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.