Transglutaminase-catalyzed insertion of a fluorescent probe into the protease-sensitive region of rhodopsin.
Transglutaminase-catalyzed insertion of a fluorescent probe into the protease-sensitive region of rhodopsin.
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转谷氨酰胺酶催化荧光探针插入视紫红质的蛋白酶敏感区域。
DOI：
10.1021/bi00604a021
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发表时间：
1978
期刊：
影响因子：
2.9
通讯作者：
L. Stryer
中科院分区：
文献类型：
--
作者：
J. Pober;V. Iwanij;E. Reich;L. Stryer
豚鼠肝脏中的转氨酶将丹磺尸胺和腐胺插入视网膜盘膜中的牛视紫红质中。标记的化学计量是每个视紫红质分子一个胺。标记腐胺防止随后插入丹磺尸胺，这表明相同的谷氨酰胺残基标记这些胺。标记位点的确定利用了这样一个事实，即盘膜中的视紫红质被不同特异性的各种蛋白水解酶切割成两个片段，称为FI和F2 [Pober，JS，and Stryer，L.等（1975），J. Mol. 95，全部]。三条证据表明，转氨酶标记的位点位于FI和F2片段之间的蛋白酶敏感区。首先，先前的酶促蛋白水解抑制了转氨酶催化的标记。第二，先前的转氨酶催化标记抑制酶促蛋白水解。第三，在枯草杆菌蛋白酶水解标记视紫红质的初始阶段，FI显示丹磺酰荧光。来自豚鼠肝脏的转氨酶催化伯胺插入肽和蛋白质中的N-乙酰氨基残基（Clarke et al.，1959; Folk和Chung，1973; Connellan等人，1971年）。这种酶已被用于标记膜蛋白，并且在不存在外源胺的情况下，催化它们之间的α-（γ-谷氨酰基）-赖氨酰交联的形成（Brewer和Singer，1974;达顿和Singer，1975;达顿等人，1976; Iwanij，1977）。标记反应的特异性和反应条件的温和性促使我们探索使用转氨酶将光谱探针插入完整盘膜中的视紫红质。所用探针在不存在转氨酶的情况下不反应。此外，只有蛋白质可以被标记，因为这种酶需要谷氨酰胺侧链作为其底物之一。只有残基的视紫红质位于
Transglutaminasefrom guinea pig liver inserts dansylcadaverine and putrescine into bovine rhodopsin in retinal disk membranes. The stoichiometry of labeling is one amine per rhodopsin molecule. Labeling with putrescine prevents the subsequent insertion of dansylcadaverine, suggesting that the same glutamine residue is labeled by these amines. The site of labeling was determined by taking advantage of the fact that rhodopsin in disk membranes is cleavedby a variety of proteolytic enzymes of differingspecificity into two fragments, called FI and F2 [Pober, JS, and Stryer, L.(1975), J. Mol. Biol. 95, All]. Three lines of evidence indicate that the site labeled by transglutaminase is located in the protease-sensitive region between the FI and F2 fragments. First, prior enzymatic proteolysis inhibits transglutaminase-catalyzed labeling. Second, prior transglutaminase-catalyzed labeling inhibits enzymatic proteolysis. Third, dansyl fluorescence is exhibited by FI during the initial stage of proteolysis of labeled rhodopsin by subtilisin. The dansyl label is then excised from FI with littleTransglutaminase from guinea pig liver catalyzes the in-sertion of primary amines into glutaminyl residues in peptides and proteins (Clarke et al., 1959; Folk and Chung, 1973; Connellan et al., 1971). This enzyme has been used tolabel membrane proteins and, in theabsence of exogenous amines, to catalyze the formation of€-(y-glutamyl)-lysyl cross-links between them (Brewer and Singer, 1974; Dutton and Singer, 1975; Dutton et al., 1976; Iwanij, 1977). The specificity of the labeling reaction and the mildness of the reaction conditions stimulated us to explore the use of transglutaminase to insert spectroscopic probes into rhodopsin in the intact disk mem-brane. The probes used are not reactive in the absence of transglutaminase. Furthermore, only proteins can be labeled because this enzyme requires a glutamine side chain as one of its substrates. Only residues of rhodopsin located in the