Assignment III, Chapters 10 to 13 Chapter 10 10-18 Predict which of the following organisms will have the highest percentage of unsaturated fatty acid chains in their membranes. Explain your answer. A. Antarctic fish B. Desert iguana C. Human being D. Polar bear E. Thermophilic bacterium 10-25 The asymmetric distribution of phospholipids in the two monolayers of the plasma membrane implies that very little spontaneous flip-flop occurs or, alternatively, that any spontaneous flip-flop is rapidly corrected by phospholipid translocators that return phospholipids to their appropriate monolayer. The rate of phospholipid flip flop in the plasma membrane of intact red blood cells has been measured to decide between these alternatives. One experimental measurement used the two spin-labeled phospholipids described Figure 10 3. To measure the rate of flip-flop from the cytoplasmic monolayer to the outer monolayer, red blood cells with spin-labeled phospholipids exclusively in the cytoplasmic monolayer were incubated for various times in the presence of ascorbate and the loss of ESR signal was followed. To measure the rate of flip flop from the outer to the cytoplasmic monolayer, red blood cells with spin-labeled phospholipids exclusively in the outer monolayer were incubated for various times in the absence of ascorbate and the loss of ESR signal was followed. The results of these experiments are illustrated in Figure was 10-4. A. From the results in Figure 10 6, estimate the rate of flip flop from the cytoplasmic to the outer monolayer, and from the outer to the cytoplasmic monolayer. A convenient way to express such rates is as the half time of flip-flop that is, the time it takes for half the phospholipids to flip-flop from one monolayer to the other. B. From what you learned about the behavior of the two spin labeled phospholipids (Figure 10 3), deduce which one was used to label the cytoplasmic monolayer of the intact red blood cells, and which one was used to label the outer monolayer. C. Using the information in this problem, propose a method to generate intact red blood cells that contain spin labeled phospholipids exclusively in the cytoplasmic monolayer, and a method to generate cells spin-labeled exclusively in the outer monolayer.
Figure 10-3. Structures of two nitroxide labeled lipids. The nitroxide radical is shown at the top, and its position of attachment to the phospholipids is shown below. The label is redox sensitive and its sensitivity depends on its accessibility. Figure 10-6. Decrease in ESR signal intensity of red blood cells containing spin-labeled phospholipids in the outer monolayer (outside) and cytoplasmic monolayer (inside) of the plasma membrane (Problem 10 25 There are differences in the loss of the ESR signal due to the location of the nitroxide radical on the two phospholipids. The nitroxide radical in phospholipid 1 is on the head group and is therefore in direct contact with the external environment. Thus, it is quickly deactivated, eg. by a reducing agent. The nitroxide radical in phospholipid 2 is attached to the fatty acid chain and is therefore partially buried in the interior of the membrane. Thus, it is less accessible by reducing agents and therefore more stable. In addition, the interior of red blood cell is a reducing environment. Figure 10-4 Decrease in ESR signal intensity as a function of time in red cells and red cell ghosts in the presence and absence of ascorbate. (A and B) Phospholipid 1 and phospholipid 2 in red cells.
Chapter 11 11-14 Cells use transporters to move nearly all metabolites across membranes. But how much faster is a transporter than simple diffusion? There is sufficient information available for glucose transporters to make a comparison. The normal circulating concentration of glucose in humans is 5 mm, whereas the intracellular concentration is usually very low. (For this problem assume the internal concentration of glucose is 0 mm.) A. At what rate (molecules/sec) would glucose diffuse into a cell if there were no transporter? The permeability coefficient for glucose is 3 x 10-8 cm/ sec. Assume a cell is a sphere with a diameter of 20 µm. The rate of diffusion equals the concentration difference multiplied by the permeability coefficient and the total surface area of the cell (surface area = 4πr 2 ). (Remember to convert everything to compatible units so that the rate is molecules/ sec). B. If in the same cell there are 10 5 GLUT3 molecules (Km = 1.5 mm) in the plasma membrane, each of which can transport glucose at a maximum rate of 10 4 molecules per second, at what rate (molecules/ sec) will glucose enter the cell? How much faster is transporter-mediated uptake of glucose than entry by simple diffusion? Chapter 12 12-108 A classic paper describes a genetic method for determining the organization of a bacterial protein in the membrane of E. coli. The hydropathy plot of the protein in Figure 12-21 indicated three potential membrane spanning segments. Hybrid fusion proteins of different lengths, some with internal deletions, were made with the membrane protein at the N terminus and alkaline phosphatase at the C-terminus (Figure 12-22). Alkaline phosphatase is easy to assay in whole cells and has no significant hydrophobic stretches. Moreover, when it is on the cytoplasmic side of the membrane its activity is low, and when it is on the external side of the membrane (in the periplasmic space) its activity is high. The assayed levels of alkaline phosphatase activity are indicated (HIGH or LOW) in Figure 12 22. A. How is the protein organized in the membrane? Explain how the results with the fusion proteins indicate this arrangement. B. How is the organization of the membrane protein altered by the deletion? Are your measurements of alkaline phosphatase activity in the internally deleted plasmids consistent with the altered arrangement?
Figure 12-21. Hydropathy plot of a membrane protein (Problem 12 108). The three hydrophobic peaks indicate the positions of three potential membrane spanning segments. Figure 12-22. Structures of hybrid proteins used to determine the organization of a membrane protein (Problem 12 108). The membrane protein (orange segment) is at the N- terminus and alkaline phosphatase (blue segment) is at the C-terminus of the protein. The inverted V indicates the site from which amino acids were deleted from modified hybrid proteins. The most C-terminal amino acid of the membrane protein is numbered in each hybrid protein. The activity of alkaline phosphatase in each hybrid protein is shown on the right. Chapter 13 13-42 If you were to remove the ER retrieval signal from protein disulfide isomerase (PDI), which is normally a soluble resident of the ER lumen, where would you expect the modified PDI to be located?
13-48 Two extreme models vesicle transport and cisternal maturation have been proposed to account for the movement of molecules across the polarized structure of the Golgi apparatus In the vesicle transport model, the individual Golgi cisternae remain in place as proteins move through them (Figure 13 9A). By contrast, in the cisternal maturation model, the individual Golgi cisternae move across the stack, carrying the proteins with them (Figure 13-9B). Transport vesicles serve critical functions in both models, but their roles are distinctly different. Describe the roles of the transport vesicles1n each of the two models. Comment specifically on the roles of vesicles in the forward movement of proteins across the Golgi stack, in the retention of Golgi resident proteins in individual cisternae, and on the return of escaped ER proteins to the ER. Figure 13-9 Two models for the movement of molecules through the Golgi apparatus (Problem 13 48). (A) The vesicle transport model. (B) The cisternal maturation model. In (B), the individual cisternae have been separated for illustration purposes. ER, endoplasmic reticulum; CGN, cis Golgi network; TGN, trans Golgi network.