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vicster vicster
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10 years ago
1.   Role of Acarbose as an Invertase Inhibitor

This experiment is designed to help you determine whether acarbose functions as a competitive or noncompetitive inhibitor of invertase.

Set up an experiment at 50° C, buffer pH 4.0, at a of 50 mM with no acarbose. Run the experiment, determine the slope of the line, record data, and then repeat this type of experiment increasing concentration (keeping at 0.0 m M) in 10 mM increments until you have run experiments at 60 mM, 70 mM, 80 mM, and 90 mM. These uninhibited measurements will be important for studying the activity of invertase when it is inhibited by acarbose.

Repeat this series of measurements in the presence of 0.2 m M acarbose. Keep all other conditions the same as you did for the uninhibited measurements.

Plot a Lineweaver—Burk plot and/or an Eadie—Hofstee plot with both the uninhibited and inhibited data on the same plot as follows. Shift-click to select the five uninhibited measurements, select the plot you want (either Lineweaver—Burk or Eadie—Hofstee), then click Plot Selected Data. Return to the Data view by clicking the Data tab at the top of the screen. Shift-click to select the five inhibited measurements, then change the Data for Curve value to 2 (this indicates that the inhibited data will be plotted as the second curve on plot 1). Change the shape of the symbol to be plotted for these data by clicking on the popup menu for Symbol and choosing a different symbol for the inhibited data. Change the color of the inhibited data from the default, black, to another color using the Color popup menu, then click Plot Selected Data. You will now see a plot with two plotted lines. Your uninhibited data will be plotted in black and your inhibited data will be plotted in the color that you chose. Print this plot.

Determine Vmax and KM for the uninhibited and inhibited studies, then answer the following questions.

Compare your data from the inhibited reactions to your data from the uninhibited experiments. What did you find? Explain what happened to invertase activity as you increased . Why did this occur? What happened to Vmax in the presence of the inhibitor? What happened to KM? If either Vmax or KM changed, explain why.

Based on these results and what you already know about inhibitors of enzyme activity, is this inhibitor functioning as a competitive inhibitor or a noncompetitive inhibitor? How do you know? Explain your answers.


2.Role of DRI Inhibitor B as an Invertase Inhibitor

To investigate how DRI inhibitor B inhibits invertase, carry out the same sets of experiments, both uninhibited and inhibited, with DRI inhibitor B that you set up for acarbose. For inhibited measurements, set DRI inhibitor B to 25 mM. Note: If you did not delete the uninhibited measurements from your experiment with acarbose, you do not need to repeat the uninhibited experiments; you can use your data from the acarbose experiment.

Compare your data from the inhibited reactions to your data from the uninhibited experiments. What did you find? Explain what happened to invertase activity as you increased . Why did this occur? What happened to Vmax in the presence of the inhibitor? What happened to KM? If either Vmax or KM changed, explain why.

Based on these results and what you already know about inhibitors of enzyme activity, is this inhibitor functioning as a competitive inhibitor or a noncompetitive inhibitor? How do you know? Explain your answers.

Compare and contrast what happened as you increased with acarbose to what happened with DRI inhibitor B. Were the results the same or different? If the effect of on invertase was different in the presence of each inhibitor, explain why.
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