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 language | English (US) |
|---|---|
| Pages (from-to) | 34-41 |
| Number of pages | 8 |
| Journal | Molecular Pharmacology |
| Volume | 39 |
| Issue number | 1 |
| State | Published - Jan 1991 |
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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 journal › Article
}
TY - JOUR
T1 - 3′-Phosphoadenosine-5′-phosphosulfate
T2 - Photoaffinity ligand for sulfotransferase enzymes
AU - Otterness, Diane M.
AU - Powers, Stephen P.
AU - Miller, Laurence J
AU - Weinshilboum, Richard M
PY - 1991/1
Y1 - 1991/1
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0026034639&partnerID=8YFLogxK
M3 - Article
C2 - 1987450
AN - SCOPUS:0026034639
VL - 39
SP - 34
EP - 41
JO - Molecular Pharmacology
JF - Molecular Pharmacology
SN - 0026-895X
IS - 1
ER -