Partial purification and characterization of collagen galactosyltransferase from chick embryos.
Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.
D000645:Ammonium Sulfate; D000818:Animals; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008626:Mercuribenzoates / Q000494:pharmacology; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000494:pharmacology; D011489:Protein Denaturation; D013347:Subcellular Fractions / Q000378:metabolism
D008626:Mercuribenzoates; D011092:Polyethylene Glycols; D003094:Collagen; D005700:Galactosyltransferases; D000645:Ammonium Sulfate; D004229:Dithiothreitol