N-ACETYLNEURAMINIC ACID HYDROXYLASE

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。

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.