We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of V-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-D-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, Suggesting that alloxan indeed was inhibiting (OGT), In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0-10 mM alloxan, and OGT activity was measured directly by quantitating UDP-[H-3]-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions. OGT activity was completely inhibited by I mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT. and as Such, is the first OGT inhibitor described. (C) 2002 Elsevier Science (USA). All rights reserved.