Purification of a phenobarbital-inducible morphine UDP-glucuronyltransferase isoform, absent from Gunn rat liver.
Purification of a phenobarbital-inducible morphine UDP-glucuronyltransferase isoform, absent from Gunn rat liver.
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Gunn 大鼠肝脏中不存在的苯巴比妥诱导吗啡 UDP-葡萄糖醛酸转移酶亚型的纯化。
DOI：
10.1006/abbi.1994.1510
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发表时间：
1994
影响因子：
3.9
通讯作者：
Kazuta Oguri
中科院分区：
文献类型：
--
作者：
Yuji Ishii;K. Tsuruda;Mitsuko Tanaka;Kazuta Oguri
从用苯巴比妥治疗的 Sprague-Dawley 大鼠的肝微粒体中纯化吗啡 UDP-葡萄糖醛酸转移酶（吗啡 UGTPB）。肝微粒体中的 UDP-葡萄糖醛酸转移酶用 Emulgen 911 溶解，并通过我们实验室开发的 omega-(β-羧基丙酰基-氨基)辛基 Sepharose 4B 柱色谱分离。吗啡 UDP-葡萄糖醛酸转移酶被洗脱成两个组分：峰 I 和峰 II，它们具有不同的底物特异性。吗啡 UGTPB 通过对吗啡更具特异性的 Peak II 进行两次色谱聚焦纯化。纯化的吗啡 UGTPB 的色谱聚焦表观等电点为 8.0，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后显示亚基分子量为 55 kDa。纯化的酶催化吗啡的3-羟基和少量的4-羟基联苯的葡萄糖醛酸化，但不催化雄酮、胆红素、氯霉素、可待因、4-甲基伞形酮、4-硝基苯酚、睾酮和吗啡的6-羟基的葡萄糖醛酸化。吗啡UGTPB的N端氨基酸序列与纯合Gunn大鼠缺陷的UGT1*01P的N端氨基酸序列相同。用苯巴比妥处理的 Gunn 大鼠肝微粒体的 omega-(β-羧基丙酰氨基)辛基 Sepharose 4B 柱色谱中不存在峰 II，而峰 I 中的吗啡 UGT 在 Gunn 大鼠中是 PB 诱导的。目前的结果表明，吗啡 UDP-葡萄糖醛酸转移酶的同工型属于 UGT1 家族，并且是苯巴比妥诱导型。
A morphine UDP-glucuronyltransferase (morphine UGTPB) was purified from liver microsomes of Sprague-Dawley rats treated with phenobarbital. UDP-glucuronyl-transferases in the liver microsomes were solubilized with Emulgen 911 and separated by omega-(beta-carboxypropionyl-amino)octyl Sepharose 4B column chromatography, which has been developed in our laboratory. Morphine UDP-glucuronyltransferases were eluted into two fractions, Peak I and Peak II, which have different substrate specificities. Morphine UGTPB was purified by two times of Chromato-focusing from Peak II which was more specific to morphine. The purified morphine UGTPB gave an apparent pI of 8.0 on chromatofocusing and displayed a subunit molecular weight of 55 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the glucuronidation of 3-hydroxyl group of morphine and small extent of 4-hydroxybiphenyl, but not of androsterone, bilirubin, chloramphenicol, codeine, 4-methylumbelliferone, 4-nitrophenol, testosterone, and 6-hydroxyl group of morphine. The N-terminal amino acid sequences of morphine UGTPB were identical to those of UGT1*01P which is deficient to homozygous Gunn rat. Peak II was absent from the fraction of omega-(beta-carboxypropionylamino)octyl Sepharose 4B column chromatography of liver microsomes of Gunn rats treated with phenobarbital, whereas morphine UGT in Peak I was PB-inducible in Gunn rats. Present results suggest that an isoform of morphine UDP-glucuronyltransferase belongs to the UGT1 family and is phenobarbital-inducible.