Characterization of a protein:glucosyltransferase activity in human platelets.
Human platelets exhibited significant glucosyltransferase activity, that transfer [14C]glucose from UDP-Glc to an endogenous protein acceptor. The enzyme protein:glucosyltransferase responsible for the catalysis was characterized and compared with glycogen:glucosyltransferase. We describe a partial separation of both activities, the ratio of protein:glucosyltransferase/glycogen:glucosyltransferase varied from 7:1 in a crude homogenate of platelets to 36:1 in the Sephadex G-100 column. This procedure failed to separate the protein:glucosyltransferase 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 glycogen:glucosyltransferase, 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 protein:glucosyltransferase, whereas at 0.001-0.007% concentration it was acceptor substrate for glycogen:glucosyltransferase 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.
Rodríguez
P
P
Laboratorio de Trombosis Experimental, Instituto Venezolano de Investigaciones Científicas, Caracas.
Durante
P
P
D001786:Blood Glucose / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D005964:Glucosyltransferases / Q000097:blood*; D006003:Glycogen / Q000097:blood / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D014532:Uridine Diphosphate Glucose / Q000097:blood
D001786:Blood Glucose; D001798:Blood Proteins; D008345:Manganese; D006003:Glycogen; C036655:1,3-alpha-D-glucan synthase; D005964:Glucosyltransferases; D008274:Magnesium; D002118:Calcium; D014532:Uridine Diphosphate Glucose