Identification of an active site peptide of skeletal myosin after photoaffinity labeling with N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate.
Identification of an active site peptide of skeletal myosin after photoaffinity labeling with N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate.
复制标题
用 N-(4-叠氮基-2-硝基苯基)-2-氨乙基二磷酸进行光亲和标记后，鉴定骨骼肌球蛋白的活性位点肽。
DOI：
10.1073/pnas.82.6.1575
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发表时间：
1985
影响因子：
11.1
通讯作者：
Yount,RG
中科院分区：
文献类型：
--
作者：
Okamoto,Y;Yount,RG
骨肌蛋白的活性位点被ADP类似物N-（4-氮杂基-2-硝基苯基）-2-氨基乙基三磷酸（NANDP）光亲和标记（大约等于50%），遵循Wells和young的钴菲罗啉活性位点捕获程序[Wells， J. A. & young， R. G. (1979) Proc. Natl。学会科学。[j]。对[3H] nandp标记的肌球蛋白亚片段1进行广泛的蛋白水解消化，得到两个主要肽，P1和P2，通过反相高效液相色谱纯化。这些肽代表了所有标记氨基酸的50%，并含有1 mol不寻常的氨基酸ε - n-三甲基赖氨酸。通过Edman技术分析P2得到Val-Asn-Pro-Tyr-Lys(Me3)-X-Leu-Pro-Val-Tyr序列，该序列与Tong和Elzinga测定的残基125-134序列一致[Tong， S. W. & Elzinga， M. (1983) J. Biol]。兔骨肌球蛋白重链中X为Trp-130的片段。化学，258,13100-13110。P1与P2完全相同，除了在cooh末端含有额外的三个氨基酸，Asn-Pro-Gln。氨基酸组成、序列数据、光谱测量以及P1和P2中放射性标记的位置均表明，Trp-130是NANDP标记的主要位点。邻近的epsilon- n-三甲基赖氨酸可以为ATP的三磷酸部分提供部分结合位点。
The active site of skeletal myosin has been photoaffinity labeled (approximately equal to 50%) by the ADP analog N-(4-azido-2-nitrophenyl)-2-aminoethyl triphosphate (NANDP) following the cobalt phenanthroline active site trapping procedure of Wells and Yount [Wells, J. A. & Yount, R. G. (1979) Proc. Natl. Acad. Sci. USA 76, 4966-4970]. Extensive proteolytic digestion of [3H]NANDP-labeled myosin subfragment one yielded two major peptides, P1 and P2, which were purified by reversed-phase high-performance liquid chromatography. These peptides represented 50% of all labeled amino acids and contained 1 mol of the unusual amino acid epsilon-N-trimethyllysine. Analysis of P2 by Edman techniques gave a sequence Val-Asn-Pro-Tyr-Lys(Me3)-X-Leu-Pro-Val-Tyr, which corresponds to an identical sequence for residues 125-134 determined by Tong and Elzinga [Tong, S. W. & Elzinga, M. (1983) J. Biol. Chem. 258, 13100-13110] for a segment of rabbit skeletal myosin heavy chain in which X is Trp-130. P1 was identical to P2 except it contained an additional three amino acids, Asn-Pro-Gln, at the COOH-terminal end. Amino acid composition, sequence data, spectral measurements, and location of radioactive label in both P1 and P2 all indicate Trp-130 is the major site of labeling by NANDP. The adjacent epsilon-N-trimethyllysine may provide part of the binding site for the triphosphate portion of ATP.