GLUTATHIONE S-TRANSFERASE AND ITS REGULATION OF CARCINOGENIC ELECTROPHILES

这项工作的目标是了解在生物化学和分子 术语谷胱甘肽S-转移酶（GST）催化的 共轭反应自然发挥作用，或者可以通过 增强，在减少毒性烷基化事件的发生率， 细胞 使用细菌产生的GST pi同工酶的初步研究将 氨基酸残基、表位的联合收割机定点突变 一组抑制性单克隆抗体的图谱，以及 合作，其中涉及x射线晶体分析，以确定 负责结合GSH和外源性底物的残基 GST π酶。 所有氨基酸的随机寡核苷酸诱变 将完成GST π和GST γ c同工酶内的氨基酸，并且是新颖的， 将通过选择GST- 表达大肠大肠杆菌细胞中的抗BPDE（pi）或美法仑（Yc）。 的 天然存在的GST同工酶的能力以及动力学- 赋予亲电体抗性的有效突变体衍生物将 通过将重组表达载体引入几个 哺乳动物系统 这些将包括：i）FVB的转基因系， 在真皮中表达串联设计的人GST pi构建体的小鼠 成纤维细胞抑制苯并（a）芘诱导的纤维肉瘤形成， ii）人mu-类GST构建体在来自以下的淋巴母细胞中的表达： 携带遗传性GST 1无效表型的人受试者，和iii） 培养的啮齿动物细胞（10T1/2，MatB乳腺癌）， 表达重组GST突变体，以确定 可以实现的对亲电体的抗性。 的最后一个方面 这项工作包括使用新的克隆策略来分离cDNA， 来自小鼠肝cDNA表达文库，其编码i）血浆- 从哺乳动物细胞输出GSH-缀合物的膜泵，和ii） 一种介导抗氧化剂诱导剂诱导GST的蛋白质 分子如丁基化羟基苯甲醚。

The goal of this work is to understand in biochemical and molecular terms the role which glutathione S-transferase (GST)-catalyzed conjugation reactions naturally play, or can be made to play by augmentation, in reducing the incidence of toxic alkylation events in cells. Initial studies using a bacterially-produced GST pi isozyme will combine site-directed mutagenesis of amino acid residues, epitope mapping of a panel of inhibitory monoclonal antibodies, and a collaboration which involves x-ray crystal analysis, to identify residues which are responsible for binding GSH and xenobiotic substrates to the GST pi enzyme. Random oligonucleotide mutagenesis of all amino acids within the GST pi and GST Yc isozymes will be done, and novel, increased-efficiency mutant enzymes will be identified by selecting GST- expressing E. coli cells in anti-BPDE (pi) or melphalan (Yc). The ability of naturally-occurring GST isozymes as well as kinetically- efficient mutant derivatives to confer resistance to electrophiles will be examined by introducing recombinant expression vectors into several mammalian systems. These will include: i) a transgenic line of FVB mice expressing a tandem-design human GST pi construct in dermal fibroblasts to suppress benzo(a)pyrene-induced fibrosarcoma formation, ii) expression of human mu-class GST constructs in lymphoblasts from human subjects carrying an inherited GST1 null-phenotype, and iii) cultured rodent cells (10T1/2, MatB mammary carcinoma) which are expressing recombinant GST mutants to determine the maximal degree of resistance to electrophiles that can be achieved. The final aspect of this work involves the use of novel cloning strategies to isolate cDNAs from a mouse liver cDNA-expression library that encode i) a plasma- membrane pump which exports GSH-conjugates from mammalian cells, and ii) a protein which mediates the induction of GST by antioxidant inducer molecules such as butylated hydroxyanisole.