Submolecular Structure of Subfragment-1 of the Myosin Molecule
1972 Volume 72 Issue 1 Pages 83-100
Details
1972 Volume 72 Issue 1 Pages 83-100
1. An electrophoretogram of rabbit skeletal myosin on SDS-polyacrylamide gel showed that it contains three kinds of light components with molecular weights of 25, 000 (g1), 18, 000 (g2), and 14, 000 (g3), besides f-subunit with a molecular weight of about 210, 000 (1-5). The weight ratio of the f-subunit to the g-subunit was 88: 12, indicating that one molecule of myosin (mol. wt., about 480, 000 (1, 6-8)) may contain each of the three kinds of g-subunit and the two f-subunits.
2. Small polypeptide chains with molecular weights of less than 10, 000 were removed from S-l by treatment with alkali. After purification by combination with actin, the Mg2+-ATPase [EC 3. 6. 1. 3] activity of alkli-treated S-l was almost the same as that of intact S-l. The molecular weight of alkali-treated S-l was estimated to be 110, 000±5, 000 by osmometry and gel-filtration. S-l was separated into fractions a and b with molecular weights of about 55, 000 and 26, 000, respectively, in a weight ratio of 0.92: 1 by gel-filtration with 5M guanidine-HCl.
3. S-l was found to consist of the five components with molecular weights of 52, 000-55, 000, 27, 000, 21, 000, 16, 000, and 14, 000 by SDS-gel electrophoresis. The weight ratio of the largest component to the others was 0.92: 1.
4. The changes in the patterns of SDS-gel electrophoretograms were followed during digestion of myosin and HMM with trypsin [EC 3. 4. 4. 4]. The results indicated that the components with molecular weights of 52, 000-55, 000 (named the f'-component) and 27, 000 (f'') were derived from the f-subunit and the component with a molecular weight of 21, 000 was derived from g1, whereas that with a molecular weight of 16, 000 was from g2 and that with a molecular weight of 14, 000 was g3 itself.
5. The S1-sulfhydryl group was not located on the g-subunit, but on the f-subunit. Activation of the Ca2+-ATPase activity due to blocking of the S1-sulfhydryl group was not affected by further blockage of 4.0 moles of sulfhydryl groups per 4.2×l05g of the f-subunit and 2.2 moles per 5.7×lO4g of the g-subunit with IAA.
6. The amino acid composition of the f'-component was similar to that of the g-subunit rather than those of helical portions of the f-subunit.
7. Based on these results, the non-identical structure of the two S-l from the bipartite heads of the myosin molecule were discussed.
