CHARACTERIZATION OF N-ACETYLNEURAMINIC ACID HYDROXYLASE

N-乙酰神经氨酸羟化酶的表征

• 批准号: 3467892
• 负责人: Elaine Anselmo Muchmore
• 金额: $ 6.28万
• 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 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467892
• 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; protein purification

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，许多具有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，病毒的结合。细菌囊 抗原性和生长的调节。 唾液酸，通常是 细胞表面糖蛋白的末端碳水化合物部分和 糖脂质，对生物产生影响的独特位置 功能。 o-乙酰化的唾液酸修饰，N-糖苷神经氨基 在胎儿人体组织和 人类肿瘤数量的增加。 因为它引入了正常 成人人类会产生强烈的免疫原性反应，这是一个真正的onco- 胎儿抗原。 NEU5GC在正常细胞事件中起的作用是 尚不清楚，其在恶性转化中的作用也不理解。 Neu5GC通过酶N-从N-乙酰神经氨酸转化 乙酰神经酶单氧酶。 详细的酶动力学尚未 完成了，这种酶也从未被隔离和纯化。 该项目的目的是通过 常规和亲和力膜片。 动力学和特性 将确定分离的酶。 纯化的抗体 将产生蛋白质，以便可以开发免疫测定。 这 免疫测定法将用于三个主要调查领域。 第一的， 它将用于将酶表达与NEU5GC的表达相关联 在大鼠结肠中，有发育调节的组织 neu5gc的表达。 其次，确切的亚细胞位置 将确定酶。 第三，酶的细胞调节 可以确定表达。 纯化的酶也将用于 确定蛋白质的结构和动力学特性。 在 长期，来自肽序列或抗体的寡核苷酸探针将 用于克隆编码酶的基因。 最终，目标是 了解正常的胎儿表达和恶性重新表达 NEU5GC通过与活动和转化酶的存在相关性 在基因，信息和活动水平上。