Sevier E D, Shannon L M
Biochim Biophys Acta. 1977 Apr 27;497(2):578-85. doi: 10.1016/0304-4165(77)90214-8.
A particulate enzyme preparation from horseradish root tissue was shown to catalyze the transfer of 2-acetamido-2-deoxy-D-[14C1]glucose from uridine diphosphate 2-acetamido-2-deoxy-D-[14C1]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 radio-labeled 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 degrees C for 3 min prior to assay. Trypsin treatment caused a 14% decrease in label incorporated while pronase treatment caused a 93% decrease,
已证明，从辣根根组织中提取的一种颗粒状酶制剂可催化2-乙酰氨基-2-脱氧-D-[14C1]葡萄糖从尿苷二磷酸2-乙酰氨基-2-脱氧-D-[14C1]葡萄糖转移至一种源自辣根过氧化物酶的外源受体分子。该受体是通过与琼脂糖共价结合的糖苷水解酶混合物作用于纯化的过氧化物酶而产生的。含有转移酶的膜制剂通过水相两相分配和不连续蔗糖密度梯度离心法纯化了约12倍。反应产物的水解产生氨基葡萄糖作为唯一的放射性标记物质。用抗过氧化物酶抗血清沉淀反应产物表明，放射性标记物已掺入过氧化物酶中。当仅从受体中去除12%的2-乙酰氨基-2-脱氧-D-葡萄糖时，转移酶对受体的利用效率最高。在测定前将受体加热至100℃ 3分钟，其接受能力没有丧失。胰蛋白酶处理使掺入的放射性标记物减少14%，而链霉蛋白酶处理使掺入的放射性标记物减少93%。
