A LYMPHOBLAST MODEL FOR DISEASES OF PURINE METABOLISM

嘌呤代谢疾病的淋巴细胞模型

• 批准号: 3226836
• 负责人: MICHAEL S HERSHFIELD
• 金额: $ 23.02万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-01-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3226836
• 关键词: S adenosylmethionine; adenine; adenosine; adenosine deaminase; binding proteins; complementary DNA; cyclic AMP; deoxyadenosines; electrofocusing; enzyme inhibitors; enzyme mechanism; genetic library; genetic manipulation; high performance liquid chromatography; human subject; human tissue; hydrolase; laboratory mouse; lymphoblast; messenger RNA; molecular cloning; molecular pathology; monoclonal antibody; mutant; nucleic acid probes; orphan disease /drug; protein sequence; purine /pyrimidine metabolism disorder; purine analog; radiotracer; severe combined immunodeficiency; tissue /cell culture; transmethylation

Biochemical effects of adenosine (Ado) and 2'-deoxyadenosine (dAdo) block lymphoid development and cause severe immune dysfunction in genetic deficiency of Ado deaminase (ADA). One target of these nucleosides is the essential metabolic enzyme S-adenosylhomocysteine hydrolase (AdoHcyase), which we identified as a high affinity Ado and cyclic AMP binding protein. Ado inhibits AdoHcy hydrolysis in ADA deficient cells. We discovered that dAdo irreversibly inactivates AdoHcyase by causing reduction of enzyme associated NAD. In ADA deficiency these effects of Ado and dAdo result in secondary deficency of AdoHcyase and accumulation of AdoHcy, which is an inhibitor of S-adenosylmethionine (AdoMe+) dependent transmethylation reactions. We recently showed that the genetic loci for AdoHcyase and ADA are linked, suggesting an evolutionary relationship between these enzymes. We propose to examine the possible relatedness of AdoHycase to ADA or to other proteins that bind NAD, Ado and cAMP; to provide information, which is currenty unavailable, regarding the primary sequence and structure of AdoHcyase and the nature of its catalytic and ligand binding sites; to investigate the influence of ADA deficiency on the turnover of AdoHcyase; and to continue studies now in progess of the metabolic consequences to lymphoid cells of the inhibition of AdoHcy hydrolysis. Specifically, we will attempt to isolate and sequence the cDNA for human AdoHcyase mRNA, using monoclonal antibodies to AdoHcyase and AdoHcyase specific oligonucleotides as probes for screening human liver cDNA libraries. We will evaluate the possibility that Adohcyase may be composed of two kinds of subunits. Essential amino acid residues involved in ligand binding and in catalysis will be located within the sequence of AdoHcyase. We will attempt to crystallize AdoHcyase in the presence and absence of ligands so that its structure may be established definitively. Antibodies and cDNA probes will be used to determine whether inactivation of AdoHcyase by dAdo in ADA deficiency alters the rate of AdoHCyase degradation or AdoHcyase turnover. We will characterize lymphoblast mutants that we have selected for resistance to Ado analogues that specifically inhibit Adohcyase, with the aim of better defining the effect of AdoHcy accumulation on the synthesis and turnover of AdoMet.

腺苷（Ado）和2 '-脱氧腺苷（dAdo）阻断的生化效应 淋巴系统发育并导致遗传性严重免疫功能障碍 腺苷脱氨酶（ADA）缺乏。 这些核苷的一个靶点是 必需的代谢酶S-腺苷高半胱氨酸水解酶（S-腺苷高半胱氨酸水解酶）， 我们将其鉴定为高亲和力的Ado和环AMP结合蛋白。 Ado抑制ADA缺陷细胞中的β-Hcy水解。 我们发现 dAdo通过引起酶的还原而不可逆地使谷胱甘肽半胱氨酸酶失活 相关的NAD 在ADA缺乏症中，Ado和dAdo的这些作用导致 继发性β-Hcy酶缺乏和β-Hcy蓄积， S-腺苷甲硫氨酸依赖性转甲基化抑制剂 反应. 我们最近发现，腺苷酸酶和腺苷脱氨酶的基因位点， 这表明了这些酶之间的进化关系。 我们建议检查可能的相关性，以ADA或 结合NAD、Ado和cAMP的其他蛋白质;提供信息， 目前还没有关于 半胱氨酸酶及其催化和配体结合位点的性质; 探讨ADA缺乏对谷胱甘肽半胱氨酸酶周转的影响; 并继续研究现在的代谢后果， 抑制淋巴样细胞中同型半胱氨酸的水解。 我们特别 将尝试分离和测序人谷胱甘肽酶mRNA的cDNA， 使用抗α-Hcyase和α-Hcyase特异性的单克隆抗体 寡核苷酸作为探针筛选人肝cDNA文库。 我们 我将评估谷胱甘肽酶可能由两种物质组成的可能性 的subunits。 参与配体结合的必需氨基酸残基， 在催化作用中的位置将位于谷胱甘肽半胱氨酸酶的序列内。 我们将 尝试在存在和不存在配体的情况下结晶脱氢酶， 它的结构可以确定。 抗体和cDNA 将使用探针来确定dAdo是否会使β-Hcyase失活 在ADA缺乏症中， 周转 我们将描述我们选择的淋巴母细胞突变体， 对于特异性抑制腺苷酸酶的Ado类似物的抗性， 目的是更好地确定同型半胱氨酸积累对 合成和周转。