Cross-linking of three heavy-chain domains of myosin adenosinetriphosphatase with a trifunctional alkylating reagent.

The chemotherapeutic alkylating reagent tris(2-chloroethyl)amine (TCEA) was used as a trifunctional cross-linking reagent with a cross-linking span of 5 A for myosin subfragment 1 (S-1). When S-1 was incubated with TCEA, all three domains of 20, 26, and 50 kDa in the S-1 heavy chain were cross-linked via the highly reactive sulfhydryl group SH1 (Cys-707) on the 20-kDa domain. The cross-linking was accelerated by nucleotides. The present observation is consistent with the proposal that SH1 is close to both the 26- and 50-kDa domains of S-1 and that movement within S-1 associated with the nucleotide binding occurs around SH1 as well as around another reactive thiol, SH2 & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396; Hiratsuka, T. (1987) Biochemistry 26, 3168-3173].