CHARACTERIZATION OF N-ACETYLNEURAMINIC ACID HYDROXYLASE

N-乙酰神经氨酸羟化酶的表征

• 批准号: 3467895
• 负责人: Elaine Anselmo Muchmore
• 金额: $ 7.99万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467895
• 关键词: N acetylneuraminate; affinity chromatography; antibody formation; colon; developmental genetics; embryo /fetus antigen; enzyme mechanism; enzyme structure; gel electrophoresis; gene expression; immunologic techniques; laboratory rat; molecular cloning; monoclonal antibody; nucleic acid probes; oxygenases; protein purification; protein sequence

In recent years, many examples of cell surface carbohydrates which have important biological functions have been described. These include recognition by antibodies, binding of viruses. bacterial capsular antigenicity and regulation of growth. Sialic acids, which are often the terminal carbohydrate moieties on cell surface glycoproteins and glycolipids, are in a unique position to have effects on biological functions. O-acetylated sialic acid modification, N-glycolyl neuraminic aced (Neu5Gc), has been found in both fetal human tissue and in an increasing number of human tumors. Because its introduction to a normal adult human results in an intense immunogenic response, it is a true onco- fetal antigen. The role which Neu5Gc plays in normal cellular events is not known, nor is its role in malignant transformation understood. Neu5Gc is converted from N-acetyl neuraminic acid by the enzyme N- acetylneuraminic mono-oxygenase. Detailed enzyme kinetics have not been done, nor has this enzyme ever been isolated and purified. The goal of this project is to isolate and purify the mono-oxygenase by conventional and affinity chromotography. The kinetics and properties of the isolated enzyme will be determined. Antibodies to the purified protein will be generated so that an immunoassay can be developed. The immunoassay will be used for three major areas of investigation. First, it will be used to correlate enzyme expression with expression of Neu5Gc in the rat colon, a tissue which has developmentally-regulated changes in expression of Neu5Gc. Second, the precise subcellular location of the enzyme will be determined. Third, the cellular regulation of enzyme expression can be determined. The purified enzyme will also be used to determine the structural and kinetic properties of the protein. In the long run, oligonucleotide probes from peptide sequences or antibodies will be used to clone the gene encoding the enzyme. Ultimately, the goal is to understand the normal fetal expression and malignant re-expression of Neu5Gc by correlation with activity and presence of the converting enzyme at the gene, message, and activity levels.

近年来，许多细胞表面碳水化合物的例子， 已经描述了重要的生物学功能。 这些包括 抗体识别，病毒结合细菌荚膜 抗原性和生长调节。 唾液酸，通常是 细胞表面糖蛋白上的末端碳水化合物部分， 糖脂，在一个独特的地位，有影响的生物 功能协调发展的 O-乙酰化唾液酸修饰，N-羟乙酰神经氨酸 已在胎儿人体组织和胎儿组织中发现了Aced（Neu 5Gc）。 越来越多的人类肿瘤。 因为它的引入是正常的 成年人导致强烈的免疫原性反应，这是一个真正的肿瘤， 胎儿抗原 Neu 5Gc在正常细胞事件中的作用是 其在恶性转化中的作用也不清楚。 Neu 5Gc由N-乙酰神经氨酸通过N-乙酰神经氨酸酶转化而来。 乙酰神经氨酸单加氧酶 详细的酶动力学尚未被 这种酶从未被分离和纯化过。 本课题的目的是分离纯化单加氧酶， 常规和亲和色谱法。 动力学和性质 将测定分离的酶。 抗纯化的 将产生蛋白质，从而可以开发免疫测定。 的 免疫分析将用于三个主要研究领域。 第一、 它将用于将酶表达与Neu 5Gc表达相关联 在大鼠结肠中，一种具有发育调节变化的组织， Neu 5Gc的表达。 其次，细胞的精确亚细胞位置 酶将被确定。 第三，酶的细胞调节 表达可以确定。 纯化的酶也将用于 确定蛋白质的结构和动力学性质。 在 从长远来看，来自肽序列或抗体的寡核苷酸探针将 用于克隆编码该酶的基因。 最终的目标是 了解胎儿正常表达和恶性再表达 Neu 5Gc与转化酶活性和存在相关 在基因、信息和活动水平上。