Characterization of a protein:glucosyltransferase activity in human platelets.

ABSTRACT

Human platelets exhibited significant glucosyltransferase activity, that transfer [14C]glucose from UDP-Glc to an endogenous protein acceptor. The enzyme proteinglucosyltransferase responsible for the catalysis was characterized and compared with glycogenglucosyltransferase. We describe a partial separation of both activities, the ratio of proteinglucosyltransferase/glycogenglucosyltransferase varied from 71 in a crude homogenate of platelets to 361 in the Sephadex G-100 column. This procedure failed to separate the proteinglucosyltransferase from its endogenous acceptor. Glucosylation of protein demonstrated dependence with respect to time and both protein and UDP-Glc concentration, and was saturated by very low concentration of donor and acceptor substrates. It was inhibited 76% by 5 mM Mn2+ concentration and was activated 23 and 11% by 5 mM concentrations of Ca2+ and Mg2+, respectively. With respect to glycogenglucosyltransferase, when the effect of time, protein, and substrate concentration were determined under identical conditions, it did not show the same dependence. At 5 mM concentration, Mn2+, Ca2+, and Mg2+ were activators of the enzyme 43, 80, and 200%, respectively. On the basis of these characteristics, we conclude that the synthesis of glucoprotein and glycogen are catalyzed by two distinct enzymes. Addition of exogenous glycogen (range 0.002-1%) inhibited the proteinglucosyltransferase, whereas at 0.001-0.007% concentration it was acceptor substrate for glycogenglucosyltransferase activity. At higher concentrations this activity was strongly inhibited. The concentration of glycogen in platelets could play a regulatory role in forming the glucoprotein and the glycogen molecules.