Figure 2.

APS kinase activity and APS binding properties of human PAPS synthases. A, APS kinase activity was measured in a coupled enzymatic assay where ADP production is linked to NADH consumption via pyruvate kinase and lactate dehydrogenase. 15 individual velocity measurements from two different batches are shown. Please refer to Table 1 for the averaged specific activity. B, APS binding studies where fluorescently labeled APS (1 μm mant-APS) was titrated with increasing concentrations of PAPS synthase protein. Data were fitted assuming one binding site. C, back-titration of 1 μm mant-APS and 50 μm PAPSS protein with increasing concentrations of APS. As mant-APS can be displaced by APS, the fluorescent mant moiety did not interfere with binding to the protein.