Photoaffinity labeling of skeletal myosin with 2-azidoadenosine triphosphate.
Photoaffinity labeling of skeletal myosin with 2-azidoadenosine triphosphate.
复制标题
用 2-叠氮腺苷三磷酸对骨骼肌球蛋白进行光亲和标记。
DOI：
10.1021/bi00073a001
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Yount,RG
中科院分区：
文献类型：
--
作者：
Grammer,JC;Kuwayama,H;Yount,RG
1993年3月23日接收的修订版Mandarin pt摘要：用2-叠氮腺苷二磷酸（2-N3 ADP）对骨骼肌肌球蛋白上ATP的嘌呤结合位点进行了光亲和标记。2-N_3ADP通过两个重链活性巯基（Cys-697和Cys-707）与Co（III）菲咯啉的络合作用稳定地捕获在活性位点（r1/2~ 5天，0 ℃）。光掺入仅发生在重链的23-kDa NH_2-末端胰蛋白酶片段。用胰蛋白酶和枯草杆菌蛋白酶对光标记的肌球蛋白亚片段1进行广泛的连续消化，得到一系列标记肽，这些标记肽经HPLC纯化。序列和放射性标记分析的8个光标记的肽都表明，色氨酸-130是唯一的标记残基。该标记位点证实了用非核苷酸ADP类似物2-[（4-叠氮基-2-硝基苯基）-氨基]乙基二磷酸（NANP）的早期光标记研究，其也标记了Trp-130 [Okamoto，Y.，& Yount，R. G.等人（1985）Proc. Acad. Sci. USA 82，1575-1579]。2-N3 ADP和NANDP的结构的比较表明，它们的叠氮基可以叠加，如果这两个类似物结合到活性位点的扩展构象的方式类似于ATP的反构象。
Revised Manuscript Received March 23, 1993 abstract: The purine binding site of ATP on skeletal muscle myosin has been photoaffinity labeled with 2-azidoadenosine diphosphate (2-N3ADP). 2-N3ADP was stably trapped at the active site (r 1/2~ 5 days, 0 C) by complexation of the two heavy chain reactive thiols (Cys-697 and Cys-707) with Co (III) phenan-throline. Photoincorporation occurred only in the 23-kDa NH2-terminal tryptic fragment of theheavy chain. Extensive serial digestion of photolabeled subfragment 1 of myosin by trypsin and subtilisin yielded a series of labeled peptides which were purified by HPLC. Sequence and radiolabeling analysis of eight photolabeled peptides all indicated that tryptophan-130 was the only labeled residue. This site of labeling confirms earlier photolabeling studies with the non-nucleotide ADP analogue, 2-[(4-azido-2-nitrophenyl)-amino] ethyl diphosphate (NANDP), which also labeled Trp-130 [Okamoto, Y., & Yount, R. G.(1985) Proc. Natl. Acad. Sci. USA 82, 1575-1579]. Comparison of the structures of 2-N3ADP and NANDP indicate that their azido groups can be superimposed if both analogues bind to the active site in an extended conformation in a manner analogous to the anti conformation of ATP.