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- Title
A versatile polyacrylamide gel electrophoresisbased sulfotransferase assay.
- Authors
Wu, Zhengliang L.; Ethen, Cheryl M.; Larson, Sara; Prather, Brittany; Weiping Jiang
- Abstract
Background: Sulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes. Results: A universal sulfotransferase assay method based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is described. This assay has been successfully applied to substrates as small as anaphthol and as big as proteoglycans. As examples, we present the assays for recombinant human CHST4, TPST1, CHST3 and HS6ST1. In order to assess whether a small molecule can be applicable to this type of assay, a method to estimate the relative mobility of a molecule to PAPS is also presented. The estimated relative mobilities of various sulfated small molecules generated by SULT1A1, SULT1E1, SULT2A1 and CHST4 are in the range of ± 0.2 of the actual relative mobilities. Conclusion: The versatility of the current method comes from the ability that SDS-PAGE can separate proteins and small molecules according to different parameters. While mobilities of proteins during SDS-PAGE are inversely related to their sizes, mobilities of small molecules are positively related to their charge/mass ratios. The predicted relative mobility of a product to PAPS is a good indicator of whether a sulfotransferase can be assayed with SDSPAGE. Because phosphorylation is most similar to sulfation in chemistry, the method is likely to be applicable to kinases as well.
- Subjects
POLYACRYLAMIDE gel electrophoresis; ENZYMES; FIRE assay; SULFOTRANSFERASES; COLLOIDS
- Publication
BMC Biotechnology, 2010, Vol 10, p11
- ISSN
1472-6750
- Publication type
Article
- DOI
10.1186/1472-6750-10-11