Photoaffinity labeling of human recombinant sulfotransferases with 2-azidoadenosine 3',5'-[5'-32P]bisphosphate.
Photoaffinity labeling with 2-azidoadenosine 3', 5'-[5'-32P]bisphosphate was used to identify and characterize adenosine 3',5'-bisphosphate-binding proteins in human liver cytosol and recombinant sulfotransferase proteins. The sulfotransferases investigated in these studies were the human phenol sulfotransferases, HAST1, -3, and -4, dehydroepiandrosterone sulfotransferase, and estrogen sulfotransferase. The cDNAs for these enzymes have been previously cloned and expressed in COS-7 cells or Escherichia coli. Photoaffinity labeling of all proteins was highly dependent on UV irradiation, was protected by co-incubation with unlabeled adenosine 3',5'-bisphosphate and phosphoadenosine phosphosulfate, and reached saturation at concentrations above 10 microM. To verify that the 31 35-kDa photolabeled proteins were indeed sulfotransferases, specific antibodies known to recognize human sulfotransferases were used for Western blot analyses of photolabeled proteins. It was shown unequivocally that the proteins in the 31-35-kDa region recognized by the antibodies also photoincorporated 2-azidoadenosine 3',5'-[5'-32P]bisphosphate. This is the first application of photoaffinity labeling with 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for the characterization of recombinant human sulfotransferases. Photoaffinity labeling will be also useful in the purification and functional identification of other adenosine 3',5'-bisphosphate-binding proteins and to determine amino acid sequences at or near their active sites.
Radominska
A
A
Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Drake
R R
RR
Zhu
X
X
Veronese
M E
ME
Little
J M
JM
Nowell
S
S
McManus
M E
ME
Lester
R
R
Falany
C N
CN
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D015239:Arylsulfotransferase / Q000302:isolation & purification / Q000378:metabolism*; D001345:Autoradiography; D001386:Azides / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004926:Escherichia coli; D006801:Humans; D008099:Liver / Q000201:enzymology; D010761:Phosphorus Radioisotopes; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015238:Sulfotransferases / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D000345:Affinity Labels; D001386:Azides; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D015238:Sulfotransferases; C019998:dehydroepiandrosterone sulfotransferase; D015239:Arylsulfotransferase; C020122:estrone sulfotransferase