3'-Phosphoadenosine-5'-phosphosulfate: photoaffinity ligand for sulfotransferase enzymes.
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 microM, 2.1 mM, 7.7 mM, and 91 microM, 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.
Otterness
D M
DM
Department of Pharmacology, Mayo Clinic/Mayo Foundation, Rochester, Minnesota 55905.
Powers
S P
SP
Miller
L J
LJ
Weinshilboum
R M
RM
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000345:Affinity Labels; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D001345:Autoradiography; D001386:Azides; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D008099:Liver / Q000201:enzymology*; D009596:Nitrophenols; D010724:Phosphoadenosine Phosphosulfate; D010782:Photolysis; D012965:Sodium Chloride; D013223:Statistics as Topic; D013462:Sulfur Radioisotopes
D000345:Affinity Labels; D001386:Azides; D009596:Nitrophenols; D013462:Sulfur Radioisotopes; C054323:2,6-dichloro-4-nitrophenyl phosphate; C062179:2-iodo-4-azidophenol; D012965:Sodium Chloride; D010724:Phosphoadenosine Phosphosulfate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D015239:Arylsulfotransferase