Effect of Nucleotides and Actin on the Orientation of the Light Chain-Binding Domain in Myosin Subfragment 1†

Biochemistry ◽  
1997 ◽  
Vol 36 (43) ◽  
pp. 13201-13207 ◽  
Author(s):  
Cybelle Smyczynski ◽  
Andrzej A. Kasprzak
Keyword(s):  
Light Chain ◽  
Binding Domain ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

scholarly journals The molecular organization of myosin in stress fibers of cultured cells.

1986 ◽  
Vol 102 (1) ◽  
pp. 200-209 ◽  
Author(s):  
G Langanger ◽  
M Moeremans ◽  
G Daneels ◽  
A Sobieszek ◽  
M De Brabander ◽  
...  
Keyword(s):  
Light Chain ◽  
Cultured Cells ◽  
Staining Pattern ◽  
Central Zone ◽  
Molecular Organization ◽  
Stress Fibers ◽  
General Distribution ◽  
Immunogold Staining ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

scholarly journals Localization of a myosin subfragment-1 interaction site on the C-terminal part of actin

1992 ◽  
Vol 284 (1) ◽  
pp. 75-79 ◽  
Author(s):  
J P Labbé ◽  
M Boyer ◽  
C Roustan ◽  
Y Benyamin
Keyword(s):  
Conformational Changes ◽  
Light Chain ◽  
Myosin Head ◽  
Terminal Region ◽  
Terminal Part ◽  
Rigor State ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Chain Interaction ◽  
Rule Out

The actin-myosin head complex in the rigor state reveals several high-affinity sites on the actin molecule in sequences 18-28 and 40-113. In the presence of Mg(2+)-ATP, participation of the actin N-terminal 1-7 sequence is known to occur. The proximity of the C-terminal region of actin to the A1 light chain of the myosin head [S-1(A1)] (where S-1 is myosin subfragment-1) was described previously. We observed that C-terminal antigenic structures located near Met-305, Met-325 and Met-355 and the C-terminal end (Cys-374) of actin are markedly modified in the presence of S-1(A1), S-1(A2) and scallop S-1 and in the absence of Mg(2+)-ATP. This seems to rule out any important specific involvement of the A1 light chain in the described conformational changes. An S-1-binding site was located in this actin C-terminal region by testing the tryptic CB9 peptide (360-372 sequence) previously implicated in the A1 light chain interaction. This peptide was able to bind well to S-1(A1), S-1(A2) and scallop S-1, but not in the presence of Mg(2+)-pyrophosphate. These results strengthen the hypothesis of a multisite interface between S-1 and actin located in the actin subdomain I.

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