Direct measurement and regulation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) generation in vitro.
3'-Phosphoadenosine 5'-phospho[35S]sulfate (PAPS) biosynthesized from inorganic [35S]sulfate and ATP was separated from its radiolabeled precursor by reversed-phase paired-ion HPLC and quantified by on-line radiometric detection. This single-step procedure circumvents several problems inherent in conventional sulfotransferase-coupled assays employed in the measurement of PAPS formation. A good correlation was observed between the rate of PAPS generation assayed in several mammalian tissues measured by direct HPLC-radiometry and by coupling to the sulfation of minoxidil or 4-methylumbelliferone. Both AMP and ADP inhibited the rat liver sulfate-activating enzymes competitively with respect to MgATP2-, and the rate of PAPS production was decreased with decreasing ratios of [ATP]:[ADP] and [ATP]:[AMP]. It is possible that these adenine nucleotides regulate sulfate activation by kinetic control and by negative feedback modulation.
Wong
K O
KO
Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore. bchsitkp@nus.sg
Wong
K P
KP
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006168:Guinea Pigs; D000882:Haplorhini; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D009711:Nucleotides / Q000494:pharmacology; D010724:Phosphoadenosine Phosphosulfate / Q000032:analysis* / Q000096:biosynthesis*; D011874:Radiometry / Q000379:methods; D051381:Rats; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases
D009711:Nucleotides; D013431:Sulfates; D000249:Adenosine Monophosphate; D010724:Phosphoadenosine Phosphosulfate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D015238:Sulfotransferases