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. 2015 Jun 17;290(31):19133–19145. doi: 10.1074/jbc.M115.662866

FIGURE 7.

FIGURE 7.

Catalytic activity of GP12 with bis-pNPP and pNPP. A, reactions (100 μl) were performed with 5 mm bis-pNPP or pNPP and GP12 at the concentrations indicated. Left and middle panels show the pNP produced after 30 min. Data points are the mean from three independent experiments; errors bars are the S.D. Right panel shows that the initial rate of pNP production from bis-pNPP (black circles) and pNPP (open circles) was linear with GP12 concentration. B, HPLC analysis of the products from GP12 hydrolysis of bis-pNPP. After 30 min, reaction mixtures from A were injected onto a C18 reversed phase column. Separation was achieved by a 10-min gradient of 2–95% CH3CN + 0.1% TFA in ddH2O + 0.1% TFA. The product mixture was determined to contain 12.1 nmol of pNPP and 15.4 nmol of pNP, based on interpolation to standard curves. C, reactions of GP12 with bis-pNPP were assayed for pNP (black circles) and Pi (open circles), at the time points indicated. Data points are the mean from three independent experiments; error bars are the S.D. The rates of pNP and Pi production, determined from the slope of the linear regression line between 30 and 120 min, were 2.57 and 0.13 μm/min, respectively.