Characterization of products synthesized by galactosyltransferase purified from the ascites of ovarian cancer patients.
Galactosyltransferase purified from the ascites of ovarian cancer patients can use to an equal extent N-glycosidic glycoproteins, such as asialoagalactofetuin, and O-glycosidic mucin, such as asialo bovine submaxillary mucin (BSM), as acceptors. Thermal treatment and substrate competition experiments demonstrated that the same enzyme catalyzed the transfer of galactose to both types of acceptors. Alkaline borohydride treatment showed that, while the product with asialoagalactofetuin was totally resistant, about 90% of the product with asialo BSM was hydrolyzed by this treatment. Gel filtration of the released oligosaccharides on a calibrated Biogel P-2 column showed three peaks. One major oligosaccharide (O-2) of size 5.7 glucose U and two minor peaks (O-1 and O-3) of sizes 8.7 and 3.7 glucose U, respectively, were obtained. The oligosaccharides were doubly labeled, first by incubation with uridine-diphosphate [14C]galactose, followed by alkali treatment in the presence of [3H]borohydride. The doubly labeled oligosaccharides were separately purified by gel filtration and ion-exchange chromatography and digested with various exoglycosidases. The digested products were characterized by gel filtration and paper chromatography in three different systems. From these results, the structures of these oligosaccharides were computed as follows: O-1 = beta-galactosyl-beta-N-acetylglucosamine-galactosaminitol (sialic acid); O-2 = beta-galactose-beta-N-acetylglucosamine-galactosaminitol; O-3 = beta-galactose-galactosaminitol. These results suggest that the galactosyltransferase from the ascites of ovarian cancer patients catalyzes the transfer of galactose to N-acetylglucosamine, irrespective of whether it is a part of an N-glycan or an O-glycan.
D000117:Acetylglucosamine / Q000378:metabolism; D001201:Ascites / Q000201:enzymology*; D001212:Asialoglycoproteins; D001667:Binding, Competitive; D001894:Borohydrides; D002845:Chromatography; D002850:Chromatography, Gel; D005260:Female; D060748:Fetuins; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D009077:Mucins / Q000378:metabolism; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000201:enzymology*; D013379:Substrate Specificity; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D001894:Borohydrides; D060748:Fetuins; D009077:Mucins; D009844:Oligosaccharides; D000509:alpha-Fetoproteins; C017029:asialofetuin; D014531:Uridine Diphosphate Galactose; C025364:sodium borohydride; D005700:Galactosyltransferases; D006026:Glycoside Hydrolases; D000117:Acetylglucosamine; D005690:Galactose