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Sulfotransferase Activity Assays

Introduction

Sulfation is an important post-translational modification that occurs on glycans, proteins, steroids, hormones, neurotransmitters, and xenobiotics. It can result in changes in their chemical, physical, and biological properties such as solubility, activity, and affinity. In humans, there are 48 sulfotransferases. More than half of them are Golgi resident enzymes involved in sulfation on carbohydrates. R&D Systems currently offers several recombinant carbohydrate-specific sulfotransferases (CHST; Table 1). Activities of these recombinant sulfotransferases have been tested on various oligosaccharides, polysaccharides, and proteoglycans.

Table 1. R&D Systems Enzymes
 
Recombinant Enzyme Catalog #
Human CHST1 5316-ST
Human CHST2 5107-ST
Human CHST4 5357-ST
Human CHST6 5326-ST
Human GalNAc4S-6ST 3365-ST
Human HS6ST1 5057-ST
Mouse CHST1 5355-ST
Mouse CHST5 5210-ST
Mouse CHST7 5108-ST
Mouse HS6ST3 5406-ST

Warnings and Precautions

These assays involve the use of radioactive material. Use appropriate precautions.

Materials

Note: The assay buffers, enzyme dilutions, and substrates vary depending on the enzyme that is being assayed. Consult the specific assay protocol (Steps 1 and 2) for the assay buffer and enzyme and substrate dilutions that should be used in each assay.

Assay Buffers

  • Assay Buffer 1: 25 mM MES, 0.5% (w/v) Triton® X-100, 2.5 mM MgCl2, 2.5 mM MnCl2, 1.25 mM CaCl2, 0.75 mg/mL BSA, pH 7.0
  • Assay Buffer 2: 25 mM MES, 0.5% (w/v) Triton X-100, 2.5 mM MgCl2, 2.5 mM MnCl2, 1.25 mM CaCl2, 0.75 mg/mL BSA, 0.3 mg/mL Protamine (Sigma, Catalog # P4005), pH 7.0
  • Assay Buffer 3: 0.1 M Sodium Acetate, pH 5.0

 

  • Gel Running Buffer: 40 mM Tris, 40 mM Acetic Acid, 1 mM EDTA, pH 8.0
  • Recombinant Enzyme (See table below)
  • Substrate (See table below)
  • Adenosine 3’-phosphate 5’-phosphosulfate lithium salt hydrate (PAPS) (Sigma, Catalog # A5508), 1 mM stock solution in 5% ethanol, 95% deionized water
  • 35S-PAPS, ~0.1 mM, 1 mCi/mL, prepared in-house (1-3)
  • 2X or 5X SDS gel loading buffer
  • 8% SDS-PAGE (approximately 15 cm x 20 cm, 20 lanes per gel)
  • Blotting paper

 

  • Gel dryer
  • Glogos® II autorad markers (Stratagene, Catalog # 420202) or equivalent
  • Blue sensitive medical X-ray film
  • X-ray film cassette
  • Film developer (Konica SRX-101A Medical Film Processor) or equivalent
  • Liquid scintillation counter (Beckman Coulter, Model # LS5000TD) or equivalent (Note: Sensitivity of the scintillation counter may affect linearity when measuring low levels of activity)
  • Liquid scintillation fluid (Beckman Coulter, Catalog # 141349)

Assay Protocols

Recombinant Enzyme Alternate Name Catalog # Substrate
Human CHST1 KSGal6ST 5316-ST Recombinant human Aggrecan
(Catalog # 1220-PG)
Human CHST2 GlcNAc6ST1, GST2 5107-ST GlcNAcbeta1-6Man
Human CHST4 GlcNAc6ST2, LSST, GST3 5357-ST GlcNAcbeta1-6Man
Human CHST6 GlcNAc6ST-5,
C-GlcNAc6ST		 5326-ST GlcNAcbeta1-6Man
Human GalNAc4S-6ST BRAG 3365-ST Chondroitin Sulfate
Human HS6ST1   5057-ST Heparan Sulfate
Mouse CHST1 KSGal6ST 5355-ST Recombinant human Aggrecan
(Catalog # 1220-PG)
Mouse CHST5 GlcNAc6ST3, I-GlcNAc6ST 5210-ST GlcNAcbeta1-6Man
Mouse CHST7 GlcNAC6ST4, GST5, C6ST2 5108-ST GlcNAcbeta1-6Man
Mouse HS6ST3   5406-ST Recombinant human Syndecan-4
(Catalog # 2918-SD)

References

  1. Robbins, P.W. (1962) Methods in Enzymology, Vol. V, New York: Academic Press, Inc., p. 964.
  2. MacRae, I.J. et al. (2000) Biochemistry 39:1613.
  3. Wu, Z.L. et al. (2002) Faseb J. 16:539.

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