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Zusammenfassung:
A series of thiol-specific cross-linking reagents were prepared for studying the kinetics of cross-linking between SH1 (Cys707) and SH2 (Cys697) in rabbit skeletal muscle myosin subfragment 1. The reagents were of the type RSS(CH2)nSSR, with R = 3-carboxy-4-nitrophenyl and n = 3, 6, 7, 8, 9, 10, and 12, spanning distances from 9 to 20 ?. The reactions were monitored spectrophotometrically by measuring the release of 2-nitro-5-thiobenzoate. Reaction rates for modification of SH1 (k1) and for cross-linking (k2) were measured by the decrease of the K+(EDTA)-ATPase activity and the decrease of the Ca2+-ATPase activity, respectively, and corrected for the different reactivities of Cn. Cross-linking rates in the presence and absence of MgADP showed similar dependence on the length of the reagents: While the cross-linking rates for n = 3 or n = 6 were close to those for n = 0 (Ellmans reagent), those for n = 7 and 8 were significantly increased. Thus the distance between SH1 and SH2 appears to be equal in both states and can be estimated as 15 ?, based on the length of the reagent with n = 8 in stretched conformation. Under rigor conditions, reactivity of SH1 differed significantly from that in the presence of MgADP, presumably because of shielding through a lipophilic domain. Similarly, the cross-linking rates k2 for C3, C6, and C7 in the absence of MgADP were ca. 15 times lower than in the presence of MgADP, suggesting a change in the structure of the SH2 region that depends on nucleotide binding. The results are discussed in terms of recent X-ray structures of S1 and S1-MgADP [Rayment et al. (1993) Science 261, 50-58; Gulick et al. (1997) Biochemistry 36, 11619-11628]
