Sutoh K, Lu R C
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Biochemistry. 1987 Jul 14;26(14):4511-6. doi: 10.1021/bi00388a051.
The thiol-specific photoactivatable reagent 4-(2-iodoacetamido)benzophenone (BPIA) can be selectively incorporated into the SH-1 of myosin subfragment 1 (S1), and upon photolysis an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa or with the central 50-kDa region [Lu, R. C., Moo, L., & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396]. Heavy chains with these two types of intramolecular cross-links and un-cross-linked heavy chain have different mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels and therefore can be purified electrophoretically. Each type of heavy chain was cleaved with Staphylococcus aureus protease, chymotrypsin, or lysyl endopeptidase. The cleavage points were determined on the basis of the molecular weights of weights of peptides containing the N-terminus, which was identified with the use of an antibody. Locations of the cross-links were deduced by comparing the peptide maps of cross-linked and un-cross-linked heavy chains. The results indicate that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide, whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide. With use of the avidin-biotin system, it has been shown that SH-1 is located 13 nm from the head/rod junction [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1984) J. Mol. Biol. 178, 323-339]. Since BPIA spans less than 1 nm, our results show that two regions, separated by approximately 400 amino acid residues and located in the 25- and 50-kDa domains of S1, respectively, are also part of the head structure about 12-14 nm from the head/rod junction.
巯基特异性光活化试剂4-（2-碘乙酰胺基）二苯甲酮（BPIA）可选择性地掺入肌球蛋白亚片段1（S1）的SH-1中，光解后，在SH-1与S1的N端25 kDa区域之间形成分子内交联。如果在光解过程中存在Mg2+核苷酸，则交联可与25 kDa区域或中央50 kDa区域形成[Lu, R. C., Moo, L., & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392 - 6396]。具有这两种分子内交联的重链和未交联的重链在十二烷基硫酸钠（NaDodSO4）-聚丙烯酰胺凝胶上具有不同的迁移率，因此可以通过电泳进行纯化。每种类型的重链都用金黄色葡萄球菌蛋白酶、胰凝乳蛋白酶或赖氨酰内肽酶进行切割。根据含有N端的肽段的分子量确定切割点，该N端用抗体进行鉴定。通过比较交联和未交联重链的肽图来推断交联的位置。结果表明，重链N端约12 - 16 kDa处的片段可通过BPIA与SH-1交联，与核苷酸的存在无关，而N端57 - 60 kDa处的片段仅在存在Mg2+核苷酸时可与SH-1交联。利用抗生物素蛋白-生物素系统已表明，SH-1位于头部/杆部交界处13 nm处[Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1984) J. Mol. Biol. 178, 323 - 339]。由于BPIA跨度小于1 nm，我们的结果表明，分别位于S1的25 kDa和50 kDa结构域中、相隔约400个氨基酸残基的两个区域，也是距头部/杆部交界处约12 - 14 nm的头部结构的一部分。
