Transmission of ADP.vanadate-induced conformational changes to three peptide segments of myosin subfragment-1.
In order to study the conformational changes associated with formation of the stable ternary complex of myosin subfragment-1 (S-1) with ADP and orthovanadate (Vi), S-1 was fluorescently labeled with 9-anthroylnitrile, 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole, and 5-(iodoacetamido) fluorescein at the 23-, 50-, and 20-kDa peptide segments of S-1, respectively (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188-18194; Hiratsuka, T. (1986) J. Biol. Chem. 261, 7294-7299; Takashi, R. (1979) Biochemistry 18, 5164-5169). The extrinsic fluorescence of these S-1 derivatives was sensitive not only to binding of ADP but to formation of the stable ternary complex with ADP and Vi. By using these fluorescent properties, the kinetics of formation of the stable ternary complexes of these S-1 derivatives with ADP and Vi, M. ADP.Vi, were analyzed according to the scheme proposed by Goodno (Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2620-2624). [Formula; see text] The values obtained for KVi )0.2-0.4 mM) and k (0.03-0.05 s-1) of these S-1 derivatives were similar regardless of the peptide segments of S-1 where the fluorophore had been covalently labeled. These results suggest that the conformational changes, which are induced by formation of the stable ternary complex of S-1 with ADP and Vi, are transmitted to all three peptide segments of S-1 at a similar rate. The present results also encourage us to confirm that the ATPase site of S-1 resides at or near the region where all three peptide segments of S-1 are contiguous.
Hiratsuka
T
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Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014638:Vanadates / Q000378:metabolism* / Q000494:pharmacology
D000873:Anthracenes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010446:Peptide Fragments; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D014638:Vanadates; D000244:Adenosine Diphosphate; C015296:5-iodoacetamidofluorescein; C066291:9-anthroylnitrile; D009327:4-Chloro-7-nitrobenzofurazan