Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1.
Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1.
复制标题
肌球蛋白亚片段 1 蛋白水解敏感区域中核苷酸诱导的变化。
DOI：
10.1021/bi00315a038
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
Reisler,E
中科院分区：
文献类型：
--
作者：
Applegate,D;Reisler,E
(Sl)用弹性蛋白酶、枯草杆菌蛋白酶、木瓜蛋白酶和嗜热菌蛋白酶产生的片段在1-2K道尔顿内对应于胰蛋白酶产生的25 K、50 K和20 K片段。木瓜蛋白酶和嗜热菌蛋白酶优先切割26 K/70 K交界处，而弹性蛋白酶和枯草杆菌蛋白酶切割S1中的26 K/70 K和75 K/22 K交界处。使用上述蛋白酶作为构象探针，我们先前已经证明，肌动蛋白的结合在26 K/50 K和50 K/22 K连接处都被感知[Applegate，D.，& Reisler，E.等人（1983）Proc. Acad. Sci. USA 80，7109-7112]，我们在此报道，在活性位点处的核苷酸的结合也在两个连接处被感测。2 mM MgADP和5 mM MgATP均减慢95 K重链的弹性蛋白酶和枯草杆菌蛋白酶切割速率。对于弹性蛋白酶，在26 K/50 K和50 K/22 K连接处证明了核苷酸诱导的切割速率降低3倍。对枯草杆菌蛋白酶的分析是复杂的Mg大量有据可查的证据表明，ATP和核苷酸类似物与肌球蛋白亚片段1（S1）1的结合诱导了这种蛋白质的构象变化。核苷酸诱导的光谱探针在不同位点的扰动和蛋白质上某些残基的反应性的变化表明在S1的宽区域上感测到结合（Morales等人，1982年）。仅举几个例子，Mg核苷酸诱导Sl的内在色氨酸荧光的变化（Werber et al.，1972），在SH 1和SH 2硫醇基团对烷基化的反应性中（Watterson & Schaub，1973; Reisler等人，1977）、与反应性赖氨酰残基连接的三硝基苯基部分的吸收光谱（Muhlrad，1977）和轻链的光谱性质（Marsh等，1982年）。最新增加的光学和化学工具的SI分析是有限的胰蛋白酶消化这种蛋白质。胰蛋白酶切割S1的95 K重链以产生在非变性条件下保持缔合的三个离散片段（25 K、50 K、20 K）。各种证据表明25 K肽含有活性位点（Szilagyi等人，一九七九年; Okamoto& Yount，1983），而两个单独的肌动蛋白结合位点位于另外两个肽上，一个在20 K肽上，另一个在50 K肽上（Mornet等人，1981; Yamamoto & Sekine，1979 a; Sutoh，1983）。在50 K/20 K交界处的胰蛋白酶攻击被肌动蛋白减慢（Mornet等人，一九七九年; Yamamoto和Sekine，1979 b），并且在25 K和50 K片段之间的连接处检测到核苷酸的结合（Hozumi和Muhlrad，1981; Muhlrad和Hozumi，1982）。最近，我们开始使用蛋白酶而不是胰蛋白酶来进一步研究S-1的亚结构。我们已经证明
(Sl) with elastase, subtilisin, papain, and thermolysin yield fragments that correspond within 1-2K daltons to the 25K, 50K, and 20K fragments produced by trypsin. While papain and thermolysin cut preferentially at the 26K/70K junction, elastase and subtilisin cleave both the 26K/70K and the 75K/22K junctions in Sl. Using the above proteases as conformational probes, we have previously dem-onstrated that the binding of actin is sensed at both the 26K/50K and the 50K/22K junctions [Applegate, D., & Reisler, E.(1983) Proc. Natl. Acad. Sci. USA 80, 7109-7112], We report here that the binding of nucleotides at the active site is also sensed at both junctions. Both 2 mM MgADP and 5 mM MgATP slow the rate of elastase and subtilisin cleavage of the 95K heavy chain. With elastase, the 3-fold decrease in the rate of cleavage induced by nucleotides is evidenced at both the 26K/50K and the 50K/22K junctions. The analysis of subtilisindigestions is complicated by Mg large body of well-documented evidence suggests that the binding ofATP and nucleotide analogues to myosin sub-fragment 1 (Sl) 1 induces conformational changes in this protein. Nucleotide-induced perturbations of spectroscopic probes at various sites and changes in the reactivity of certain residues on the protein indicate that the binding is sensed over broad regions of Sl (Morales et al., 1982). To mention just a few examples, Mg nucleotides induce changes in the intrinsic tryptophan fluorescence of Sl (Werber et al., 1972), in the reactivities of SHj and SH2 thiol groups to alkylation (Watterson & Schaub, 1973; Reisler et al., 1977), in the absorption spectrum of the trinitrophenyl moietyattached to the reactive lysyl residue (Muhlrad, 1977), and in the spectral properties of light chains (Marsh et al., 1982). The most recent addition to the optical and chemical tools of Sl analysis is the limited tryptic digestion of this protein. Trypsin cleaves the 95K heavy chain of Sl to produce three discrete fragments (25K, 50K, 20K) that remain associated under nondenaturing conditions. Various lines of evidence suggest that the 25K peptide contains the active site (Szilagyi et al., 1979; Okamoto& Yount, 1983), whereas two separate actinbinding sites are located on the other two peptides, one on the 20K peptide and the other on the 50K peptide (Mornet et al., 1981; Yamamoto & Sekine, 1979a; Sutoh, 1983). Tryptic attack at the 50K/20K junction is slowed byactin (Mornet et al., 1979; Yamamoto & Sekine, 1979b), and the binding of nucleotides is sensed at the junction between the 25K and 50K fragments (Hozumi & Muhlrad, 1981; Muhlrad & Hozumi, 1982). Recently, we initiated the use of proteases other than trypsin to further studythe substructure of S-1. We have shown that