J. Biochem, 1977, Vol. 81, No. 4 977-988
© 1977 Japanese Biochemical Society
research-article |
Preparation of Myosin Subfragment-1 from Pig Cardiac Muscle Myosin by Chymotryptic Digestion1
Department of Chemistry, Faculty of Science, Hokkaido University Kita-ku, Sapporo, Hokkaido 060
Subfragment-1 was prepared from pig cardiac myosin by chymotryptic digestion at low ionic strength at 0–2°C for 20 h. Two components were distinguished in polyacrylamide gel electrophoresis of subfragment-1 preparations, and were designated as S-1 (CT) and S-1 (CT)'. The isoelectric points were 6.70 and 6.45, respectively. The two components could not be separated by Sephadex G-200 gel filtration, but they were separated on a DEAE-cellulose (DE 32) column or a 6-aminohexylPP1-Sepharose 4B conjugate column. S-1 (CT) consisted of one large polypeptide chain (the head portion of the f-chain) and one small polypeptide chain (g1). In SDS gel electrophoresis, the large polypeptide of S-1 (CT) migrated at the same rate as that of S-1 (CT)', but the small polypeptide of S-1 (CT)' migrated at a faster rate than that of S-1 (CT) The small chain of S-1 (CT)' was thus designated as g1'. The migration rate of the small chain of S-1 (CT) was the same as that of g1 of cardiac myosin, but the small chain of S-1 (CT)' migrated faster than g1 and slower than g2 of cardiac myosin. It was therefore suggested that S-1 (CT)' was produced by overdigestion of S-1 (CT).
The yield of S-1 (CT) plus S-1 (CT)' estimated on the basis of absorption at 280 nm was 36% of the weight of myosin employed. The molecular weight was 1.19×105 and the maximum ADP binding number was 0.48 mol using S-1 (CT) 30% contaminated by S-1 (CT)'. The pH-activity relationships in the presence of Ca2+ and EDTA were investigated. The ATPase activity in the presence of Ca2+ at pH 7.5 was 0.3 µmol mg–1 min–1 for both S-1 (CT) and S-1 (CT)'. The maximum ATPase activity of S-1 (CT) in the presence of F-actin was 8.3 µmol mg–1.min–1 and the affinity for F-actin was about 1/20 of that of skeletal S-1 (CT).
1This work was aided by grants from the Muscular Dystrophy Associations of America, Inc. and from the Ministry of Education, Science and Culture of Japan.