Identification of photolabeled peptides for the acceptor substrate binding domain of beta 1,4-galactosyltransferase.
Identification of photolabeled peptides for the acceptor substrate binding domain of beta 1,4-galactosyltransferase.
复制标题
鉴定 β 1,4-半乳糖基转移酶受体底物结合域的光标记肽。
DOI：
10.1248/cpb.47.667
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发表时间：
1999
影响因子：
1.7
通讯作者：
Y. Hatanaka
中科院分区：
文献类型：
--
作者：
M. Hashimoto;Y. Hatanaka
我们成功地将带有生物素基团的卡宾生成 N-乙酰氨基葡萄糖衍生物应用于牛 UDP-半乳糖：N-乙酰氨基葡萄糖 β 1,4-半乳糖基转移酶（GalT，EC 2.4.1.38）催化结构域内肽的无放射性同位素鉴定。由于交联产率低，传统的光亲和标记实验在试图从非常复杂的混合物中分离标记组分时通常会遇到棘手的问题。我们的光亲和探针引入的生物素标签使我们能够将光标记的蛋白质与大量共存的未标记的 GalT 分离。生物素的引入对于基于高灵敏度化学发光技术的标记蛋白质的无放射性同位素检测也很有用。我们开发了一种新型聚偏二氟乙烯膜，用于在简单的斑点印迹测定中鉴定标记的肽。使用这种膜，我们成功地在许多 HPLC 分离的片段中鉴定出了生物素肽，这些片段源自光标记 GalT 蛋白的蛋白酶消化。序列分析显示生物素标签掺入 Y197-R208 的胰蛋白酶 GalT 片段内。我们的方法首次获得了该酶中受体底物结合位点片段的信息，这是使用其他方法很难获得的。这些数据与之前有关 GalT 受体位点的建议一致，清楚地证明了我们的方法快速鉴定光标记肽的有效性。
We successfully applied a carbene-generating N-acetylglucosamine derivative carrying a biotinyl group to the radioisotope-free identification of peptides within bovine UDP-galactose: N-acetylglucosamine beta 1,4-galactosyltransferase (GalT, EC 2.4.1.38) catalytic domain. Owing to the low yield of cross-linking, conventional photoaffinity labeling experiments usually encounter a thorny problem in attempting to isolate labeled components from very complex mixtures. A biotin tag introduced with our photoaffinity probe enabled us to separate the photolabeled protein from a large amount of coexisting unlabeled GalT. The introduction of biotin was also useful for the radioisotope-free detection of a labeled protein based on a highly sensitive chemiluminescent technique. We developed a novel poly(vinylidene difluoride) membrane for the identification of labeled peptides in a simple dot blot assay. Using this membrane, we successfully identified biotinyl peptides among a number of HPLC separated fragments derived from the protease digestion of photolabeled GalT proteins. The sequence analysis revealed that the biotin tag was incorporated within a tryptic GalT fragment of Y197-R208. Our approach yields, for the first time, information on the acceptor substrate binding-site fragment in this enzyme, that has been difficult to obtain using other approaches. These data are consistent with previous suggestions concerning the GalT acceptor site and clearly demonstrate the effectiveness of our approach for rapid identification of photolabeled peptides.