PURIFICATION AND PROPERTIES OF UDP-GLUCURONYLTRANSFERASE FROM KIDNEY MICROSOMES OF BETA-NAPHTHOFLAVONE TREATED RAT
PURIFICATION AND PROPERTIES OF UDP-GLUCURONYLTRANSFERASE FROM KIDNEY MICROSOMES OF BETA-NAPHTHOFLAVONE TREATED RAT
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DOI：
10.1093/oxfordjournals.jbchem.a122839
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发表时间：
1989-08-01
影响因子：
2.7
通讯作者：
YUASA, A
中科院分区：
文献类型：
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作者：
YOKOTA, H;OHGIYA, N;YUASA, A
大鼠肾微粒体UDP-葡糖醛酸基转移酶对酚类异生物质的活性通过用β-葡糖醛酸基转移酶处理动物而增强约4-5倍。萘酮。对血清素，内源性底物的转移酶活性，也提高了约7.5倍。一种形式的UDP-葡糖醛酸基转移酶从β-葡糖醛酸基转移酶的肾微粒体中纯化。通过用胆酸钠增溶和两步柱色谱法，第一步用DEAE-Toyopolymer（快速流动速率液相色谱法：FFLC），第二步用UDP-己醇胺Sepharose 4 B（亲和色谱法），对经萘啶酮处理的大鼠进行纯化。这些程序得到约39倍的纯化和11.5%的产率的1-萘酚转移酶活性。从十二烷基硫酸钠（SDS）-聚丙烯酰胺平板凝胶电泳上的单一蛋白条带（Mr 54，000）判断，该制剂高度纯化，暂命名为“GT-2”。它不仅能催化1-萘酚、4-硝基苯酚和4-甲基伞形酮等酚类外源物质的葡萄糖醛酸化，还能催化5-羟色胺的葡萄糖醛酸化。从GT-2的表观分子量通过内切-β-葡聚糖酶降低至50，000的结果可知，用N-乙酰葡糖胺糖苷酶H（Endo H）处理后，发现GT-2是一种携带“高甘露糖”型寡糖链的50，000 Da多肽。GT-2氨基酸序列为Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu-Ile-Val。与从3-甲基胆蒽处理的大鼠肝微粒体中纯化的GT-1相比，观察到在7个NH 2-末端残基中有两个氨基酸取代。GT-2的氨基端序列与其他研究者克隆的各种UDP-葡萄糖醛酸转移酶的氨基端第26 - 46位氨基酸残基的序列同源。因此，本研究证实了新合成的转移酶的第25位氨基酸残基的NH 2-末端区域含有信号肽的概念。
Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5 fold by treatment of the animal with .beta.-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of .beta.-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography: FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed "GT-2", was highly purified as judges from the single protein band (Mr 54,000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50,000 by endo-.beta.-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50,000 Da polypeptide carrying "high mannose" type oligosaccharide chain(s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu-Ile-Val. It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators. The notion that the NH2-terminal region to the 25th amino acid residue of newly synthesized transferase contains the signal peptide was thus confirmed by this study.