Key to exam 1

Key to exam 1 - 1 Bio 167 Exam Fall 2000 Name: F. Scott Key...

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Bio 167 Exam Name: F. Scott Key Fall 2000 Below are 3 questions, each worth the indicated number of points. Good Luck 1) (40 points) You have identified the receptor for a new peptide hormone called gamma α . You know from previous work that gamma α causes macrophages to divide in culture. a) How would you determine the number of gamma α binding sites present on macrophage cells? Describe in broad outline how you would perform the experiment and include a generic diagram of what you would expect you results to look like. You would mix radio-labeled gamma α with macrophage cells, incubate for a short time. You would centrifuge the cells, wash off unlabeled ligand and count the amount of radioactivity left on the cells. You would also perform this binding experiment with an excess of unlabeled ligand to determine the relative contribution of non-specific binding. After subtracting the non-specific binding from the total binding, you would plot the amount of specific binding as a function of the concentration of the added gamma α . The maximal binding observed (asymptote of the curve) would be equal to the total number of binding sites on the macrophages, and the value at 50% of this is equal to the Km. b) You now wish to purify the receptor for gamma α from macrophages. Briefly describe what approach would you take? The best approach would be to solubilize the cells in a non-ionic detergent and then to purify the receptor over a column containing the gamma α ligand covalently attached to a Sepharose bead. c) You are successful in cloning the gamma α receptor. Analysis of the sequence reveals 7 transmembrane domains. Where in the cell in this receptor likely to be located and what is the likely immediate downstream target of this receptor? The receptor is likely to be a G-protein coupled receptor, and is thus likely to be localized in
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Key to exam 1 - 1 Bio 167 Exam Fall 2000 Name: F. Scott Key...

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