Biosynthesis of PAPS in vitro by human liver : Measurement by two independent assay procedures

Abstract

The biosynthesis of 3'-phosphoadenosine-5'-phosphosulphate (PAPS) by extracts of human liver from inorganic sulphate and APS was assayed by transferring the “active sulphate” to two different acceptors, namely N-acetyldopamine (NADA) and 4-methylumbelliferone (4-MU). NADA-sulphate was quantified by an HPLC-ECD method while the decrease in 4-MU was monitored continuously by a fluorimetric procedure. The optimum pH was 8.0 for both the PAPS generation and APS-kinase reaction. The apparent K m value for APS determined by the fluorimetric and HPLC-ECD procedures was 17.6 and 16.8 μM, respectively. PAPS-generation measured in 13 samples of human liver showed excellent correlation between the two assay procedures, with correlation coefficients ( r) of 0.96 and 0.95 for PAPS generation from inorganic sulphate and APS, respectively. The fluorimetric assay was preferred as it is simple, equally sensitive and more reproducible. There was also a good correlation between the APS-kinase reaction and the two-step PAPS-generation from inorganic sulphate, with r = 0.97 and 0.91, as determined by the fluorimetric and HPLC-ECD procedures. The rate of PAPS generation from inorganic sulphate was only marginally less than that from APS in each of the 13 human liver samples, suggesting that the coupling of ATP-sulphurylase to APS-kinase facilitates “sulphate activation” and releases it from the constraints imposed by the unfavourable ATP sulphurylase reaction.