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胚胎鸡肝脏中一种诱导型葡萄糖醛酸转移酶的新检测方法及细胞定位

A new assay and cellular localization for an inducible glucuronyltransferase in the embryonic chick liver.

作者信息

Roth S, Levine J B

出版信息

Exp Cell Res. 1983 Jan;143(1):217-25. doi: 10.1016/0014-4827(83)90122-2.

Abstract

A direct, radioisotopic assay is described for the uridine diphosphate glucuronic acid (UDPGA): p-aminophenol glucuronyltransferase. The assay uses solid phase p-aminophenol-Sephadex as the glucuronyl acceptor and UDP-[14C]GA as the glucuronyl donor. After incubation with the enzyme, the derivatized Sephadex beads are washed in SDS-urea or with high salt concentrations to remove all labeled material except for that covalently attached to the beads. Sonicated livers from chick embryos exposed to phenobarbital for at least 5 days transfer more than ten times the glucuronic acid to derivatized beads than do uninduced livers of the same developmental age. Glucuronyl-transferase activity can be detected on intact, living cells after 5 days of phenobarbital induction, whereas sonicate activity is detectable within 3 days of induction. Suspensions of living cells can show 25% the activity found in the same suspension after all the cells are lysed by sonication.

摘要

本文描述了一种用于尿苷二磷酸葡萄糖醛酸(UDPGA):对氨基苯酚葡萄糖醛酸转移酶的直接放射性同位素测定法。该测定法使用固相对氨基苯酚 - 葡聚糖凝胶作为葡萄糖醛酸受体,UDP - [14C]GA作为葡萄糖醛酸供体。与酶孵育后,用SDS - 尿素或高盐浓度洗涤衍生化的葡聚糖凝胶珠,以除去所有标记物质,除了共价连接到珠子上的物质。暴露于苯巴比妥至少5天的鸡胚超声破碎肝脏比相同发育年龄的未诱导肝脏将多十倍以上的葡萄糖醛酸转移到衍生化珠子上。苯巴比妥诱导5天后,完整活细胞上可检测到葡萄糖醛酸转移酶活性,而超声破碎物活性在诱导3天内可检测到。活细胞悬液在所有细胞通过超声破碎裂解后,其活性仅为相同悬液中活性的25%。

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