Abstract
A thermostable phenol sulfotransferase, SULT1A1, has been implicated in numerous detoxification and bioactivation pathways; however, little is known regarding its endogenous function or its putative role in mediating risk for human environmental disease. A simple endpoint colorimetric assay is described that can be used for rapid phenotyping of SULT1A1 activity in human populations. The assay utilizes a microtiter-plate format and relatively small amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of 2-naphthylsulfate from 2-naphthol and 5′-phosphoadenosine 3′-phosphosulfate (PAPS), whereas addition ofp-nitrophenyl sulfate to the assay contributes to an effective PAPS-regenerating system. In contrast to other sulfotransferase assay methods, 3′-phosphoadenosine 5′-phosphate (PAP) does not accumulate during the incubation to interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitrophenyl sulfate to regenerate PAPS. This reaction concomitantly results in generation ofp-nitrophenol that can be quantified colorimetrically at 405 nm (ε = 18,200 M−1) to give an indirect measure of sulfotransferase activity. Using platelet enzyme preparations from adult human subjects, sulfation rates of two prototypical thermostable phenol sulfotransferase substrates (2-naphthol andp-nitrophenol) and one thermolabile phenol sulfotransferase substrate (dopamine) were determined using standard radiochemical protocols. These data were then compared with results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the phenotyping assay and radiochemical assays for both 2-naphthol sulfotransferase and p-nitrophenol sulfotransferase activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine sulfotransferase activity (r = 0.07) in these human platelet preparations. This inexpensive and rapid method for phenotyping SULT1A1 activity may help investigators assess a role for this enzyme in disease susceptibility.
Footnotes
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Send reprint requests to: Dr. Fred F. Kadlubar, Division of Molecular Epidemiology, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079. E-mail: fkadlubar{at}nctr.fda.gov
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↵1 Present address: Institute of Environmental and Human Health, Texas Tech University, Lubbock, TX 79416.
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↵2 Present address: Department of Pharmacology, National Institute of Health Sciences, Tokyo 158, Japan.
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This work was supported by National Cancer Institute Grants CA58697 and CA55751, by Environmental Protection Agency Grant R825280, and by National Institute on Aging Grant AG15722.
- Abbreviations used are::
- PST
- phenol sulfotransferase
- SULT1A1
- SULT1A2, isozymes of the thermostable phenol sulfotransferases
- SULT1A3
- isozyme of the thermolabile phenol sulfotransferase
- PAP
- 3′-phosphoadenosine 5′-phosphate
- PAPS
- 3′-phosphoadenosine 5′-phosphosulfate
- Received June 7, 1999.
- Accepted June 1, 2000.
- U.S. Government
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