Abstract
A rapid and sensitive radiometric assay was developed for the measurement of 3'- phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis in rat skin extract. The formation of PAP 35S from sodium 35sulfate and ATP was quantified by the transfer of the 35sulfate to minoxidil by rat liver minoxidil sulfotransferase (MST). The assay is sensitive enough for the detection of as little as 2 pmol of PAP 35S. The PAPS-generating system showed a pH optimum of 8.6, with an apparent K m value of 1 mM for the ATP-Mg 2+ complex and 68 μM for sodium 35sulfate. ATP and Mg 2+, present individually or together in equimolar concentrations, were inhibitory above 8 mM. Excess (or free) ATP was a competitive inhibitor with respect to the ATP-Mg 2+ complex; the apparent K i , measured was 0.32 mM. The specific activity of the PAPS-generating system, measured in rat skin cytosol was 0.15 nmol PAPS/ min/mg protein. The importance of PAPS generation in detoxification and bioactivation of xenobiotics in skin is discussed.
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