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Preparation of myosin subfragment 1 from pig cardiac muscle myosin by chymotryptic digestion


Journal of Biochemistry (Tokyo) 81(4): 977-988
Preparation of myosin subfragment 1 from pig cardiac muscle myosin by chymotryptic digestion
Subfragment-1 was prepared from pig cardiac myosin by chymotryptic digestion at low ionic strength at 0-2.degree. 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 2 components were not separated by Sephadex G-200 gel filtration, but they were separated on a DEAE-cellulose (DE 32) column or a 6-aminohexyl-PPi-Sepharose 4B conjugate column. S-1 (CT) consisted of 1 large polypeptide chain (the head portion of the f-chain) and 1 small polypeptide chain (g1). In SDS [sodium dodecyl sulfate] 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. 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 MW was 1.19 .times. 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 .mu.mol/mg per min 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 .mu.mol/mol per min and the affinity for F-actin was about 1/20 of that of skeletal S-1 (CT).


Accession: 006173548

PMID: 18456



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