Photoaffinity labeling of scallop myosin with 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate: identification of an active site arginine analogous to tryptophan-130 in skeletal muscle myosin.
Photoaffinity labeling of scallop myosin with 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate: identification of an active site arginine analogous to tryptophan-130 in skeletal muscle myosin.
复制标题
用 2-[(4-叠氮基-2-硝基苯基)氨基]乙基二磷酸对扇贝肌球蛋白进行光亲和标记：鉴定骨骼肌肌球蛋白中类似于色氨酸 130 的精氨酸活性位点。
DOI：
10.1021/bc00016a012
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发表时间：
1992
影响因子：
4.7
通讯作者：
Yount,RG
中科院分区：
文献类型：
--
作者：
Kerwin,BA;Yount,RG
用ADP光亲和类似物2-[(4-叠氮-2-硝基苯基)氨基]乙基二磷酸(NANDP)标记扇贝肌球蛋白的ATP结合部位。通过与钒和锰的络合，每摩尔肌球蛋白约有1摩尔的NANDP被捕获在活性部位。ADP而不是AMP抑制NANDP的捕获。被捕获的NANDP在紫外光照射下可以标记高达37%的肌球蛋白。用胰酶消化光标记肌球蛋白制备的Papain1亚片段，用反相高效液相色谱分离得到主要的光标记胰蛋白酶多肽。主标记肽的氨基酸序列为X-Leu-Pro-Ile-Tyr-Thr-Asp-Ser-Val-Ile-Ala-Lys，，其中X代表光标记氨基酸Arg128。以前，兔骨骼肌肌球蛋白的Trp130已经被证明是由NANDP[Okamoto，Y.，and Young t，RG(1985)Proc]光标记的。娜塔莉。阿卡德。SCI。美国82,1575-1580]。扇贝和兔骨骼肌肌球蛋白在该区域显示出高度的序列相似性，与Arg128在Trp130的相同位置。这些结果表明，两种肌球蛋白的嘌呤结合部位的组成是相似的，Arg和Trp在结合ATP方面发挥着相似的作用，尽管它们的侧链有显著的差异。
The ADP photoaffinity analogue 2-[(4-azido-2-nitrophenyl) amino] ethyl diphosphate (NANDP) was used tophotolabel the ATP binding site of scallop myosin. Approximately 1 mol of NANDP per mol of myosin was trapped at the active site by complexation with vanadate andmanganese. ADP, but not AMP, inhibited trapping of NANDP. The trapped NANDP photolabeled up to 37% of the myosin upon UV irradiatioin. Papainsubfragment-1 prepared from the photolabeled myosin was digested with trypsin, and the major photolabeled tryptic peptides were isolated by reversed-phase HPLC. The amino acidsequence of the major labeled peptide was X-Leu-Pro-Ile-Tyr-Thr-Asp-Ser-Val-Ile-Ala-Lys, where X represents the photolabeled amino acid Arg128. Previously, Trp130 of rabbit skeletal muscle myosin has been shown to be photolabeled by NANDP [Okamoto, Y., and Yount, RG (1985) Proc. Natl. Acad. Sci. USA 82, 1575-1580]. Scallop and rabbit skeletal muscle myosin display a high degree of sequence similarity in this region with Arg128 in an equivalent position as Trp130. These results suggest that the composition of the purine binding site is analogous in both myosins and that Arg and Trp play a similar role in binding ATP, despite the marked differences of their side chains.