14. The assay may require to set up the measuring time points to
establish the linearity region of PPi production versus time.
15. The no-substrate control absorbance value should not increase
during the one-hour assay, as an indication that there is not too
much Pi or PPi contamination that could be transformed after
the 10 min “Pi-mop” preincubation. When the absorbance of
the no-substrate control is stable, the mean of the values
obtained in each time point can be calculated and subtracted
from the values of the experimental reactions.
16. Use the linear equation from a PPi standard curve (see Subheading 3.2) to quantify the PPi produced in each time point
and, after plotting it, select the linear range of PPi production
to calculate the enzyme activity (μM PPi min
À1
μg
À1
) for each
condition.
17. To build the pH curve represent the enzyme activities versus
the pH. Relative values can be calculated after giving a 100% to
the optimal performance.
18. Other softwares may be used to build the Michaelis–Menten
curve and calculate the enzyme kinetics. Using Prism, we
created an XY table selecting “Enzyme kinetics—Michaelis–
Menten” as sample data and added the triplicates of the
enzyme activities obtained for each substrate concentration.
To obtain the regression curve and the kinetic parameters
from the generated graph we performed a “Nonlinear regression”
analysis
selecting
“Enzyme
Kinetics—
Substrate vs. Velocity” and “Michaelis–Menten equation.” As
a result of the analysis, the regression curve is superimposed on
the graph and a table with the values of V max and K m together
with the statistical parameters is retrieved.
19. A standard curve with nine different PPi concentrations (0, 5,
10, 20, 30, 40, 50, 60, 75 μM) is enough to obtain the linear
regression equation with a square of the Pearson correlation
coefficient (R
2 ) > 0.99. Prepare at least three replicates per PPi
concentration to build the standard curve.
20. The manufacturer’s instructions indicate an incubation of
30–60 min, enough time to transform all the PPi in the reaction mixture into 2-amino-6-mercapto-7-methylpurine. We
incubated the Standard Curve 60 min adjusting the time to
the Standard Reaction for GGPPS activity measurement.
Around an hour is needed to (1) synthesize the GGPP and
release the PPi molecules and then to (2) consume the PPi by
the kit enzymes.
36
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n
establish the linearity region of PPi production versus time.
15. The no-substrate control absorbance value should not increase
during the one-hour assay, as an indication that there is not too
much Pi or PPi contamination that could be transformed after
the 10 min “Pi-mop” preincubation. When the absorbance of
the no-substrate control is stable, the mean of the values
obtained in each time point can be calculated and subtracted
from the values of the experimental reactions.
16. Use the linear equation from a PPi standard curve (see Subheading 3.2) to quantify the PPi produced in each time point
and, after plotting it, select the linear range of PPi production
to calculate the enzyme activity (μM PPi min
À1
μg
À1
) for each
condition.
17. To build the pH curve represent the enzyme activities versus
the pH. Relative values can be calculated after giving a 100% to
the optimal performance.
18. Other softwares may be used to build the Michaelis–Menten
curve and calculate the enzyme kinetics. Using Prism, we
created an XY table selecting “Enzyme kinetics—Michaelis–
Menten” as sample data and added the triplicates of the
enzyme activities obtained for each substrate concentration.
To obtain the regression curve and the kinetic parameters
from the generated graph we performed a “Nonlinear regression”
analysis
selecting
“Enzyme
Kinetics—
Substrate vs. Velocity” and “Michaelis–Menten equation.” As
a result of the analysis, the regression curve is superimposed on
the graph and a table with the values of V max and K m together
with the statistical parameters is retrieved.
19. A standard curve with nine different PPi concentrations (0, 5,
10, 20, 30, 40, 50, 60, 75 μM) is enough to obtain the linear
regression equation with a square of the Pearson correlation
coefficient (R
2 ) > 0.99. Prepare at least three replicates per PPi
concentration to build the standard curve.
20. The manufacturer’s instructions indicate an incubation of
30–60 min, enough time to transform all the PPi in the reaction mixture into 2-amino-6-mercapto-7-methylpurine. We
incubated the Standard Curve 60 min adjusting the time to
the Standard Reaction for GGPPS activity measurement.
Around an hour is needed to (1) synthesize the GGPP and
release the PPi molecules and then to (2) consume the PPi by
the kit enzymes.
36
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n