• Author / Uploaded
• Guhan

Citation preview

MEMBRANE CONFERENCE

1

PROBLEM #1 The primary role of platelets is to control blood clotting. When they encounter the exposed basement membrane (collagen fibers) of a damaged blood vessel or a newly forming fibrin clot, they change their shape from round to spiky and stick to the damaged area. At the same time they begin to secrete serotonin and ATP, which accelerate similar changes in newly arriving platelets, leading to the rapid formation of a clot. The platelet response is regulated by protein phosphorylation. Significantly, platelets contain high levels of two protein kinases: C-kinase (protein kinase C), which initiates serotonin release, and myosin light-chain kinase, which mediates the change in shape. When platelets are stimulated with thrombin, the light chain of myosin and an unknown protein of 40,000 daltons are phosphorylated. When platelets are treated with a calcium ionophore, only the myosin light chain is phosphorylated; when they are treated with diacylglycerol, only the 40 kd protein is phosphorylated. Experiments using combinations of calcium ionophore and diacylglycerol show that the extent of phosphorylation of the 40 kd protein depends only on the concentration of diacylglycerol (Figure 1510A); however, serotonin release depends on diacylglycerol and the calcium ionophore (Figure 15-10B). A. Based on these experimental observations, describe the normal sequence of molecular events that leads to phosphorylation of the myosin light chain and the 40 kd protein. Indicate how the calcium ionophore and diacylglycerol treatments interact with the normal sequence of events. B. Why do you think serotonin release requires both calcium ionophore and diacylglycerol?

2

PROBLEM #2 After prolonged exposure to hormones that bind to β-adrenergic receptors, cells become refractory and cease responding. To examine this desensitization phenomenon, you use a newly developed reagent, CGP-12177, which is a hydrophilic molecule that specifically binds to β-adrenergic receptors. In contrast to the binding of dihydroalprenolol, which is hydrophobic, CGP-12177 binding exactly parallels the decrease in hormone-dependent adenylyl cyclase activity observed in extracts from cells treated with isoproterenol for increasing times (Figure 15-16). To understand the difference in receptor binding by these two molecules, you lyse untreated cells and isoproterenol-treated (desensitized) cells, fractionate them by centrifugation through sucrose-density gradients and measure binding by dihydroalprenolol and CGP-12177 (Figure 15-17). In addition to ligand binding, you also measure 5'-nucleotidase activity, which is a marker enzyme for the plasma membrane. A. Give an explanation for the differences in binding by dihydroalprenolol and CGP-12177. B. What do you think might be the basis for isoproterenol-induced desensitization in these cells?

3

PROBLEM #3 Insulin is a small protein hormone that binds to a receptor in the plasma membrane of fat cells. This binding dramatically increases the rate of uptake of glucose into the cells. The increase occurs within minutes and is not blocked by inhibitors of protein synthesis or glycosylation. Therefore, insulin must increase the activity of the glucose transporter in the plasma membrane without increasing the total number of transporters in the cell. The two experiments described below suggest a possible mechanism for the insulin effect. In the first experiment, the initial rate of glucose uptake in control and insulintreated cells was measured, with the results shown in Figure 1. In the second experiment, the concentration of glucose transporter in fractionated membranes from control and insulin cells was measured, using the binding of radioactive cytochalasin B as the assay as shown in Table 1. A. Deduce the mechanism by which glucose transport is increased in insulin-treated cells. (Hint: cytochalasin B binds specifically to glucose transporters). B. Transport proteins, like enzymes, can be characterized by the kinetic parameters Km (the concentration of substrate at which the rate of transport is half-maximal) and Vmax (the rate of transport achieved at saturating substrate concentration). Does insulin stimulation alter either of these kinetic properties of the glucose transporter? How can you tell from these data?

Table 11-2 Amount of Glucose Transporter Associated with the Plasma Membrane and Internal Membranes in the Presence and Absence of Insulin (Problem 3) Bound 3H-Cytochalasin B (Cpm/mg vesicle protein) _______________________________ Untreated cells Treated cells Membrane Fraction (- insulin) (+ insulin) Plasma membrane 880 4480 Internal membranes 4070 480

4

PROBLEM #4 Much of our knowledge of the topography of plasma membrane components has been gained by study of the red blood cell. Since these cells contain no intracellular organelles, the plasma membrane can be easily isolated free from intracellular components by lysis in a hypotonic solution. The membranous sacs or “ghosts” so obtained can be sealed under certain experimental conditions (sealed right-side-out ghosts). They can also be turned “inside out” and sealed, thereby making the original cytosolic side accessible from the outside. Enzymatic digestion of sealed right-side-out red cell ghosts was originally used to determine the sidedness of the major membrane-associated proteins: spectrin, band 3, and glycophorin. These experiments made use of sialidase, which removes sialic acid residues from protein, and pronase, which cleaves peptide bonds. The proteins from normal ghosts and enzyme-treated ghosts were separated from SDS polyacrylamide-gel electrophoresis and then stained for protein and carbohydrate (Figure 105). A. How does the information in Figure 10-5 allow you to decide whether the carbohydrate of glycophorin is on the cytoplasmic or external surface, and how does it allow you to decide which of the red cell proteins are exposed on the external side of the cell? B. When you show your deductions to a colleague, she challenges your conclusion that some proteins are not exposed on the external surface and suggests instead that these proteins may be resistant to pronase digestion. What control experiment can you propose to test this possibility? C. How would you modify this enzymatic approach in order to determine which red cell proteins span the plasma membrane?

5

PROBLEM #5 You are studying the binding of proteins to the cytoplasmic face of cultured neuroblastoma cells and have found a method that gives a good yield of inside-out vesicles from the plasma membrane. Unfortunately, your preparations of inside-out vesicles are contaminated with variable amounts of right-side-out vesicles. Nothing you have tried avoids this variable contamination. A friend suggests that you pass your vesicles over an affinity column made of lectin coupled to solid beads. What is the point of your friend’s suggestion?

6