Roy Chowdhury J, Roy Chowdhury N, Falany C N, Tephly T R, Arias I M
Biochem J. 1986 Feb 1;233(3):827-37. doi: 10.1042/bj2330827.
UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct 'maps', indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining.
UDP-葡糖醛酸基转移酶（EC 2.4.1.17）活性从雄性Wistar大鼠肝脏微粒体部分在乳化剂911中溶解，通过在PBE 94（pH 9.4至6.0）上进行色谱聚焦分离出六个具有转移酶活性的组分。组分I通过在UDP-己醇胺-琼脂糖4B上进行亲和色谱进一步分离为同工型Ia、Ib和Ic。组分III中的UDP-葡糖醛酸基转移酶通过再色谱聚焦（pH 8.7至7.5）进一步纯化。组分IV和V中的UDP-葡糖醛酸基转移酶通过UDP-己醇胺-琼脂糖色谱纯化。组分II、III、IV和V中的转移酶同工型最终通过在TSK G 3000 SW柱上进行高效液相色谱纯化。纯化的UDP-葡糖醛酸基转移酶同工型Ia（Mr 51,000）、Ib（Mr 52,000）、Ic（Mr 56,000）、II（Mr 52,000）、IV（Mr 53,000）和V（Mr 53,000）在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳上显示出单一的考马斯亮蓝染色带。同工型III酶显示出Mr 52,000和53,000的两条带。通过Cornish-Bowden方法[(1980) Anal. Biochem. 105, 233 - 238]比较氨基酸组成表明，所有UDP-葡糖醛酸基转移酶同工型在结构上相关。各个同工型的胰蛋白酶肽段的反相高效液相色谱显示出不同的“图谱”，表明蛋白质一级结构存在差异。同工型III的两条带在有限的酶促蛋白水解后显示出不同的电泳肽图谱。在用磷脂酰胆碱脂质体重组后，纯化的同工型表现出不同但重叠的底物特异性。同工型V对胆红素葡糖醛酸化具有特异性，不受其他苷元底物的抑制。除Ia外，每种同工型通过过碘酸/席夫染色鉴定为糖蛋白。
