UDP-galactose: N-acetylglucosamine galactosyltransferase was isolated from pooled human milk, pooled amniotic fluid and from two different individual samples of malignant ascites. The purification procedure involving two successive affinity chromatography steps on N-acetylglucosamine -agarose and a-lactalbuniin -agarose yielded an enzyme preparation homogeneous by size. Under non-denaturing conditions the ascites and amniotic fluid enzymes had identical electrophoretic mobility, but they moved faster than the milk enzyme. Isoelectric analysis in the presence and absence of urea resolved the milk enzyme into at least 13 different forms, nine of which had the same isoelectric points after refocusing. All enzyme forms showed similar activity when free N-acetylglucosamine, ovalbumin, sialic-acid-free ovine submaxillary mucin and glucose, in the presence of a-lactalbumin, were used as acceptor substrates. Comparative isoelectric focusing of the three galactosyltransferases revealed identical patterns of the amniotic and ascites enzymes, but only partial overlap with the milk enzyme, which was less negatively charged. Neuraminidase treatment of ascites and milk galactosyltransferases produced very similar focusing patterns. The possible structural basis for this charge heterogeneity is briefly discussed.Biosynthesis of the heteropolysaccharide moieties of complex carbohydrates is catalyzed by glycosyltransferases (for review see [l]). The information on their structure is scarce. One of the best known glycosyltransferases is UDP-galactose : N-acetylglucosamine galactosyltransferase or A protein of the lactose synthetase complex (for review see [ 2 ] ) . This enzyme has been purified from various animal and human body fluids [3-81 but, with the exception of amino acid and carbohydrate content of the bovine milk enzyme [9], no additional information on the structure is available. Preliminary work indicated charge heterogeneity of galactosyltransferase as revealed by activity measurements of crude enzyme preparations which were subjected to isoelectric focusing [lo]. The present investigation was carried out in order to characterize the charge heterogeneity of pure galactosyltransferase. Since reports dealing with electrophoretic variants associated with cancer have recently been published [11--131, it seemed of interest to correlate the patterns obtained for the Abbreviations. Asialo-mucin, sialic-acid-free ovine submaxillary mucin; butyl-PBD, 2-(~-tert-butylphenyl)-5-(4-bisphenylyl)-1,3,4-oxadiazole.Enzynw. UDP-galactose :N-acetylglucosamine galactosyltransferase (EC 2.4.1.22). milk enzyme to the enzymes isolated from other sources, such as malignant ascites and amniotic fluid. The latter was chosen in order to investigate the possible occurrence of an onco-fetal variant in ascites (for review see [14]). The present work describes a higher electrophoretic mobility of galactosyltransferases from ascites and amniotic fluid under nondenaturing conditions when compared to the milk enzyme. This difference was due solely to ...

Two different galactosyltransferase activities have been found in normal sera from A and 0 donors. Galactosyltransferase A incorporated galactose from UDP-Gal into sialic-acid-free ovine submaxillary mucin (asialo-mucin), whereas galactosyltransferase B transferred galactose from UDP-Gal to free N-acetylglucosamine or N-acetylglucosamine-glycoproteins. Specificity, kinetic and stability differences permitted the distinction of the activity of galactosyltransferase A from that of galactosyltransferase B ; the only substrate found for galactosyltransferase A was asialo-mucin, whereas galactosyltransferase B showed only low activity towards asialo-mucin and free N-acetylgalactosamine, but had a main specificity for either free N-acetylglucosamine or N-acetylglucosamineprotein. Galactosyltransferase B was more stable on heat inactivation than galactosyltransferase A ; galactosyltransferase B could be separated from galactosyltransferase A by affinity chromatography on N-acetylglucosamine-derivatized agarose.The products of both enzyme activities have been analyzed. The galactosyltransferase A product was cleaved from asialo-mucin by alkaline-borohydride treatment. The acceptor used to identify the galactosyltransferase B product was free N-acetylglucosamine. Periodate oxidation studies performed on the reduced disaccharides indicated the linkage type of the products. The anomeric configuration of the respective galactosyltransferase products were determined with specific galactosidases. Using these methods, galactosyltransferase A was found to form a Galfl(1+3)GalNAcprotein linkage and galactosyltransferase B was found to form a Galfl(1+4)GlcNA~-linkage.Galactosyltransferase activity in human and animal sera has been extensively studied in relation to different pathophysiological conditions [I -51. Among the various correlations, galactosyltransferase activity has been found increased [6] or qualitatively changed in cancer [7-91. Most of the above cited work has been carried out with asialo-agalacto-fetuin as acceptor for galactose, under the tacit assumption that this acceptor exhibits only N-acetylglucosamine residues; however Spiro and Bhoyroo [lo] recently published a more complete study of the carbohydrate structure of fetuin, showing that 21 % of all of the carbohydrate is O-glycosically linked. Since Schachter Abbreviations. GlcNAc, N-acetylglucosamine; GalNAc, Nacetylgalactosamine; GalNAcol, N-acetylgalactosaminitol; asialomucin, sialic-acid-free ovine submaxillary mucin; butyl-PBD, 2 -(~-tert-butylphenyl)-5-(4-biphenylyl)-l,3,4-Oxadiazole; UDP-Gal, uridine diphosphate galactose.Enzymes. Galactosyltransferase B (EC 2.4.1.22); galactosyltransferase A (EC 2.4.1. -).et al. [ l l ] have found a 'mucin-type' of galactosyltransferase in porcine submaxillary glands, the question arose whether serum contains different galactosyltransferase activities which cannot be distinguished when asialo-agalacto-fetuin is used as acceptor. Kim et al. [12] already observed significant incorporation of galactose into asialo-...

No abstract

No abstract