BCH6206exam1-2006 - BCH 6206 First Exam Thursday, September...

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BCH 6206 First Exam Thursday, September 28, 2006 Frost (72) _________________ O’Brien (14) _______________ Stevens (7) ________________ Kilberg (7)_________________ Total (100) ________________
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Name:______________________________ 2 1.) (5 points) Shown to the right is picture of centrifugation tube, loaded with different concentrations of sucrose. Describe the type of centrifugation that this represents, the principles behind the technique, and an example for which this application would be useful. 2.) The Figure that accompanies this question represents a western blot (immunoblot) of a membrane protein (protein X, shown as a doublet) whose expression is thought to regulate a signaling system that is associated with improved response to cancer drugs. In this experiment, three different drugs were tested for their effect on protein X. Answer the two questions below. 2a.) (3 points) Three different membrane fractions from a liver cancer cell line were collected to assess the expression of protein X. How would you determine the identity of each of these membrane fractions? (You may pick specific subcellular compartments to assist in your discussion, if you chose). 2b.) (3 points) Experimentally, do you have sufficient information about the effect of the drug to recommend one over the others? Please state why you have come to your conclusion, whether your answer is yes or no. 1 2 3 ABC ABC ABC protein X expressed in fraction Membrane fraction Drug
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Name:______________________________ 3 3.) (6 points) The P-glycoprotein multidrug transporter (Pgp) acts as an ATP-powered efflux pump for a large variety of hydrophobic drugs, natural products, and peptides. To understand its normal biological function, the following experiment was performed. Pgp was isolated from plasma membrane of a highly drug resistant cancer cell line (CH R B30). The resulting preparation was >85% pure (meaning that 85% of the protein was Pgp). Pgp was then reconstituted into lipid vesicles containing a fluorescently-labeled phosphatidylethanolamine (among of the phospholipids). This fluorescently-labeled PE is abbreviated NBD- PE. The experiment actually has two parts. In the first part, phospholipids (including NBD-PE) were reconstituted in the absence of Pgp. As in other “liposomes”, a bilayer of phospholipids was formed. Fluorescence (from NBD-PE) was measured in an instrument (called a fluorimeter) at a wavelength specific for NBD. Sodium dithionate was then added to the vesicles which eliminates the fluorescence of only those NBD- PE molecules that are in the outer leaflet. A second fluorescence reading was taken. In the second part of the experiment, phospholipids (including NBD-PE) were reconstituted in the presence of Pgp (along with ATP which is required for its function). Fluorescence was measured before and after addition of sodium dithionate.
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This note was uploaded on 06/07/2011 for the course BCH 6206 taught by Professor Staff during the Spring '08 term at University of Florida.

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BCH6206exam1-2006 - BCH 6206 First Exam Thursday, September...

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