Purification and Characterization of a Lymph Node Sulfotransferase Responsible for 6-O-Sulfation of the Galactose Residues in 2′-Fucosyllactose and Other Sialyl LewisX-Related Sugars

Shailubhai, Kunwar; Huynh, Quang Khai; Boddupalli, Hymavathi; Yu, Hong; Jacob, Gary S.

doi:10.1006/bbrc.1999.0258

Cited by 9 publications

(3 citation statements)

References 30 publications

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“…Two recent examples, pertinent to lymphocyte homing (Table 2), concern the identification of GlcNAc-6-O-sulfotransferase [26] and Gal-6-O-sulfotransferase [27] activities in extracts of porcine lymph nodes. Similarly, only a few examples of enzyme purifications will be mentioned.…”

Section: Biochemical Demonstration Of Carbohydrate Sulfotransferasesmentioning

confidence: 99%

“…Invariably, one of the purification steps employs the use of 3'5' ADP-agarose as an affinity matrix. One method for verifying the final product is to perform photoaffinity labeling on the product with [3'P]3'5'-ADP (32) or 8-azido[ ''PP]-PAPS (27). Typically, the various sulfotransferases are relatively minor proteins in the starting extracts, since purification factors range from 2,000-fold for conditioned medium [34] to 65,000-fold for a crude detergent extract [28].…”

Section: Biochemical Demonstration Of Carbohydrate Sulfotransferasesmentioning

confidence: 99%

See 1 more Smart Citation

Carbohydrate Sulfotransferases

Rosen¹,

Bistrup²,

Hemmerich³

2000

Carbohydrates in Chemistry and Biology

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Section: Biochemical Demonstration Of Carbohydrate Sulfotransferasesmentioning

confidence: 99%

Section: Biochemical Demonstration Of Carbohydrate Sulfotransferasesmentioning

confidence: 99%

Carbohydrate Sulfotransferases

Rosen¹,

Bistrup²,

Hemmerich³

2000

Carbohydrates in Chemistry and Biology

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“…Usually, sulfotransferase assays rely on chromatography steps, such as HPLC [12-14] and TLC [15-17], to separate substrates and products. However, HPLC is difficult to process multiple samples and TLC is unsuitable to separate proteins and polysaccharides.…”

Section: Introductionmentioning

confidence: 99%

A versatile polyacrylamide gel electrophoresis based sulfotransferase assay

Ethen

Larson

et al. 2010

BMC Biotechnology

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BackgroundSulfotransferases 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.ResultsA 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 α-naphthol 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.ConclusionThe 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 SDS-PAGE. Because phosphorylation is most similar to sulfation in chemistry, the method is likely to be applicable to kinases as well.

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