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作者在研究大白鼠肝脏化学致癌过程中,发现尿苷二磷酸葡萄糖醛酸基转移酶(UDP-GT)的分子形式有胚胎型变化。化学致癌剂2-甲基-胆蒽(3-MC)、二乙基亚硝酸胺(DEN)、2-乙酰氨基芴(FAA)以及抗氧化剂2,3-丁基-4-羟基苯甲醚(BHA)均能诱导炎 o-GT(以2-氨基苯酚作为底物的“胚胎型”UDP-GT),苯巴比妥则诱导 p-GT(以酚酞作为底物的“新生儿型”UDP-GT),在肝脏癌前病变(增生性结节)或在高分化型肝癌时,o-GT 活性显著升高,但在低分化型肝癌时o-GT 活性下降;p-GT 活性虽在癌前病变时有所增高,但在肝癌时,无论在高分化型或低分化型,其活性均下降。采用以氨基已烷基琼脂糖凝胶4B(Aminohexyl Sepharose 4B)为载体的疏水性层析法提纯以 DEN 处理的大白鼠肝脏 o-GT 和 p-GT,其纯化度分别为100倍和50倍,两者的回收率分别为17%和8%。用 SDS-聚丙烯酰胺凝胶电泳法所测得o-GT 和 p-GT 亚单位的分子量分别为54000和58000;用聚丙烯酰胺凝胶等电聚焦电泳法所测得的 o-GT 和 p-GT 的等电点分别为6.2和6.6。 0.05～0.1%Triton X-100对肝微粒体膜结合性 o-GT 有激活作用,但对纯化的该酶却有抑制作用。0.1～0.5%胆酸钠以及5～10mM DEN 无论是对肝微粒体膜结合性的 o-GT,还是对纯化的 o-GT 均有激活作用,但对 p-GT 均有抑制作用。DEN对 o-GT 活性的影响,主要表现在对 V_(max) 的增减上。用以 DEN 诱导产生的 UDP-GT 制成抗体,进行琼脂双向免疫扩散试验,结果o-GT 和 p-GT 之间形成了融合的沉淀线,但是用该抗体所做的酶活性抑制试验表明,o-GT 的抑制率高于 p-GT。在肝脏致癌过程中,胚胎型 UDP-GT的诱导可作为肝脏癌前病变或高分化型肝脏的生物化学标志,具有诊断价值,而且对化学致癌机理和抑癌机理的研究具有重要意义。
The authors found that the molecular form of uridine diphosphate glucuronosyltransferase (UDP-GT) is embryonic in the course of chemical carcinogenesis in rat liver. Chemical carcinogens such as 2-methylcholanthrene (3-MC), diethylnitrosamine (DEN), 2-acetylaminofluorene (FAA) and antioxidants 2,3 -butyl-4-hydroxyanisole (BHA) could induce inflammation o-GT (“embryonic” UDP-GT with 2-aminophenol as substrate) and phenobarbital induced p-GT (“neonate” with phenolphthalein as substrate UDP-GT), o-GT activity was significantly increased in precancerous lesions of the liver (hyperplastic nodules) or in well-differentiated hepatocellular carcinoma, but decreased o-GT activity in poorly differentiated hepatocellular carcinoma In precancerous lesions has increased, but in liver cancer, both in well-differentiated or poorly differentiated, the activity decreased. Purification of rat liver o-GT and p-GT treated with DEN by hydrophobic chromatography with Aminohexyl Sepharose 4B was carried out at 100-fold and 50-fold respectively , The recovery rates of both are 17% and 8% respectively. The molecular weights of o-GT and p-GT subunits measured by SDS-polyacrylamide gel electrophoresis were 54000 and 58000, respectively; o-GT and p measured by polyacrylamide gel isoelectric focusing electrophoresis The isoelectric point of -GT is 6.2 and 6.6, respectively. 0.05 ~ 0.1% Triton X-100 can activate the membrane-bound o-GT of liver microsome, but inhibit the purified enzyme. 0.1 to 0.5% sodium cholate and 5 to 10 mM DEN have either an effect on hepatic microsomal membrane-bound o-GT or on purified o-GT, but all inhibit p-GT. DEN on o-GT activity, mainly in the increase or decrease of V_ (max). Using UDP-GT produced by DEN as an antibody, two-dimensional agar immunodiffusion assay was performed. As a result, a fusion sedimentation line was formed between o-GT and p-GT. However, the enzyme activity inhibition test using this antibody showed that, The inhibition rate of o-GT is higher than that of p-GT. Induction of embryonic UDP-GT can be used as a biochemical marker of precancerous lesions or well-differentiated liver in liver carcinogenesis, which is of diagnostic value, and is of great significance for the study of mechanism of carcinogenesis and tumor suppressor mechanism.