STUDIES ON UDP-GLUCURONOSYLTRANSFERASES

治疗上有用的药物和内生菌的葡糖苷酸化， 类固醇和胆红素调节作用的持续时间和毒性 这些物质，尤其是在人类身上。 微粒体UDP家族- 葡萄糖醛酸基转移酶（UDPGTs）催化这些反应，并且在某些情况下， 例，人肝中存在独特的UDPGTs。 本提案的目的 是揭示UDPGTs介导的功能和结构特性， 吗啡的3-0和6-0-葡萄糖醛酸化，除 吗啡（例如，曲哌那明、阿米替林）。 特别是吗啡 UDPGT将从大鼠和人肝微粒体中纯化至均一 并就其底物特异性、NH 2-末端 氨基酸序列和活性位点肽氨基酸序列。 cDNA 将使用聚合酶进行大鼠和人肝脏UDPGTs的克隆 使用寡核苷酸的链式反应技术，其合成将是 基于氨基酸序列知识。 比活性部位光亲和性 使用[3 H]-氟硝西泮（FNZ）标记将有助于实现 这些目标的数量。 活性和FNZ标记的吗啡UDPGT将被 使用Fractogel、色谱聚焦和亲和层析纯化至均匀 层析 还将研究人肝叔胺UDPGT 使用光亲和标记，选择的抗体免疫识别， 色谱分离，NH 2-末端氨基酸测序和cDNA 克隆技术 与性别有关或药物引起的人类 还将研究肝雌三醇UDPGT。 这项研究将提供 这些信息将有助于预测潜在的药物-药物 相互作用、研究方法或可能的人类UDPGTs多态性， 以及对葡萄糖醛酸化调节代谢的理解。

Glucuronidation of therapeutically useful drugs and endobiotics such as steroids and bilirubin regulates the duration of action and toxicity of these substances, especially in humans. A family of microsomal UDP- glucuronosyltransferases (UDPGTs) catalyze these reactions and, in certain cases, unique UDPGTs exist in human liver. The objective of this proposal is to reveal the functional and structural properties of UDPGTs mediating the 3-0 and 6-0-glucuronidation of morphine, tertiary amines other than morphine (e.g., tripelennamine, amitryptyline). Specifically, morphine UDPGTs will be purified to homogeneity from rat and human liver microsomes and compared with respect to their substrate specificity, NH2-terminal amino acid sequences and active site peptide amino acid sequences. cDNA cloning of rat and human liver UDPGTs will be performed using polymerase chain reaction techniques with oligonucleotides whose synthesis will be based on amino acid sequence knowledge. Specific active site photoaffinity labeling using [3H]-flunitrazepam (FNZ) will be useful in accomplishing a number of these aims. Active and FNZ-labeled morphine UDPGT will be purified to homogeneity using Fractogel, chromatofocusing and affinity chromatography. Human liver tertiary amine UDPGT will also be studied using photoaffinity labeling, selected antibody immunorecognition, chromatographic separations, NH2-terminal amino acid sequencing and cDNA cloning techniques. Gender-related or drug-induced increases in human liver estriol UDPGT will also be studied. This research will provide information which will allow for predictions of potential drug-drug interaction, methods for study or possible human polymorphisms of UDPGTs, and an understanding of the regulation of metabolism by glucuronidation.