MOLECULAR FORMS OF HUMAN LIVER ALCOHOL DEHYDROGENASE

人肝乙醇脱氢酶的分子形式

• 批准号: 3108789
• 负责人: TING-KAI K LI
• 金额: $ 18.49万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-07-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3108789
• 关键词: Alaskan Native American; Asians; Native Americans; affinity chromatography; alcohol dehydrogenase; alcoholism /alcohol abuse; biological polymorphism; chemical kinetics; complementary DNA; electrofocusing; enzyme structure; enzyme substrate; ethanol; gel electrophoresis; gene expression; genetic regulation; genotype; high performance liquid chromatography; human population genetics; human tissue; ion exchange chromatography; isozymes; liver metabolism; molecular genetics; molecular pathology; monoclonal antibody; oxidation; postmortem; stop flow technique

The long-term objective of this research project is to elucidate the enzymatic regulation of alcohol metabolism. It is our hypothesis that genetic variations in the isoenzymes of human liver alcohol dehydrogenase (ADH) account to a large extent for individual differences in alcohol elimination rate and may underlie, in part, the observed individual differences in the physiological, psychological and pathological consequences of ethanol consumption. Studies in the past 12 years have revealed that ADH in human liver can be differentiated into 3 classes on the basis of their closeness in molecular and catalytic properties. Class I contains a large group of isoenzymes composed of various dimeric combinations of Alpha, Beta and Gamma subunits, coded by three genes, designated ADH1, ADH2 and ADH3, respectively. Genetic polymorphism at the ADH2 and ADH3 loci have been detected, giving rise to Beta 1, Beta 2 and BetaIndianapolis subunits, and Gamma 1 and Gamma 2 subunits, respectively. Class II are the pi-ADH and class III in chi-ADH molecular forms. The isoenzyme forms within class I differ in Vmax and Km values for substrates and coenzymes, and in pH-optima for activity. The goals of tis continuing research program in the next 5 years are the isolation and characterization of the BetaInd-containing isoenzyme forms, the multiple forms of Pi-ADH, and the high and low activity Beta 1 Beta 1 forms; the kinetic characterization of the negative cooperativity of ethanol oxidation by Gamma 1 Gamma 1 and Gamma 2 Gamma 2; elucidation of the structural and kinetic basis for the observed differences among the Beta 1 Beta 1, Beta 2 Beta 2 and BetaInd Beta 1 Ind alleloenzymes; determination if the amounts of the Alpha, Beta, Gamma, Pi and Chi subunits produced by the five ADH genes differ in different individuals; and the determination of ADH genotypes in livers and white blood cells by use of recombinant DNA technology. Affinity and ion-exchange chromatography, HPLC, starch-gel electrophoresis, isoelectric focusing and quantitative immunoblotting will be employed to isolate and identify the specific isoenzymes and to quantify the amounts of the different subunit forms. Steady-state and transient stopped-flow kinetics will be employed to compare the effects of pH, anions and deuterated ethanol on the kinetic and rate constants of the alleloenzymes. Protein and peptide sequence analysis and chemical modification of catalytically essential Cys, Arg and His residues will be employed to elucidate structural differences. Finally, human ADH-cDNA probes will be developed to search for restriction-length-polymorphism in liver specimens of known phenotypes and in leukocytes.

该研究项目的长期目标是阐明 酒精代谢的酶调节。 我们的假设是 人肝乙醇脱氢酶同工酶的遗传变异 (ADH) 在很大程度上解释了酒精的个体差异 消除率，可能部分取决于观察到的个体 生理、心理、病理的差异 乙醇消耗的后果。 过去12年的研究 揭示人肝脏中的 ADH 可分为 3 类 它们在分子和催化特性上的相似性的基础。 班级 I 含有一大群由各种二聚体组成的同工酶 由三个基因编码的 Alpha、Beta 和 Gamma 亚基的组合， 分别指定为 ADH1、ADH2 和 ADH3。 基因多态性位于 已检测到 ADH2 和 ADH3 基因座，产生 Beta 1、Beta 2 和 分别为 BetaIndianapolis 亚基、Gamma 1 和 Gamma 2 亚基。 II 类是 pi-ADH，III 类是 chi-ADH 分子形式。 这 I 类同工酶形式的底物 Vmax 和 Km 值不同 和辅酶，并处于最佳活性 pH 值。 持续的目标 未来5年的研究计划是分离和表征 含有 BetaInd 的同工酶形式，Pi-ADH 的多种形式， 以及高活性Beta 1 和低活性Beta 1 形式；动力学 乙醇氧化负协同性的表征 伽玛 1 伽玛 1 和伽玛 2 伽玛 2；结构的阐明和 Beta 1 Beta 1、Beta 2 之间观察到的差异的动力学基础 Beta 2 和 BetaInd Beta 1 Ind 等位酶；确定金额是否 由五个 ADH 产生的 Alpha、Beta、Gamma、Pi 和 Chi 亚基 不同个体的基因不同；以及ADH的测定 使用重组 DNA 检测肝脏和白细胞的基因型 技术。 亲和层析和离子交换层析、HPLC、淀粉凝胶 电泳、等电聚焦和定量免疫印迹将 用于分离和鉴定特定同工酶并定量 不同亚基形式的数量。 稳态和瞬态 将采用停流动力学来比较 pH 值、阴离子的影响 和氘代乙醇对动力学和速率常数的影响 等位基因酶。 蛋白质和肽序列分析和化学 催化必需的 Cys、Arg 和 His 残基的修饰将 用来阐明结构差异。 最后，人类ADH-cDNA 将开发探针来搜索限制性长度多态性 已知表型的肝脏标本和白细胞。