An enzymatic procedure for the preparation and purification of 3'-phosphoadenosine 5'-phospho-[35S]sulfate ([35S]PAPS): applications in syntheses of 8-azido and 8-bromo derivatives of [35S]PAPS.
This paper describes a rapid and an efficient procedure for the enzymatic synthesis of 3'-phosphoadenosine 5'-phospho[35S]sulfate ([35S]PAPS). [35S]PAPS was synthesized by incubating ATP and a carrier-free [35S]-Na2(35)SO4 with ATP sulfurylase, a recombinant APS kinase and inorganic pyrophosphatase. The transfer of 35SO4 group from [35S]Na2SO4 to [35S]PAPS proceeded more efficiently in the presence of an ATP-regenerating system composed of pyruvate kinase and phosphoenol pyruvate. About 90% of the radioactivity present in the starting material [35S]Na2SO4 was transferred to [35S]PAPS within a 2-h reaction incubation. The reaction products were applied to a Mono Q column, and [35S]PAPS was eluted by a step-wise gradient of triethylamine bicarbonate buffer (pH 7.5). Under these conditions, [35S]PAPS eluted as a sharp peak at 0.7 M triethylammonium bicarbonate and it was very well separated from other contaminants. The purified [35S]PAPS (yield 85%, purity > 95%) was functional in donating sulfate to an oligosaccharide acceptor in a standard sulfotransferase reaction. The enzymatic procedure described above was particularly useful for the synthesis of [35S]PAPS at a wide range of concentrations and specific activities (up to 1500 Ci/mmol). This generally useful approach was also found to be successful in the syntheses of 8-azido and 8-bromo derivatives of [35S]PAPS. Applications of these two derivatives of PAPS, for purification and identification of sulfotransferases, have also been discussed.
Shailubhai
K
K
Department of Immunology, G. D. Searle Company (a subsidiary of Monsanto Company), St. Louis, Missouri 63167, USA, KKSHAI@ccmail.monsanto.com
Singh
R K
RK
Schmuke
J J
JJ
Jacob
G S
GS
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D007700:Kinetics; D010724:Phosphoadenosine Phosphosulfate / Q000302:isolation & purification*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D013430:Sulfate Adenylyltransferase / Q000378:metabolism
D001386:Azides; C051606:8-bromoadenosine 5'-triphosphate; D010724:Phosphoadenosine Phosphosulfate; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D013430:Sulfate Adenylyltransferase