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. 2011 Aug 11;6(8):e23172. doi: 10.1371/journal.pone.0023172

Figure 6. Assay for adenosine 5′-phosphosulfate kinase (APSK) from Penicillium chrysogenum.

Figure 6

A) An APS curve. All reactions were initiated with 0.12 mM ATP, 0.1 µg of APSK and 0.3 µg CD39L2 in 150 µL assay buffer at room temperature and proceeded for 10 minutes. The OD was plotted versus the APS concentration and the apparent Km (designated as Km′) was visually estimated to be about 10 µM. B) An ATP curve. All reactions were performed in the presence of 100 µM APS, 0.2 µg of APSK and 0.3 µg of CD39L2 in 150 µL assay buffer at room temperature and proceeded for 15 minutes. The obtained OD readings (purple) were first corrected by background subtraction (blue) and then corrected by the ATP inhibition factors (red) and finally fit with the Michaelis-Menten equation to obtain a Km of 126 µM. C) An APSK enzyme curve. All reactions were performed with 1 mM ATP, 0.1 mM APS and 0.14 µg CD39L2 in 50 µL assay buffer at room temperature for 20 minutes. The OD was plotted versus APSK input. The slope of curve was converted to a specific activity, 1125 pmol/min/µg, using Eq.10 and r = 0.61.