IDENTIFICATION AND CHARACTERIZATION OF UDP-GALACTOSE: 2-ACETAMIDO-2-DEOXY-D-GLUCOSE 3BETA - GALACTOSYLTRANSFERASE

BRADLEY THURSTON SHEARES, Purdue University

Abstract

The biosynthesis of galactosyl-(beta)1,3-N-acetylglucosamine has been demonstrated for the first time by using membranes isolated from pig trachea. Unlike the UDP-galactose:N-acetylglucosamine 4(beta)-galactosyltransferase, which is inhibited by high levels of N-acetylglucosamine, the UDP-galactose:N-acetylglucosamine 3(beta)-galactosyltransferase showed no inhibition at 200 mM N-acetylglucosamine. (alpha)-Lactalbumin which inhibits galactose incorporation into galactosyl-(beta)1,4-N-acetylglucosamine had little or no effect on the activity of the UDP-galactose:N-acetylglucosamine 3(beta)-galactosyltransferase. Initial experiments with crude membranee, showed that approximately 80% of the product synthesized at 200 mM N-acetylglucosamine was base-labile, suggesting the synthesis of galactosyl-1,3-N-acetylglucosamine. (beta)-Galactosidase from Escherichia coli hydrolyzed the base-stable product, as expected for galactosyl-(beta)1,4-N-acetylglucosamine, but not the base-labile component. Analysis of the base-labile component by gas chromatography of the alditol acetates, methylation analysis by gas chromatography/mass spectrometry, and treatment with specific galactosidases clearly established that the disaccharide was galactosyl-(beta)1,3-N-acetylglucosamine. The (beta)1,3-galactosyltransferase activity was solubilized in 1.0% Triton X-100 and contaminating galactosyltransferases were removed by affinity chromatography on (alpha)-lactalbumin-agarose, N-acetylglucosamine-agarose, and asialo-ovine submaxillary mucin bound to DEAE-Sephacel. The (beta)1,3-galactosyltransferase and a yet unidentified (beta)1,4-galactosyltransferase were adsorbed to a UDP-hexanolamine-Sepharose column. These two galactosyltransferases were resolved by gel filtration on Sephacryl S-200. The purified (beta)1,3-galactosyltransferase displays an absolute requirement for a divalent cation (Mn('++) or Co('++)) and is most active at pH 6.0. Thermostability studies show that the transferase is rapidly inactivated at temperatures higher than 37(DEGREES)C. The enzyme has apparent K(,m)'s for UDP-galactose and N-acetylglucosamine at 0.23 mM and 385 mM, respectively. Acceptor studies suggest that the (beta)1,3-galactosyltransferase is responsible for the elongation of oligosaccharide chains in mucin glycoproteins and maybe glycolipids.

Degree

Ph.D.

Subject Area

Biochemistry

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