CHARACTERIZATION OF N-ACETYLNEURAMINIC ACID HYDROXYLASE

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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.