Collagen glucosyltransferase. Partial purification and characterization of the enzyme from whole chick embryos and chick-embryo cartilage.
A purification of over 2000-fold is reported for collagen glucosyltransferase from Triton X-100 extract of whole chick embryos and one of about 160-fold from similar extract of chick embryo cartilage. The addition of the detergent more than doubled the enzyme activity in the homogenates. The purified enzyme preparations from whole chick embryos showed one major band and two or three minor bands in polyacrylamide gel electrophoresis and were entirely free of collagen galactosyltransferase activity. The molecular weight of collagen glucosyltransferase from both sources was about 52000 -- 54000, as determined by gel filtration. In some enzyme preparations an additional form was observed, with an elution position corresponding to a molecular weight of about 130000. Manganese was the most effective metal co-factor for the purified enzyme, but partial replacement could be obtained with Co2+, Mg2+ and Ca2+, whereas no replacement was found with other metals. The activity of the purified enzyme was stimulated by the addition of dithiothreitol to the incubation system and inhibited by preincubation with p-mercuribenzoate. UDP-glucose or the collagen substrate partially protected the enzyme against p-mercuribenzoate inactivation in the presence of Mn2+ but not in its absence. Some protection was also noted with Mn2+ alone.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002356:Cartilage / Q000201:enzymology*; D002642:Chick Embryo; D003035:Cobalt / Q000494:pharmacology; D003094:Collagen / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005700:Galactosyltransferases / Q000302:isolation & purification; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight
D003035:Cobalt; D008345:Manganese; D003094:Collagen; D005700:Galactosyltransferases; D005964:Glucosyltransferases; D008274:Magnesium; D002118:Calcium; D004229:Dithiothreitol