Lu R C, Wong A
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Biochemistry. 1989 May 30;28(11):4826-9. doi: 10.1021/bi00437a046.
The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin 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 region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates 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 [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.
巯基特异性光活化试剂二苯甲酮碘乙酰胺（BPIA）可选择性地掺入肌球蛋白S1最具反应性的巯基SH-1中，光解后，在SH-1和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等人（1986年）美国国家科学院院刊83, 6392]。交联和未交联的S1重链肽图的比较表明，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，而重链N端57-60 kDa处的片段仅在存在Mg2+核苷酸时可与SH-1交联[Sutoh和Lu（1987年）生物化学26, 4511]。在本报告中，S1用放射性BPIA标记，在无核苷酸的情况下光解，然后用蛋白水解酶降解。含有交联的肽通过液相色谱分离并进行氨基酸序列分析。结果表明，Glu-88是通过BPIA与SH-1（Cys-707）交联的主要位点，Asp-89和Met-92是次要位点。这些残基非常靠近反应性赖氨酸残基（Lys-83），但在一级结构中与假定的核苷酸结合区域相对较远。
