3'-Phosphoadenosine-5'-phosphosulfate: Photoaffinity ligand for sulfotransferase enzymes
- Mayo Clinic/Mayo Foundation, Rochester, MN (USA)
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.
- OSTI ID:
- 6213957
- Journal Information:
- Molecular Pharmacology; (USA), Vol. 39:1; ISSN 0026-895X
- Country of Publication:
- United States
- Language:
- English
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63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ENZYME INHIBITORS
BIOCHEMICAL REACTION KINETICS
TRANSFERASES
LABELLING
ADENOSINE
ADP
AFFINITY
ATP
AUTORADIOGRAPHY
DOSE-RESPONSE RELATIONSHIPS
ELECTROPHORESIS
ENZYME ACTIVITY
LIGANDS
LIQUID COLUMN CHROMATOGRAPHY
MOLECULAR WEIGHT
SULFATES
SULFUR 35
ULTRAVIOLET RADIATION
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ELECTROMAGNETIC RADIATION
ENZYMES
EVEN-ODD NUCLEI
ISOTOPES
KINETICS
LIGHT NUCLEI
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
RADIATIONS
RADIOISOTOPES
REACTION KINETICS
RIBOSIDES
SEPARATION PROCESSES
SULFUR COMPOUNDS
SULFUR ISOTOPES
550201* - Biochemistry- Tracer Techniques
560120 - Radiation Effects on Biochemicals
Cells
& Tissue Culture