Batt A M, MacKenzie P, Hänninen O, Vainio H
Med Biol. 1979 Oct;57(5):281-6.
A simple and sensitive fluorimetric method is described for evaluation of 3-hydroxybenzo(a)pyrene conjugation with UDPglucuronic acid. It is less expensive than a radiochemical method and suitable for routine use. 3-Hydroxybenzo(a)pyrene is readily conjugated in isolated liver microsomes in the presence of UDPglucuronic acid. Activity in rat liver microsomes was 0.90--1.20 nmol.min-1.mg-1 microsomal protein. The activity in homozygous and heteroxygous Gunn rats was considerably lower than in Wistar rats. Activity in guinea pigs was 2.5--3 nmol.min-1.mg-1 protein. 3-Methylcholanthrene pretreatment (20 mg/kg of body weight for 4 consecutive days) of rats enhanced the hepatic UDPglucuronosyltransferase activity 2--5 fold. In untreated microsomal membranes of rat liver the apparent Km for 3-hydroxybenzo(a)pyrene was 0.09 mM and for Udpglucuronic acid 4.6 mM. Conjugation with UDPglucose did not occur. 4-Nitrophenol and 4-nitrophenyl-beta-D-glucuronide behaved like non competitive inhibitors. In contrast to 4-nitrophenol conjugation, both ionic (cholic acid) and non-ionic (Triton X-100, digitonin) surfactants had no effect or inhibited the glucuronic acid conjugation of 3-hydroxybenzo(a)pyrene in rat liver microsomes as also did the treatment of microsomal membranes with phospholipases A and C. Trypsin was almost without an effect on UDPglucuronosyltransferase activity when 3-hydroxybenzo(a)pyrene was used as substrate.
本文描述了一种简单且灵敏的荧光法，用于评估3 - 羟基苯并(a)芘与UDP - 葡萄糖醛酸的结合情况。该方法比放射化学法成本更低，适用于常规使用。在UDP - 葡萄糖醛酸存在的情况下，3 - 羟基苯并(a)芘很容易在离体肝微粒体中发生结合。大鼠肝微粒体中的活性为0.90 - 1.20 nmol·min⁻¹·mg⁻¹微粒体蛋白。纯合子和杂合子冈恩大鼠的活性明显低于Wistar大鼠。豚鼠的活性为2.5 - 3 nmol·min⁻¹·mg⁻¹蛋白。大鼠经3 - 甲基胆蒽预处理（连续4天，体重20 mg/kg）可使肝脏UDP - 葡萄糖醛酸基转移酶活性提高2 - 5倍。在未处理的大鼠肝微粒体膜中，3 - 羟基苯并(a)芘的表观Km为0.09 mM，UDP - 葡萄糖醛酸的表观Km为4.6 mM。未发生与UDP - 葡萄糖的结合。4 - 硝基苯酚和4 - 硝基苯基 - β - D - 葡萄糖醛酸表现为非竞争性抑制剂。与4 - 硝基苯酚结合不同，离子型（胆酸）和非离子型（Triton X - 100、洋地黄皂苷）表面活性剂对大鼠肝微粒体中3 - 羟基苯并(a)芘的葡萄糖醛酸结合没有影响或有抑制作用，用磷脂酶A和C处理微粒体膜也有同样效果。当以3 - 羟基苯并(a)芘为底物时，胰蛋白酶对UDP - 葡萄糖醛酸基转移酶活性几乎没有影响。
