosynthetic activity. The contractile proteins of vertebrate smooth muscle are extractable at low ionic strength contrarily to those of striated muscle. The partition of the salt extractable nitrogen between the low and high ionic strength extracts is very different in these two cases. Acidification of low ionic strength extracts of vertebrate smooth muscle at pH 5 allows precipitation of the contractile proteins quantitatively together with a large amount of contaminants typical of the smooth muscles. Comparison of the contractile proteins of vertebrate smooth muscle with their striated counterparts shows that actin is a very constant component of the contractile machinery, that tropomyosin holds an intermediate position, while myosin is the most variable. The smooth muscle myosin differs not only by some general properties as salting-out range and thermostability, but also by the behaviour of various parts of the molecule. The globular head has a different ATPase activity and is responsible for the very peculiar immunological behaviour of this myosin. The point along the myosin rod which is attacked by trypsin is much more resistant to proteolysis. The light meromyosin is more soluble and differs very much in amino acid composition. The comparative study of myosin reveals only minor variations from one species to the other but more or less wide ones within each species according to the type of muscle examined.

scholarly journals Protease-susceptible sites and properties of fragments of aortic smooth-muscle myosin

1995 ◽  
Vol 312 (2) ◽  
pp. 511-518 ◽  
Author(s):  
L King ◽  
M J Jiang ◽  
T S Huang ◽  
G C Sheu
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Ionic Strength ◽  
Molecular Mass ◽  
Thermal Unfolding ◽  
Aortic Smooth Muscle ◽  
Dramatic Difference ◽  
Low Ionic Strength ◽  
Muscle Myosin ◽  
Myosin Rod

We have examined the protease susceptibility of aortic myosin, the thermal unfolding profiles of myosin rod and light meromyosin (LMM) and the solubility properties of the LMM fragments. Two major protease-susceptible sites were found, located at the head-rod junction and the heavy meromyosin (HMM)-LMM junction. Both tryptic and chymotryptic digestion of aortic myosin rod produced the LMM (80-85 kDa) and short subfragment 2 (S-2) (40-45 kDa) segments, which were similar to those of gizzard myosin rod and differed from the short LMM (70 kDa) and long S-2 (58 kDa) segments produced from skeletal-muscle rod. The thermal unfolding profile of aortic myosin rods exhibited three helix-unfolding transitions, at 47.5, 51 and 54 degrees C, similar to those of gizzard rods yet different from those of skeletal-muscle rods. There was a dramatic difference in the solubility of aortic LMM fragments of various molecular mass, as for gizzard smooth-muscle LMM and rabbit skeletal-muscle LMM. LMM fragments of molecular mass 77 kDa or more were completely insoluble in low-ionic-strength buffer, whereas LMM fragments of molecular mass 73 kDa or less were completely soluble in low-ionic-strength buffer. Proteolytic digestion patterns of LMM showed two additional protease-susceptible sites located 13 and 30 kDa from the ends of the LMM molecule. This suggests the existence of flexible regions within the LMM molecule, which may be responsible for the folded form of aortic myosin.

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