Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20,000-dalton light chains

Biochemistry ◽  
1985 ◽  
Vol 24 (9) ◽  
pp. 2380-2387 ◽  
Author(s):  
M. Ikebe ◽  
David J. Hartshorne
Keyword(s):  
Staphylococcus Aureus ◽  
Smooth Muscle ◽  
Smooth Muscle Myosin ◽  
Light Chains ◽  
Heavy Meromyosin ◽  
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).

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

1993 ◽  
Vol 127-128 (1) ◽  
pp. 219-227 ◽  
Author(s):  
Robabeh S. Moussavi ◽  
Christine A. Kelley ◽  
Robert S. Adelstein
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Myosin ◽  
Light Chains ◽  
Myosin Heavy Chains ◽  
Heavy Chains ◽  
Muscle Myosin

Calcium Binding and Conformation of Regulatory Light Chains of Smooth Muscle Myosin of Scallop1

1985 ◽  
Vol 97 (2) ◽  
pp. 553-561 ◽  
Author(s):  
Fumi MORITA ◽  
Shuhei KONDO ◽  
Ken TOMARI ◽  
Osamu MINOWA ◽  
Mitsuhiko IKURA ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Binding ◽  
Smooth Muscle Myosin ◽  
Light Chains ◽  
Regulatory Light Chains ◽  
Muscle Myosin
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