A LYMPHOBLAST MODEL FOR DISEASES OF PURINE METABOLISM

嘌呤代谢疾病的淋巴细胞模型

• 批准号: 3226837
• 负责人: MICHAEL S HERSHFIELD
• 金额: $ 25万
• 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/3226837
• 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(DAO)受体阻滞剂的生化效应 淋巴样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 必需代谢酶S-腺苷同型半胱氨酸水解酶(ADO Hcyase)， 我们鉴定它是一种高亲和力的腺苷和环状AMP结合蛋白。 ADO抑制ADA缺陷细胞的ADHcy水解酶。我们发现 DAO通过引起酶的还原不可逆转地使ADO Hcyase失活 关联的NAD。在ADA缺乏时，ADO和DAO的这些作用导致 ADOHcyase的继发性缺陷和ADOHcy的蓄积是一种 S依赖的腺苷甲硫氨酸转甲基化抑制物 反应。我们最近发现了Adobe Hcyase和ADA的遗传位点 是相互关联的，这表明这些酶之间存在进化关系。 我们建议研究ADOHycase与ADA或 其他结合NAD、ADO和cAMP的蛋白质；提供信息，这些信息 关于初级序列和结构，目前还无法获得 ADOHcyase及其催化和配体结合部位的性质； 研究ADA缺乏对ADO-Hcyase周转的影响； 并继续目前正在进行的代谢后果的研究 抑制淋巴细胞的同型半胱氨酸的水解。具体来说，我们 将尝试分离和测序人ADOHcyase mRNA的cdna， 利用抗ADOHcyase和抗ADOHcyase特异性的单抗 寡核苷酸作为筛选人肝cdna文库的探针。我们 将评估adohcyase由两种类型组成的可能性 亚基的数量。参与配基结合的必需氨基酸残基和 In的催化作用将定位在ADOHcyase的序列中。我们会 尝试在存在和不存在配体SO的情况下结晶ADO-Hcyase 它的结构可以被确定下来。抗体和c DNA 将使用探测器来确定DAO是否使ADOHcyase失活 缺乏ADA会改变ADO-HCyase或ADO-Hcyase的降解速度 营业额。我们将描述我们选择的淋巴母细胞突变体的特征 对于特定抑制ADO酶的ADO类似物的抗药性， 更好地定义ADOHcy积累对 ADOMet的合成和周转。