3′-Phosphoadenosine-5′-phosphosulfate: Photoaffinity ligand for sulfotransferase enzymes

Diane M. Otterness, Stephen P. Powers, Laurence J Miller, Richard M Weinshilboum

Research output: Contribution to journalArticle

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Abstract

Sulfation is an important pathway in the biotransformation of many drugs, xenobiotic compounds, neurotransmitters, and hormones. The sulfate donor for these reactions is 3′-phosphoadenosine-5′-phosphosulfate (PAPS). We set out to determine whether PAPS might serve as a photoaffinity ligand for sulfotransferase enzymes. UV irradiation of [35S]PAPS with partially purified human liver thermostable (TS) phenol sulfotransferase (PST) radioactively labeled a protein with a molecular mass of 35 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoaffinity labeling of TS PST with [35S] PAPS did not require the presence of a phenolic substrate but rather was inhibited by p-nitrophenol, a sulfate acceptor substrate for TS PST. Inhibitors of TS PST enzymatic activity, including 3′-phosphoadenosine-5′-phosphate, ATP, ADP, and 2,6-dichloro-4-nitrophenol, also inhibited photoaffinity labeling of the 35-kDa protein with [35S]PAPS, in a concentration-dependent fashion, with IC50 values of 14 μM, 2.1 mM, 7.7 mM, and 91 μM, respectively. The 35-kDa protein that was radioactively labeled by [35S]PAPS in the presence of UV light coeluted with TS PST enzymatic activity during gel filtration high performance liquid chromatography. [35S]PAPS was then used to photoaffinity label another sulfotransferase enzyme, the thermolabile (TL) form of PST partially purified from human liver. Therefore, [35S]PAPS appears to be a photoaffinity ligand that could be used to study a variety of PAPS-dependent sulfotransferases. Photoaffinity labeling of TS and TL PST, as well as other PAPS-dependent sulfotransferases, should enhance our ability to purify this important group of enzymes and to determine amino acid sequences at or near their active sites.

Original languageEnglish (US)
Pages (from-to)34-41
Number of pages8
JournalMolecular Pharmacology
Volume39
Issue number1
StatePublished - Jan 1991

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Phosphoadenosine Phosphosulfate
Sulfotransferases
Arylsulfotransferase
Ligands
Enzymes
Photoaffinity Labels
Proteins
Liver
Xenobiotics
Ultraviolet Rays
Biotransformation
Sodium Dodecyl Sulfate
Adenosine Diphosphate
Inhibitory Concentration 50
Sulfates
Gel Chromatography
Neurotransmitter Agents
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence
Catalytic Domain

ASJC Scopus subject areas

  • Pharmacology

Cite this

3′-Phosphoadenosine-5′-phosphosulfate : Photoaffinity ligand for sulfotransferase enzymes. / Otterness, Diane M.; Powers, Stephen P.; Miller, Laurence J; Weinshilboum, Richard M.

In: Molecular Pharmacology, Vol. 39, No. 1, 01.1991, p. 34-41.

Research output: Contribution to journalArticle

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