Localization of epitopes and functional effects of two novel monoclonal antibodies against skeletal muscle myosin.
Two skeletal myosin monoclonal antibodies, raised against human skeletal myosin, were used to study the correlation between function, primary and tertiary structure of S-1 prepared from rabbit skeletal myosin. The heavy chain of S-1 is cleaved into three fragments by trypsin--27 kDa, 50 kDa and 20 kDa--aligned in this order from the N-terminus. The epitope of the first antibody was assigned to the N-terminal 1-23 amino acid stretch of S-1, since it reacted with the 27 kDa N-terminal tryptic fragment of S-1 but not with a derivative of the 27 kDa fragment, which lacks the above amino acid stretch. The epitope of the second antibody was assigned to the 3 kDa N-terminal region of the central 50 kDa domain of S-1. This assignment was based on proteolytic and photochemical cleavage of S-1 and on the labelling of its N-terminus by a specific antibody. The antibodies were visualized binding to the myosin head on electron micrographs of rotary-shadowed complexes of antibodies with myosin. Measurements on the micrographs indicated that the distances between the head-tail junction of myosin and the 'anti-27 K' and 'anti-50 K' epitopes are 14 nm and 17 nm, respectively. Both antibodies have a high affinity to S-1. The affinity of the 'anti-50 K' to S-1 decreased upon actin binding, while that of the 'anti-27 K' was not affected by binding of S-1 to F-actin. The 'anti-50 K' antibody inhibited the K+ (EDTA) and the actin-activated ATPase activity of S-1, while the 'anti-27 K' had no effect. The results indicate that either the epitope of the 'anti-50 K' is near to the actin or to the ATP-binding sites of S-1, or that there is communication, expressed as propagated conformational changes, between these sites and the epitope.
Dan-Goor
M
M
Hebrew University-Hadassah School of Dental Medicine, Department of Oral Biology, Jerusalem, Israel.
Silberstein
L
L
Kessel
M
M
Muhlrad
A
A
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009132:Muscles / Q000276:immunology* / Q000378:metabolism / Q000648:ultrastructure; D015879:Myosin Subfragments / Q000276:immunology / Q000378:metabolism; D009218:Myosins / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits
D000199:Actins; D000911:Antibodies, Monoclonal; D000939:Epitopes; D015879:Myosin Subfragments; D000251:Adenosine Triphosphatases; D009218:Myosins