Hypothesis A second messenger mediates between receptor activation at the plasma
ID: 167016 • Letter: H
Question
Hypothesis A second messenger mediates between receptor activation at the plasma membrane and enzyme activation in the cytoplasm. Results Active glycogen phosphorylase is present in the cytoplasm. Conclusion A soluble second messenger, produced by hormone-activated membranes, is present in the solution and activates enzyme in the cytoplasm. ANALYZE THE DATA The activity of previously inactive liver glycogen phosphorylase was measured with and without epinephrine incubation, with these results: A. What do these data show? B. Propose an experiment to show that the factor that activates the enzyme is stable on heating and give predicted data. C. Propose an experiment to show that cAMP can replace the membrane fraction and hormone treatment and give predicted data.Explanation / Answer
What does this data show?
Answer: Only homogenate - does not contains epinephrine and second messenger and therefore it does not activates glycogen phosphorylase. Enzyme activity value of 0.4 units is the assay baseline.
Homogenate + epinephrine: homogenate contains plasma membrane and cytoplasm fractions. When epinephrine is added to homogenate then a soluble second messenger was produced which activated glycogen phosphorylase. As a result the measured activity is 2.5units.
Cytoplasm fraction: Cytoplasm fraction alone does not contain epinephrine or second messenger. Therefore the enzyme glycogen phosphorylase is in inactive form and there is no measurable activity in the assay (0.2units is the assay baseline)
Cytoplasm + epinephrine: In this case also the second messenger needed to activate Glycogen phosphorylase is absent and there is no measurable activity.
Membrane + epinephrine: The enzyme glycogen phosphorylase is missing in this mixture, therefore activity can’t be measured.
Cytoplasm + membranes + epinephrine: In this case second messenger molecule is present (produced by epinephrine activated membranes) and therefore glycogen phosphorylase is activated and measured in assay (2.0 units)
This data suggests that production and interaction of a second messenger molecule is necessary for the activation of the glycogen phosphorylase enzyme. Only when a complete set of epinephrine + membrane containing epinephrine receptor + cytoplasm containing glycogen phosphorylase is present, then the second messenger (cAMP) would be able to diffuse into the cytoplasm and activate the enzyme. The activity can be determined and measured by the enzymatic assay.
2. Propose an experiment to show that the factor that activates the enzyme is stable on heating and give predicted data.
Answer: The second messenger molecule that activates the enzyme can be tested for heat stability in the following way: Membrane containing epinephrine is allowed to interact for some time (incubation) for the production of second messenger. Then remove the membrane by centrifugation leaving only the solution. Subject this solution to heat treatment before adding it to the cytoplasm containing the glycogen phosphorylase enzyme. Determine the activity of the enzyme and compare it with the control value. If the factor (second messenger) is heat stable then the enzyme activity should correspond to the similar value of 2.0 units.
3. Propose an experiment to show that cAMP can replace the membrane fraction and hormone treatment and give predicted data.
Answer: cAMP is the second messenger produced upon incubation of membrane (containing epinephrine receptors) and epinephrine hormone. To test whether cAMP can replace the membrane fraction and hormone treatment, simply add cAMP directly in a dose dependent manner to the cytoplasm containing glycogen phosphorylase enzyme. Test the enzyme for the activity. If cAMP is able to replace the membrane fraction and epinephrine hormone then a significant activity should be observed in the assay. The enzyme activity units in this case would depend upon the concentration of the cAMP used.
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