Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head.
We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D008970:Molecular Weight; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014357:Trypsin
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D014357:Trypsin; D009218:Myosins