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N-ACETYLNEURAMINIC ACID HYDROXYLASE

N-乙酰神经氨酸羟化酶

基本信息

批准号：
2181839
负责人：
Elaine Anselmo Muchmore
金额：
$ 9.25万
依托单位：
VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-12-01 至 1995-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2181839
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

项目摘要

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对重要的生物学功能进行了描述。这些措施包括抗体的识别，病毒的结合。细菌囊抗原性和生长调节。唾液酸，通常是细胞表面糖蛋白上的末端碳水化合物部分和糖脂，处于独特的位置对生物产生影响功能。O-乙酰化唾液酸改性，N-羟基神经氨酸 ACED(Neu5Gc)，已在胎儿组织和人类肿瘤的数量在不断增加。因为它介绍了一种正常的成人会引起强烈的免疫原性反应，是一种真正的肿瘤- 胎儿抗原。Neu5Gc在正常细胞事件中扮演的角色是不知道，也不知道它在恶性转化中的作用。 Neu5Gc是由N-乙酰神经氨酸通过N-酶转化而来的。乙酰神经氨酸单加氧酶。详细的酶动力学还没有得到这种酶也从未被分离和提纯过。本项目的目标是通过以下方法分离和纯化单加氧酶常规层析和亲和层析。的动力学和性质分离出的酶将被测定。纯化后的抗体将产生蛋白质，以便开发一种免疫分析方法。这个免疫分析将用于三个主要领域的调查。第一, 它将被用来关联酶表达和Neu5Gc的表达在大鼠的结肠中，一种具有发育调节变化的组织 Neu5Gc的表达。第二，精确的亚细胞定位酶将会被测定。第三，酶的细胞调控可以确定表达式。纯化的酶还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，我们的目标是了解胎儿的正常表达和恶性再表达 Neu5Gc与转换酶活性和存在的相关性在基因、信息和活动水平上。