Specific labelling of the hydrophobic domain of rat renal gamma-glutamyltransferase.
Specific labelling of the hydrophobic domain of rat renal gamma-glutamyltransferase.
复制标题
大鼠肾γ-谷氨酰转移酶疏水结构域的特异性标记。
DOI：
10.1002/9780470720769.ch6
复制
发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Curthoys,NP
中科院分区：
文献类型：
--
作者：
Frielle,T;Curthoys,NP
两亲形式的γ-谷氨酰转移酶（EC 2.3. 2.2)重构为单层卵磷脂囊泡，并使用膜可溶性试剂3-三氟甲基-3-（m-[125 I]碘苯基）二氮杂环丙烷（[125 I] TID）标记，其可以被光活化。标签被专门纳入大亚基的转移酶，但没有标签被发现在从囊泡中释放的酶与木瓜蛋白酶的部分蛋白水解。木瓜蛋白酶处理的囊泡在Sephadex LH-60上的层析表明，[125 I] TID结合到两个低相对分子质量的肽。[125 I]-TID标记的肽应构成疏水性膜结合结构域。转移酶也标记还原甲基化（赖氨酸残基）或固定化半乳糖氧化酶和NaB 3 H4（半乳糖残基），纳入卵磷脂囊泡和木瓜蛋白酶处理。这两个程序产生了一个单一的[3 H]-标记的疏水肽，仍然与囊泡。在Sephadex LH-60上层析后，后两种方法标记的肽对应于两种[125 I] TID标记肽中较大的肽。这些结果表明，木瓜蛋白酶可以切割成两个肽的疏水结构域。观察到的标记也与N-末端疏水结构域之前是含有赖氨酸和碳水化合物残基的初始序列的替代可能性一致。两个[125 I] TID标记的肽中较小的肽可以通过从转移酶分子中去除亲水性片段来产生，所述转移酶分子用暴露在囊泡外表面上的N-末端重建。
The amphipathic form of gamma-glutamyltransferase (EC 2.3. 2.2) was reconstituted into unilamellar lecithin vesicles and labelled using the membrane-soluble reagent, 3-trifluoromethyl-3-(m-[125I] iodophenyl) diazirine ([125I] TID), which can be photoactivated. Label was incorporated exclusively into the large subunit of the transferase, but no label was found in the enzyme released from the vesicles by partial proteolysis with papain. Chromatography of the papain-treated vesicles on Sephadex LH-60 indicated that the [125I] TID was bound to two peptides of low relative molecular mass. The [125I]-TID-labelled peptides should constitute the hydrophobic membrane-binding domain. The transferase was also labelled by reductive methylation (Lys residues) or with immobilized galactose oxidase and NaB3H4 (Gal residues), incorporated into lecithin vesicles and treated with papain. Both procedures yielded a single [3H]-labelled hydrophobic peptide that remained associated with the vesicles. After chromatography on Sephadex LH-60, the peptide labelled by the latter two procedures corresponds to the larger of the two [125I] TID-labelled peptides. These results suggest that papain may cleave the hydrophobic domain into two peptides. The observed labelling is also consistent with the alternative possibility that the N-terminal hydrophobic domain is preceded by an initial sequence that contains both lysine and carbohydrate residues. The smaller of the two [125I] TID-labelled peptides could be generated by removal of the hydrophilic segment from the transferase molecules that are reconstituted with the N-terminus exposed on the external surface of the vesicles.