Preparation and quantification of 3'-phosphoadenosine 5'-phospho[35S]sulfate with high specific activity.
The synthesis and quantitation of the sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (PAP35S), prepared from inorganic [35S]sulfate and ATP, were studied. An enzymatic transfer method based upon the quantitative transfer of [35S]sulfate from PAP35S to 2-naphthol and 4-methylumbelliferone by the action of phenolsulfotransferase activity from rat brain cytosol was also developed. The 2-naphthyl[35S]sulfate or 35S-methylumbelliferone sulfate formed was isolated by polystyrene bead chromatography. This method allows the detection of between 0.1 pmol and 1 nmol/ml of PAP35S. PAP35S of high specific activity (75 Ci/mmol) was prepared by incubating ATP and carrier-free Na2 35SO4 with a 100,000g supernatant fraction from rat spleen. The product was purified by ion-exchange chromatography. The specific activity and purity of PAP35S were estimated by examining the ratios of Km values for PAP35S of the tyrosyl protein sulfotransferase present in microsomes from rat cerebral cortex. The advantage and applications of these methods for the detection of femtomole amounts, and the synthesis of large scale quantities of PAP35S with high specific activity are discussed.
Vargas
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Laboratoire de Physiologie, Université René-Descartes, Paris, France.
D000227:Adenine Nucleotides / Q000138:chemical synthesis*; D000818:Animals; D015239:Arylsulfotransferase / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D007553:Isotope Labeling / Q000379:methods; D008297:Male; D008861:Microsomes / Q000201:enzymology; D010724:Phosphoadenosine Phosphosulfate / Q000138:chemical synthesis*; D051381:Rats; D011919:Rats, Inbred Strains; D013154:Spleen / Q000201:enzymology; D015238:Sulfotransferases / Q000378:metabolism; D013462:Sulfur Radioisotopes
D000227:Adenine Nucleotides; D013462:Sulfur Radioisotopes; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; D015239:Arylsulfotransferase; C039079:protein-tyrosine sulfotransferase