Assay of Collagen-Galactosyltransferase and Collagen-Glucosyltransferase Activities and Preliminary Characterization of Enzymic Reactions with Transferases from Chick-Embryo Cartilage.

Procedures are described for the assay of collagen galactosyltransferase and collagen glucosyltransferase activities. The methods are based on the transfer of [¹⁴C]galactose or [¹⁴C]glucose from the corresponding radioactive UDP-glycoside to hydroxylysyl or galactosylhydroxylysyl residues in calf-skin gelatin substrate, and on the specific assay of the products of the enzymic reactions. The assay of the products includes precipitation of the protein, and removal of most of the radioactivity present in the free UDP-glycoside by several washings of the precipitate. The [¹⁴C]galactosylhydroxylysine or [¹⁴C]glucosylgalactosylhydroxylysine is then liberated by alkaline hydrolysis, and purified further by a modified Dowex chromatography and by paper electrophoresis. The procedure is entirely specific when judged by amino acid analysis, and it is possible to assay a series of 36 samples in 2 days. The sensitivity of the method permits assays in extracts from several tissues, including tissues with low transferase activities, such as liver. Preliminary characterization of the transferases from chick embryo cartilage indicated that the galactosyltransferase did not catalyze the transfer of galactose to free hydroxylysine. Dialyzable peptides were found to act as substrates for both transferases. The K_m for the gelatinized collagen substrate was 150 g/1 in the reaction with the galactosyltransferase, and 14 g/1 in that with the glucosyltransferase. The K_m for UDP-galactose or UDP-glucose in the reaction with the corresponding transferase was about 30 μM. Both transferases required a metal co-factor, and manganese was found to be far more effective than any other metal tested. The optimal manganese concentration for both transferases was 10 mM. No stimulation of the glucosyltransferase activity was found with folate, which has recently been reported to stimulate collagen glucosyltransferase from rat kidney. [ABSTRACT FROM AUTHOR]