MOLECULAR FORMS OF HUMAN LIVER ALCOHOL DEHYDROGENASE

人肝乙醇脱氢酶的分子形式

• 批准号: 3108791
• 负责人: TING-KAI K LI
• 金额: $ 21.73万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3108791
• 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; laboratory mouse; liver metabolism; molecular genetics; molecular pathology; monoclonal antibody; nucleic acid probes; oxidation; postmortem; protein sequence; 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 人肝酒脱氢酶的同工酶的遗传变异 （ADH）在很大程度上说明酒精的个体差异 消除率，可能部分是观察到的个体 生理，心理和病理的差异 乙醇消耗的后果。 过去12年的研究 揭示人肝脏中的ADH可以分为 它们在分子和催化特性上的亲密关系的基础。 班级 我包含一大批由各种二聚体组成的同工酶 Alpha，Beta和Gamma亚基的组合，由三个基因编码， 分别指定为ADH1，ADH2和ADH3。 遗传多态性 已经检测到ADH2和ADH3基因座，从而引起Beta 1，Beta 2和 Betaindianapolis亚基，伽马1和伽马2个亚基。 II类是Chi-Adh分子形式的Pi-Adh和III。 这 I类中的同工酶形式在底物的VMAX和KM值方面有所不同 和辅酶，以及在ph-optima中进行活性。 tis的目标继续 未来5年的研究计划是隔离和表征 含甜菜碱的同工酶形式，多种形式的pi-adh， 以及高和低活动β1β1表格；动力学 通过表征乙醇氧化的负合作性 伽玛1伽玛1和伽玛2伽马2;阐明结构和 观察到的beta 1 beta 1，beta 2中观察到的差异的动力学基础 Beta 2和Betaind beta 1 IND化合物酶；确定金额是否 五个ADH生产的Alpha，Beta，Gamma，Pi和Chi子单位 不同个体的基因不同；和ADH的决定 通过使用重组DNA，肝脏和白细胞中的基因型 技术。 亲和力和离子交换色谱法，HPLC，淀粉 - 凝胶 电泳，等电聚焦和定量免疫印迹将 被用来隔离和识别特定的同工酶并进行量化 不同亚基形式的数量。 稳态和瞬态 将使用停止流动动力学来比较pH，阴离子的影响 并在动力学和速率常数上剥离乙醇 alleleo酶。 蛋白质和肽序列分析和化学 修改催化性必不可少的CYS，ARG及其残留物将是 用于阐明结构差异。 最后，人类ADH-CDNA 将开发探针以搜索限制性长度 - 造态性 已知表型和白细胞中的肝标本。