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cAMP 2nd Messenger Pathway and Experiments

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cAMP 2nd Messenger Pathway and Experiments

Postby mkay28 » Tue Sep 24, 2013 10:43 pm

The following experiments were performed hypothetically. My responses or thoughts are indicated after each question A through E by the ---.

A mutant cell line is obtained. The cell line is thought to not have adenylyl cyclase within it, and is called cyc-. You need to test that it doesn't have adenylyl cyclase. The following experiments are conducted:
Experiment 1: Cell membranes from cyc- cells were isolated and mixed with protein extracts from cells which exhibited adenylyl cyclase activity (cyc+ cells). Stimulating hormone, ATP, GTP, and Mg2+ were included in the mixture. The mixture was: cell membranes from cyc-, protein extracts from cyc+, and hormone, ATP, GTP, and Mg2+. Results were that upon hormone stimulation, cAMP was produced.
Experiment 2: The membranes of the cyc- cells were mixed with protein extracts from the cyc+ cells. However, the protein extract from the cyc+ cells was treated to inactiate adenylyl cyclase activity only. This adenylyl cyclase activity was definitely inactivated. Stimulating hormone, ATP, GTP, and Mg2+ were included in mixture. Mixture was: cell membranes from the cyc- cells, protein extracts from cyc+ cells with inactivated adenylate cyclase, and hormone, GTP, ATP, and Mg2+. Results: upon hormonal stimulation, cAMP was still produced.

A. If experiment 1 was the only experiment performed, what would be your conclusion?
---If experiment 1 was the only experiment, I'd say the cAMP was produced based upon there being adenylyl cyclase within the protein extracts from the cyc+ cells.
B. Given the results from experiments 1 and 2, what is your conclusion about the existence of adenylyl cyclase in the mutant cyc- cell line? Is it really absent?
---It would have to be present somewhere within the cyc- cell line for the cAMP to be produced, based on the results of experiment 2 which show production of cAMP with the adenylyl cyclase from the cyc+ cells definitely being inactivated. Based off of this, if those same cyc+ cells were used in experiment 1, the adenylyl cyclase must still be present in the cyc- cells, as cAMP cannot be produced without this enzyme.
C. Based on knowledge of hormone action, explain results of experiment 2. Why is cAMP still produced in experiment 2?
---There could be an issue with the receptor that comes before adenylyl cyclase, so ATP is unable to be converted into cAMP. cAMP may still be produced within experiment 2 because based on the previous question, we believe the cyc- cells may have adenylyl cyclase present to convert ATP to cAMP.
D. What would happen in experiment 1 AND 2 if ATP or GTP or Mg2+ were eliminated from the reaction? Why? Why is each element important?
---Mg2+ increases adenylyl cyclase activity, which increases agonist affinity for the hormone receptor. It interacts with GTP regulatory site to prohibit the GDP from binding. If ATP or GTP were eliminated from the reaction, it would not be able to produce any cAMP. GTP functions to activate the adenylyl cyclase, and if this were missing, a major portion of the pathway would be missing. Additionally, ATP is what is converted to cAMP, so without any ATP, the conversion into cAMP could not occur.
E. What is the physiological significance of these results? Why would this be important in the cAMP 2nd messenger system?
---The physiological significance of these results is that the cAMP pathway needs all of these elements present in order to carry out the reaction and take effect within the cell.
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Postby JackBean » Wed Sep 25, 2013 11:21 am

A. seems reasonable
B. unfortunately they do not specify, how was it inactivated (nor how was it mutated in the cyc- cells, but I guess they do not know that), so it's little hard to propose something. Anyway, there are several classes of adenylate cyclases, so they probably eliminated only one gene while others were unaffected. Moreover, they do not know for sure whether cyc- cells were really missing the AC acitvity, so obviously it was not. Probably the activity remained even after the mutation
C. not mutated? Other homologues?
D. ATP is substrate, Mg2+ is cofactor, GTP triggers the G-proteins
E. Other homologues are present?

Cis or trans? That's what matters.
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Postby mkay28 » Wed Sep 25, 2013 3:14 pm

I really like your explanation of B. I would think that it would be probable that the cyc- cells (which are only believed to have no adenylyl cyclase present) may actually have adenylyl cyclase present, or that there is a different class of the adenylate cyclase that can act in the place of the adenylyl cyclase if it is indeed absent from the cells. I'd take a good guess and assume that the adenylyl cyclase activity was still in the cyc- cells, as they don't know for sure or trust that the other lab really eliminated the adenylyl cyclase from the cyc- labelled cells.

Thanks for your help! I'm feeling a little more confident about my thoughts regarding this experiment.
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