Phosphatase-Coupled Sulfotransferase Assay

Brittany Prather, Cheryl M. Ethen, Miranda Machacek, & Zhengliang L. Wu

ABSTRACT

Sulfotransferases are a large group of enzymes that transfer sulfate from the donor substrate 3’-phosphoadenosine-5’-phosphosulfate (PAPS) to various acceptor substrates, generating 3’-phosphoadenosine-5’-phosphate (PAP) as a by-product. A universal phosphatase-coupled sulfotransferase assay is described here. In this method, Golgi-resident PAP-specific 3’-phosphatase (gPAPP) is used to release the 3’-phosphate from PAP, generating 5’-adenosine monophosphate (5’-AMP). In addition, CD73, a 5’-nucleotidase, can be used to release the 5’-phosphate from 5’-AMP to increase the assay sensitivity by two-fold. The released phosphate is then detected using Malachite Green phosphate detection reagents. This assay eliminates the need for both radioisotope labeling and substrate-product separation, and is high-throughput compatible. The assay allows for the kinetic analysis of all sulfotransferases that use PAPS as a donor substrate. Assay examples are given for the carbohydrate specific sulfotransferases CHST3 and CHST10, and the cytosolic sulfotransferases SULT1C4 and SULT1A1.

METHODS

SULFOTRANSFERASE REACTION

Assay Procedures

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STEP 1 In a 96-well plate, prepare a reaction mix containing the donor (PAPS), acceptor substrate, and coupling phosphatase (gPAPP). *The addition of the nucleotidase CD73 is optional. CD73 can increase phosphate release by two-fold. STEP 2 Initiate the reaction by adding a specific sulfotransferase. STEP 3 Stop the reaction and develop the color using Malachite Green Phosphate detection reagents (Catalog # DY996). Read the absorbance at 620 nm with a plate reader. Reaction Buffer: 25 mM Tris and 15 mM MgCl2 at pH 7.5.

gPAPP is a PAP-specific Phosphatase

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FIGURE 1. (A) Equal amounts of PAP (purple line) and PAPS (green line) were incubated (separately) with increasing concentrations of gPAPP for 20 minutes at 37 °C. Phosphate release is plotted versus enzyme dose. gPAPP selectively removes a phosphate group from PAP but not PAPS. (B) gPAPP (20 ng) enzyme activity is plotted versus substrate PAP.

gPAPP Kinetics.*

Activity of Mouse CHST3

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FIGURE 2. CHST3, also known as Chondroitin 6-O-Sulfotransferase, transfers sulfate to position 6 of GalNAc residues on chondroitin sulfate (CS). Activity of Recombinant Mouse Carbohydrate Sulfotransferase 3/CHST3 (Catalog # 5356-ST) was assayed in the presence of 0.5 micrograms of gPAPP. All reactions were carried out in 50 microliters for 20 minutes at 37 °C. (A) Activity versus acceptor substrate CS in the presence of 0.8 mM PAPS. (B) Activity versus donor substrate PAPS in the presence of 133 micrograms CS. (C) Activity versus enzyme dose in the presence of 0.2 mM PAPS and 250 micrograms CS.

Activity of Human CHST10

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FIGURE 3. CHST10 is the only sulfotransferase known to transfer sulfate to the terminal glucuronic acid of both protein- and lipidlinked oligosaccharides to synthesize HNK1, a sulfated glucuronyl-lactosaminyl residue found in many neural recognition molecules. Activity of Recombinant Human Carbohydrate Sulfotransferase 10/CHST10 (Catalog # 6140-ST) was assayed in the presence of 2 micrograms of gPAPP. All reactions were carried out in 50 microliters for 20 minutes at 37 °C. (A) Activity versus acceptor substrate phenolphthalein glucuronic acid (PGA) in the presence of 50 micromolar PAPS. (B) Activity versus donor substrate PAPS in the presence of 250 micromolar PGA. (C) Activity versus enzyme dose in the presence of 50 micromolar PAPS and 500 micromolar PGA.

Activity of Human SULT1C4

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FIGURE 4. SULT1C4, a cytosolic sulfotransferase, modifies steroids, neurotransmitters, and xenobiotics, and is involved in drug detoxification. Activity of Recombinant Human Cystololic Sulfotransferase 1C4/SULT1C4 (Catalog # 7095-ST) was assayed in the presence of 0.5 micrograms of gPAPP. All reactions were carried out in 50 microliters for 20 minutes at 37 °C. (A) Activity versus acceptor substrate alpha-Naphthol in the presence of 0.1 mM PAPS. Substrate inhibition (Ki) was observed with a alpha-Naphthol concentration greater than 0.2 mM. (B) Activity versus donor substrate PAPS in the presence of 1 mM alpha-Naphthol. (C) Activity versus enzyme dose in the presence of 0.4 mM PAPS and 1.4 mM alpha-Naphthol.

Activity of Human SULT1A1

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FIGURE 5. SULT1A1, a cytosolic sulfotransferase involved in drug detoxification, functions optimally at very low substrate levels, therefore, a highly sensitive assay is required to determine Km. Activity of Recombinant Human Cystololic Sulfotransferase 1A1/SULT1A1 (Catalog # 5546-ST) was assayed in the presence of 2 micrograms of gPAPP, and in some instances CD73 to increase sensitivity. All reactions were carried out in 200 microliters for 20 minutes at 37 °C. (A) Phosphate release versus enzyme dose in the presence of 250 micromolar PAPS and 250 micromolar pNP. The reaction was carried out in both the absence (orange line) and presence (purple line) of 0.2 micrograms CD73. (B) Activity versus acceptor substrate pNP in the presence of 25 micromolar PAPS. Substrate inhibition (Ki) was observed with a pNP concentration greater than 3 mM. (C) Activity versus donor substrate PAPS in the presence of 50 micromolar pNP.

CONCLUSIONS

• This phosphatase-coupled assay is universal to all sulfotransferases that use PAPS as the donor substrate.
• The assay is quantitative and allows accurate kinetic analysis.
• It is a convenient and cost effective assay because no radioisotope and no separation of products and substrates are required.
• The assay can be performed in a 96-well plate and provides a platform for high-throughput drug screening.

For research use only. Not for use in diagnostic procedures.

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