Abstract
A particulate enzyme preparation from horseradish root tissue was shown to catalyze the transfer of 2-acetamido-2-deoxy- d-[ 14C 1]glucose from uridine diphosphate 2-acetamido-2-deoxy- d-[ 14C 1]glucose to an exogenous acceptor molecule derived from horseradish peroxidase. The acceptor was produced from purified peroxidase by the action of a mixture of glycoside hydrolases covalently bound to Sepharose. The membrane preparation containing the transferase was purified approximately 12-fold by aqueous two phase distribution and by discontinuous sucrose density gradient centrifugation. Hydrolysis of the reaction product yielded glucosamine as the only radiolabeled substance. Precipitation of the reaction product by antiserum against peroxidase showed that the label was incorporated into peroxidase. The transferase utilized the acceptor most efficiently when only 12% of the 2-acetamido-2-deoxy- d-glucose was removed from the acceptor. The acceptor lost no accepting capabilities when heated to 100°C for 3 min prior to assay. Trypsin treatment caused a 14% decrease in label incorporated while pronase treatment caused a 93% decrease.
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