Affinity chromatography of collagen glucosyltransferase on a UDP-glucose derivative coupled to agarose.
UDP-glucuronic acid from the carboxyl group was coupled to agarose via a six-carbon atom spacer, and columns prepared from this material were used in an affinity chromatography of collagen glucosyltransferase. The enzyme was found to have a high affinity for such columns in the presence of Mn2+ in the buffer, whereas a considerably lower affinity was noted in the absence of such ions. The enzyme could be eluted from the column with either EDTA, UDP-glucose, or small peptides prepared from collagen, the peptides being the most effective eluting agent. After elution the enzyme was separated from the peptides by gel filtration. With this procedure a collagen glucosyltransferase putification of about 3000-fold was obtained from extract of chick embryos by relatively simple steps. Collagen galactosyltransferase was found to have no affinity for the column, suggesting that the binding was not only due to the UDP moiety, but that the uronic acid derivate of glucose also contributed to its specificity.
D000818:Animals; D002021:Buffers; D002642:Chick Embryo; D002846:Chromatography, Affinity; D003094:Collagen; D004492:Edetic Acid; D005964:Glucosyltransferases / Q000302:isolation & purification*; D008345:Manganese; D010446:Peptide Fragments; D011485:Protein Binding; D012685:Sepharose; D014532:Uridine Diphosphate Glucose; D014535:Uridine Diphosphate Glucuronic Acid; D014539:Uridine Diphosphate Sugars
D002021:Buffers; D010446:Peptide Fragments; D014539:Uridine Diphosphate Sugars; D014535:Uridine Diphosphate Glucuronic Acid; D008345:Manganese; D003094:Collagen; D012685:Sepharose; D004492:Edetic Acid; D005964:Glucosyltransferases; D014532:Uridine Diphosphate Glucose