Properties and localization of the sulfate-activating system in rat brain.
The formation of the sulfate donor [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to beta-naphthol under the action of phenolsulfotransferase activity from rat brain cytosol, with the [35S]beta-naphthyl sulfate formed being isolated by polystyrene bead chromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chromatography. [35S]PAPS formation by a high-speed supernatant of rat cerebral cortex occurred with an optimal pH of approximately 7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+-ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1-5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micromolar concentrations inhibited the reaction. The apparent Km values of Mg2+-ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+-ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 2.7.7.4), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5'-phosphosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000227:Adenine Nucleotides / Q000096:biosynthesis*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015239:Arylsulfotransferase; D001921:Brain / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008297:Male; D010724:Phosphoadenosine Phosphosulfate / Q000096:biosynthesis*; D051381:Rats; D011919:Rats, Inbred Strains; D013431:Sulfates / Q000378:metabolism*; D013466:Sulfurtransferases / Q000378:metabolism
D000227:Adenine Nucleotides; D013431:Sulfates; D010724:Phosphoadenosine Phosphosulfate; D000255:Adenosine Triphosphate; D013466:Sulfurtransferases; D015239:Arylsulfotransferase