scholarly journals Localization and topography of antigenic domains within the heavy chain of smooth muscle myosin.

1985 ◽  
Vol 101 (1) ◽  
pp. 66-72 ◽  
Author(s):  
M D Schneider ◽  
J R Sellers ◽  
M Vahey ◽  
Y A Preston ◽  
R S Adelstein
Keyword(s):  
Smooth Muscle ◽  
Immunoelectron Microscopy ◽  
Heavy Chain ◽  
Muscle Development ◽  
Solid Phase ◽  
Smooth Muscles ◽  
Smooth Muscle Myosin ◽  
Actin Binding ◽  
Heavy Meromyosin ◽  
Muscle Myosin

We have produced and characterized monoclonal antibodies that label antigenic determinants distributed among three distinct, nonoverlapping peptide domains of the 200-kD heavy chain of avian smooth muscle myosin. Mice were immunized with a partially phosphorylated chymotryptic digest of adult turkey gizzard myosin. Hybridoma antibody specificities were determined by solid-phase indirect radioimmunoassay and immunoreplica techniques. Electron microscopy of rotary-shadowed samples was used to directly visualize the topography of individual [antibody.antigen] complexes. Antibody TGM-1 bound to a 50-kD peptide of subfragment-1 (S-1) previously found to be associated with actin binding and was localized by immunoelectron microscopy to the distal aspect of the myosin head. However, there was no antibody-dependent inhibition of the actin-activated heavy meromyosin ATPase, nor was antibody TGM-1 binding to actin-S-1 complexes inhibited. Antibody TGM-2 detected an epitope of the subfragment-2 (S-2) domain of heavy meromyosin but not the S-2 domain of intact myosin or rod, consistent with recognition of a site exposed by chymotryptic cleavage of the S-2:light meromyosin junction. Localization of TGM-2 to the carboxy-terminus of S-2 was substantiated by immunoelectron microscopy. Antibody TGM-3 recognized an epitope found in the light meromyosin portion of myosin. All three antibodies were specific for avian smooth muscle myosin. Of particular interest is that antibody TGM-1, unlike TGM-3, bound poorly to homogenates of 19-d embryonic smooth muscles. This indicates the expression of different myosin heavy chain epitopes during smooth muscle development.

scholarly journals Degradation of smooth-muscle myosin by trypsin-like serine proteinases.

1982 ◽  
Vol 201 (2) ◽  
pp. 267-278 ◽  
Author(s):  
J Kay ◽  
R F Siemankowski ◽  
L M Siemankowski ◽  
D E Goll
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Atpase Activity ◽  
Heavy Chain ◽  
Light Chain ◽  
Regulatory Light Chain ◽  
Smooth Muscle Myosin ◽  
Heavy Meromyosin ◽  
Bovine Trypsin ◽  
Muscle Myosin

1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The ‘regulatory’ light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called ‘regulatory’ light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).

Two different heavy chains are found in smooth muscle myosin

1986 ◽  
Vol 250 (6) ◽  
pp. C861-C870 ◽  
Author(s):  
A. S. Rovner ◽  
M. M. Thompson ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Sodium Dodecyl Sulfate ◽  
Heavy Chain ◽  
Sodium Dodecyl ◽  
Smooth Muscles ◽  
Protein Band ◽  
Smooth Muscle Myosin ◽  
Heavy Chains ◽  
Dodecyl Sulfate ◽  
Muscle Myosin

Two putative myosin heavy chains designated SM1 and SM2 were detected on a 3.5% polyacrylamide-sodium dodecyl sulfate gel electrophoresis system loaded with homogenates of several mammalian smooth muscles. The two polypeptides were present in nearly equal amounts in all smooth muscle tissues tested and in myosin purified from swine carotid media and stomach. Both proteins were equally stained by smooth muscle-specific myosin antibodies. The smaller of the polypeptides had a mobility nearly identical to that of the single heavy chain observed in purified fast-twitch skeletal myosin. Electrophoresis of pyrophosphate extracts from swine carotid media, swine stomach, rabbit thoracic aorta, and guinea pig taenia coli on nondenaturing pyrophosphate gels revealed a single protein band. When subsequently electrophoresed on a sodium dodecyl sulfate gel, the native bands from swine tissue extracts revealed the two putative heavy chains in nearly equal amounts, as well as a large amount of a higher molecular weight peptide whose properties reflect those of filamen. Sodium dodecyl sulfate gel analysis of the myosin band from pyrophosphate gels of purified swine stomach myosin showed exclusively the two heavy chains in a nearly 1:1 ratio. Smooth muscle myosin migrates homogeneously on pyrophosphate gels, and the virtual equality of the two heavy chains may reflect the presence of large amounts of a myosin isoenzyme, which is a heavy-chain heterodimer.

scholarly journals Visualization of Head–Head Interactions in the Inhibited State of Smooth Muscle Myosin

1999 ◽  
Vol 147 (7) ◽  
pp. 1385-1390 ◽  
Author(s):  
Thomas Wendt ◽  
Dianne Taylor ◽  
Terri Messier ◽  
Kathleen M. Trybus ◽  
Kenneth A. Taylor
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Myosin ◽  
Intramolecular Interactions ◽  
Actin Binding ◽  
Structural Basis ◽  
Binding Interface ◽  
Structural Differences ◽  
Muscle Myosin ◽  
Positively Charged ◽  
Phosphoryla Tion

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.

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