Collagen glucosyltransferase from chick embryos and human foetal tissues was found to have a high affinity for columns of concanavalin-A-agarose, which was markedly reduced in the presence of α-methyl d-mannoside. The results suggest the presence of carbohydrate units in the enzyme molecule. However, high mannoside concentrations were required for an effective elution, and the peak of the enzyme activity was eluted in a retarded position compared with the protein peak. Elution was greatly enhanced by the addition of ethylene glycol to the buffer containing the mannoside, and most of the enzyme activity could be eluted with ethylene glycol even in the absence of the mannoside. These data suggest that hydrophobic interaction contributed to the affinity of the enzyme for concanavalin-A-agarose. The role of hydrophobic interaction was studied further by using valyl-agarose. Collagen glucosyltransferase became bound to this material in the presence of ammonium sulphate under conditions in which most of the other proteins did not, and it could be eluted with a decreasing ammonium sulphate concentration gradient. In addition, the enzyme could be eluted with ethylene glycol from two specific affinity columns, namely collagen-agarose and UDP-glucose-derivative-agarose, suggesting that hydrophobic interaction may stabilize the enzyme · substrate complex formed with these two columns. An application of this information on hydrophobic and carbohydrate recognition for improving the purification procedure for the enzyme is discussed.
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Anttinen et al. (1977) studied this question.