Smooth muscle myosin subfragment-1 is a kinetic analogue for heavy meromyosin in the extended conformation

1993 ◽  
Vol 26 (4) ◽  
pp. 291-300 ◽  
Author(s):  
Jean S. Drew ◽  
Marianne P. White ◽  
Leonard A. Stein
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Myosin ◽  
Heavy Meromyosin ◽  
Extended Conformation ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Muscle Myosin

Interaction between the heavy and the regulatory light chains in smooth muscle myosin subfragment 1

Biochemistry ◽  
1992 ◽  
Vol 31 (4) ◽  
pp. 1201-1210 ◽  
Author(s):  
Hirofumi Onishi ◽  
Tetsuo Maita ◽  
Genji Matsuda ◽  
Keigi Fujiwara
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Myosin ◽  
Light Chains ◽  
Regulatory Light Chains ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Muscle Myosin

Binding of gizzard smooth muscle myosin subfragment-1 to actin in the presence and absence of adenosine 5'-triphosphate

Biochemistry ◽  
1983 ◽  
Vol 22 (3) ◽  
pp. 530-535 ◽  
Author(s):  
Lois E. Greene ◽  
James R. Sellers ◽  
Evan Eisenberg ◽  
Robert S. Adelstein
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Myosin ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Presence And Absence ◽  
Muscle Myosin

scholarly journals Filamentous smooth muscle myosin is regulated by phosphorylation.

1989 ◽  
Vol 109 (6) ◽  
pp. 2887-2894 ◽  
Author(s):  
K M Trybus
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Smooth Muscle Myosin ◽  
Heavy Meromyosin ◽  
Phosphate Release ◽  
Product Release ◽  
Single Turnover ◽  
Myosin Filaments ◽  
Muscle Myosin ◽  
Low Rate

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to "fix" dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody-stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1 (Cross, R. A., K. E. Cross, A. Sobieszek. 1986. EMBO [Eur. Mol. Biol. Organ.] J. 5:2637-2641) the rate of product release from dephosphorylated filaments was only 3-12-fold higher, depending on the ionic strength. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin (Sellers, J. R. 1985. J. Biol. Chem. 260:15815-15819) thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.

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).

ADP release from myosin in permeabilized smooth muscle

1989 ◽  
Vol 256 (1) ◽  
pp. C59-C66 ◽  
Author(s):  
T. M. Butler ◽  
D. S. Pacifico ◽  
M. J. Siegman
Keyword(s):  
Smooth Muscle ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Adp Release ◽  
The Difference ◽  
Portal Veins ◽  
Cross Bridges ◽  
Muscle Myosin

The purpose of this study was to determine the nucleotide bound to myosin and its rate of release under relaxed and activated conditions in permeabilized rabbit portal veins. Incubation of the muscles in a relaxing solution containing [3H]-ATP resulted in the formation of 60-70 microM radiolabeled ADP in the muscle whether or not the myosin light chains had been thiophosphorylated. This value was similar to the estimate of the concentration of myosin subfragment 1. Upon transfer of the muscles to a chase solution containing no labeled ATP, there was a very slow loss of labeled ADP when the light chains were unphosphorylated, but a much faster release occurred when the light chains were thiophosphorylated. The results suggest that smooth muscle myosin exists primarily in a complex with ADP under both relaxed and phosphorylated conditions and that phosphorylation of all of the light chains results in a large increase in the rate of release of the products of ATP splitting from all of the myosin. Interestingly, the exponential release of ADP in relaxed muscle shows two components, one of which contains about two-thirds of the total ADP and is 5- to 10-fold faster than the other. If the difference in rates of ADP release observed in relaxed muscle persists when the myosin is phosphorylated, then it is possible that there is a 5- to 10-fold difference in rates of cycling for different phosphorylated cross bridges in smooth muscle.

scholarly journals Use of the avidin-biotin complex for the localization of actin and myosin with fluorescence microscopy.

1977 ◽  
Vol 73 (3) ◽  
pp. 783-788 ◽  
Author(s):  
M H Heggeness ◽  
J F Ash
Keyword(s):  
Smooth Muscle ◽  
Fluorescence Microscopy ◽  
Indirect Method ◽  
Smooth Muscle Myosin ◽  
Second Step ◽  
Heavy Meromyosin ◽  
Muscle Myosin ◽  
Fluorescent Derivative ◽  
Avidin Biotin Complex

A new indirect method for fluorescence localization of proteins making use of the avidin-biotin complex is described. We have prepared both a biotin-modified rabbit heavy meromyosin (BHMM) and a biotin-modified antibody to a smooth muscle myosin. After fixation, cells can be treated with either BHMM, which binds to actin, or the biotinyl antibody, which binds to myosin. In a second step the cell are treated with a fluorescent derivative of avidin (Fl-avidin) which binds to the biotinyl proteins and thus indirectly reveals the location of the cellular action or myosin.

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