UDP-N-acetylglucosaminyl transferase (OGT) is a key enzyme of a novel signal transduction pathway that regulates protein function through O-linked glycosylation. In the current study, we found that sodium vanadate potently inhibits OGT activity in brain cytosol (IC50 = 55 muM) and nucleosol (IC50 = 150 muM), but fails to alter activity of a related enzyme (UDP-galactosyl-transferase). Vanadate also inhibits OGT activity in cytosol (IC50 of 2.3 muM) and nucleosol (IC50 of 130) derived from a stable HeLa cell line that overexpresses OGT. When HeLa cytosol was immunopurified to separate OGT from other cellular proteins, vanadate still inhibited OGT activity (IC50 = 2 muM). We conclude that OGT derived from cytosol exhibits greater vanadate sensitivity than nucleosol OGT and that a large difference exists (25-fold) in vanadate sensitivity when comparing OGT activity in different cell types (IC50 of 55 muM for brain cytosol vs. 2.3 muM for HeLa cytosol). Understanding the mechanism(s) by which a tyrosine phosphatase inhibitor differentially reduces OGT activity should lead to new insights into OGT function and regulation. (C) 2004 Elsevier Inc. All rights reserved.