Structure of heads A and B of myosin studied by tryptic digestion of myosin subfragment-1.
We studied the difference in the structure of head B (P1-burst head) and head A of myosin by limited tryptic digestion of myosin subfragment-1 (S-1), and using antibodies (anti-A and anti-B) which bind specifically with each head. The antibodies were prepared using peptides with sequences identical to those around the reactive lysine residue of heads A and B. When myosin subfragment-1 (S-1) was cleaved limitedly by trypsin, S-1 heavy chain (100 kDa) was digested into fragments of 25, 50, and 20 kDa. Two fragments with molecular masses of 75 and 27 kDa were transiently produced in the initial phase of digestion. Anti-A and anti-B antibodies bound only with peptides that contained the reactive lysine residue [S-1 heavy chain (100 kDa), 75-, 27-, and 25-kDa peptides], thus showing specific binding with antigen peptide. However, the 27-kDa fragment bound more strongly with anti-B antibody than with anti-A antibody. When S-1 was separated into fractions rich in S-1A and S-1B using insoluble anti-A or anti-B antibody, each antibody bound more strongly with the S-1 heavy chain (100 kDa) of its corresponding fraction by Western immunoblotting. These results suggest that the antibodies react specifically with peptides even after SDS-PAGE and membrane-blotting, and that the structure of the 25 kDa 50 kDa junction differs between heads A and B of myosin.
Murai
S
S
Department of Biology, Faculty of Science, Osaka University.
Arata
T
T
Inoue
A
A
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D008239:Lysine / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits; D014357:Trypsin
D000906:Antibodies; D015879:Myosin Subfragments; D014357:Trypsin; D009218:Myosins; D008239:Lysine