Maita T, Onishi H, Yajima E, Matsuda G
Department of Biochemistry, Nagasaki University School of Medicine.
J Biochem. 1987 Jul;102(1):133-45. doi: 10.1093/oxfordjournals.jbchem.a122025.
Chicken gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine (IAEDANS) in the presence of ATP and in 0.15 M KCl, where the myosin assumed 10S conformation. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodaltons) was isolated by gel filtration on a Sephadex G-100 column followed by chromatography on a CM 52 column. The amino acid sequence of the fragment was analyzed by conventional methods, and was: (S,Z)K-P-L-S-D-D-E-K-F-L-F-V-D-K-N-F-V-N-N-P-L-A-Q-A-D-W-S-A-K-K- L-V-W-V-P-S-E-K-H-G-F-E-A-A-S-I-K-E-E-K-G-D-E-V-T-V-E-L-Q-E-N-G-K-K- V-T-L-S-K-D-D-I-Q-K-M-N-P-P-K-F-S-K-V-E-D-M-A-E-L-T-C-L-N-E-A-S-V-L- H-N-L-R-E-R-Y-F-S-G-L-I-Y-T-Y-S-G-L-F-C-V-V-I-N-P-Y-K-Q-L-P-I-Y-S-E-K-I- I-D-M-Y-K-G-K-K-R-H-E-M-P-P-H-I-Y-A-I-A-D-T-A-Y-R-S-M-L-Q-D-R-E-D-Q- S-I-L-C-T-G-E-S-G-A-G-K-T-E-N-T-K-K-V-I-Q-Y-L-A-V-V-A-S-S-H-K-G-K. The amino-terminus was blocked, and the fragment was assigned as an amino-terminal part of the heavy chain of gizzard myosin. Position 127 was occupied by epsilon-N-trimethyllysine. Trp-130 of rabbit skeletal myosin heavy chain, which was reported to cross-link to an azide derivative of ATP by Okamoto and Yount (Proc. Natl. Acad. Sci. U.S. 82, 1575-1579 (1985], was replaced by glutamine in gizzard myosin. Cys-93 of the fragment is the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin (Onishi, H. (1985) J. Biochem. 98, 81-86).
在ATP存在且处于0.15M KCl条件下，鸡胗肌球蛋白在10S构象时用N - 碘乙酰 - N' -（5 - 磺基 - 1 - 萘基） - 乙二胺（IAEDANS）进行修饰。从修饰后的肌球蛋白的胰蛋白酶消化产物中，通过在Sephadex G - 100柱上进行凝胶过滤，随后在CM 52柱上进行色谱分离，分离出一个荧光片段（24千道尔顿）。通过常规方法分析该片段的氨基酸序列，结果如下：（S，Z）K - P - L - S - D - D - E - K - F - L - F - V - D - K - N - F - V - N - N - P - L - A - Q - A - D - W - S - A - K - K - L - V - W - V - P - S - E - K - H - G - F - E - A - A - S - I - K - E - E - K - G - D - E - V - T - V - E - L - Q - E - N - G - K - K - V - T - L - S - K - D - D - I - Q - K - M - N - P - P - K - F - S - K - V - E - D - M - A - E - L - T - C - L - N - E - A - S - V - L - H - N - L - R - E - R - Y - F - S - G - L - I - Y - T - Y - S - G - L - F - C - V - V - I - N - P - Y - K - Q - L - P - I - Y - S - E - K - I - I - D - M - Y - K - G - K - K - R - H - E - M - P - P - H - I - Y - A - I - A - D - T - A - Y - R - S - M - L - Q - D - R - E - D - Q - S - I - L - C - T - G - E - S - G - A - G - K - T - E - N - T - K - K - V - I - Q - Y - L - A - V - V - A - S - S - H - K - G - K。氨基末端被封闭，该片段被确定为鸡胗肌球蛋白重链的氨基末端部分。第127位被ε - N - 三甲基赖氨酸占据。据冈本和扬特（《美国国家科学院院刊》82，1575 - 1579（1985））报道，兔骨骼肌肌球蛋白重链的色氨酸 - 130与ATP的叠氮衍生物交联，在鸡胗肌球蛋白中该位点被谷氨酰胺取代。该片段的半胱氨酸 - 93是其与IAEDANS反应会改变鸡胗肌球蛋白ATP酶活性的氨基酸残基（大西，H.（1985）《生物化学杂志》98，81 - 86）。
