A simple sensitive fluorimetric assay of APS-kinase activity

Adenosine phosphosulphokinase (APS-kinase or ATP: adenylylsulphate 3′-phosphotransferase; EC 2.7.1.25) catalyses the formation of 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Its activity in various tissues was measured by transferring the sulphate from PAPS, a product of APS-kinase reaction, to 4-methylumbelliferone (4-MU) to form 4-MU-sulphate (4-MUS) using phenolsulphotransferase (PST) extracted from rat liver. Desalting with Sephadex G-25, together with the addition of EDTA effectively removed the Mg 2+ ions from the rat liver extract and thereby inhibited the APS-kinase activity therein in the subsequent PST reaction. 4-MUS formed was measured indirectly by a decrease in the fluorescence of 4-MU by a continuous fluorimetric assay. Kinetic data showed that the substrate, APS, at concentrations at and above 132 μM inhibited the APS kinase reaction. Pyrophosphate (PP) also inhibited the reaction. The apparent K m for APS was 14 μM. Two apparent K m values of 0.12 mM and 1.06 mM were obtained for ATP, while that for Mg 2+ was 0.09 mM.