A 96-well dot-blot assay for carbohydrate sulfotransferases.
Here we describe an efficient dot-blot assay for high-throughput screening of two enzymes, heparan sulfate N-deacetylase/N-sulfotransferase (NDST-1) and high-endothelial cell GlcNAc-6-sulfotransferase (HEC-GlcNAc-6-ST). The assay proceeds by transfer of 35S-labeled sulfate from [35S]-3(')-phosphoadenosine-5(')-phosphosulfate (PAPS) to the free amino groups of de-N-sulfated heparin (NDST-1), or the 6-hydroxyl groups of N-acetylglucosamine residues linked to a polyacrylamide scaffold (HEC-GlcNAc-6-ST). The 35S-labeled products are then captured on an appropriate membrane, taking advantage of their polymeric architecture. In one step, 35S-labeled by-products are then eluted from the membrane, leaving spatially separated 35S-labeled product "dots" for subsequent quantification. This assay allows for direct product detection on the membrane, obviating excessive washing and elution steps endemic to other assays. The assay was validated by measuring K(M) values for PAPS and K(I) values for PAP, the product of sulfuryl transfer. The assay method should be useful for inhibitor screens for both enzymes. In addition, the general assay architecture should be readily applicable to high-throughput screens of other carbohydrate sulfotransferases.
Verdugo
Dawn E
DE
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Bertozzi
Carolyn R
CR
D050260:Carbohydrate Metabolism; D002854:Chromatography, Paper; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007700:Kinetics; D015203:Reproducibility of Results; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000032:analysis* / Q000378:metabolism*; D013997:Time Factors; D000097811:Carbohydrate Sulfotransferases
D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C021350:heparitin sulfotransferase