Xylosyl transfer to an endogenous renal acceptor. Purification of the transferase and the acceptor and their identification as glycogenin.
A xylosyltransferase in rat kidney, tentatively identified as glycogenin (Meezan, E., Ananth, S., Manzella, S., Campbell, P., Siegal, S., Pillion, D. J., and Rodén, L. (1994) J. Biol. Chem. 269, 11503-11508), was purified by a procedure in which affinity chromatography on UDP-glucuronic acid-agarose was a particularly useful step. The purified material was nearly homogeneous, as shown by SDS-polyacrylamide gel electrophoresis and silver staining, and had an electrophoretic mobility corresponding to a M(r) of 32,000. The purified enzyme possessed both glucosyl- and xylosyltransferase activity, and incubation with UDP-[3H]xylose or UDP-[3H]glucose yielded a single macromolecular product, which had the same electrophoretic mobility as the major silver-stained component. These results indicate that the kidney transferase was indeed glycogenin and that it was functionally analogous to the larger glycogenin species previously isolated from rabbit muscle. Further examination of the properties of the rat kidney enzyme showed, i.a., that it was inhibited strongly by cytidine 5'-diphosphate. This effect was used to advantage in an alternative purification procedure, which was applied to beef kidney and involved adsorption of the enzyme to UDP-glucuronic acid-agarose and subsequent elution with cytidine 5'-diphosphate. In contrast to glycogenin, glycogen synthase did not catalyze transfer from UDP-xylose, and it is suggested that the incorporation of xylose into glycogen observed by other investigators was due to glycogenin-catalyzed xylosyl transfer and subsequent chain elongation by glycogen synthase.
Rodén
L
L
School of Medicine, University of Alabama at Birmingham 35294.
Ananth
S
S
Campbell
P
P
Manzella
S
S
Meezan
E
E
D000818:Animals; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D005964:Glucosyltransferases / Q000378:metabolism; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D009844:Oligosaccharides / Q000096:biosynthesis / Q000302:isolation & purification; D010430:Pentosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013379:Substrate Specificity; D000097608:UDP Xylose-Protein Xylosyltransferase
D005964:Glucosyltransferases; D006023:Glycoproteins; D009844:Oligosaccharides; D010430:Pentosyltransferases; D000097608:UDP Xylose-Protein Xylosyltransferase; C051887:glycogenin