Chapter 3 I MEMBRANE TRANSPORT The cell membrane, or plasma membrane, is the outermost layer of the cell. It completely surrounds the protoplasm or living portion of the cell, separating the cell s interior from the environment. Because it is so thin, the plasma membrane is not visible under the light microscope. It is visible under the electron microscope, however, where it appears as a double line structure with a total width of approximately 7.5 nm. A widely accepted model of biological membranes, the fluid mosaic model, is shown in Figure 5-2. The basic structure of the membrane is a phospholipid bilayer. Embedded into this bilayer of lipids are proteins, some of which span the width of the membrane. The membrane can be envisaged as a two-dimensional fluid in which its constituents, lipids and proteins, can move around laterally, but cannot change their inside-out orientation ( flipflop ) easily. Figure 5-2. Fluid Mosaic Model As a result of the molecular composition and physical arrangement of its components, biological membranes have some very important properties. An essential one for the maintenance of life is called differential or selective permeability. This property, which results from the structure of the lipid bilayer and the presence of specific transport proteins, allows certain molecules to pass through the membrane (either into or out of the cell) and prevents the passage of others. Hence, the primary function of the cell membrane is to maintain the integrity of the cell in relation to its environment. Before discussing in more detail the function of the cell membrane, we must discuss the ways in which molecules move from one place to another. All molecules move by one of two processes: diffusion or active transport. Diffusion is merely the physical movement of molecules along a concentration gradient, from a high concentration to a low concentration. It is passive in the sense that it does not involve an outside source of energy. The movement is the result of the kinetic energy of the molecules themselves. Active transport on the other hand is a selective process in which molecules can move against a concentration gradient at the expense of an outside source of energy. In the cell, this outside source of energy is the energy derived from the cell's metabolism. In this exercise we will only concern ourselves with the passive movement of particles. Of particular importance in biological systems is the diffusion of molecules or ions in aqueous solution. The rate of diffusion (the speed with which the particles move) increases as the temperature increases, as the concentration gradient steepens, and as the size of the particles decreases. 5-1
In the cell the process of diffusion is complicated by the presence of the membrane system. All molecules moving into or out of the cell have to pass through the cell membrane. Because of its semi-permeable nature the membrane will allow water molecules and certain other small molecules in solution to pass through it freely, but it restricts the passage of ions and larger, polar molecules such as sugars. The diffusion of water molecules through the cell membrane is called osmosis. Osmosis is the process by which all molecules of water enter or leave the cell. The direction in which the water molecules move, either into the cell or out of the cell, is dependent upon the osmotic state of the cell's environment. When the concentration of water molecules is the same both inside the cell and outside of the cell, the condition is said to be isotonic and water molecules will move into and out of the cell at the same rate; there is no net change of the water concentration on either side of the membrane. When the concentration of water is higher outside than inside, the condition is hypotonic, and the cell gains water. This condition occurs, when there are more solutes in the cytoplasm than in the environment. A cell which is in a hypotonic solution will take up water and become turgid or rigid. In contrast, a cell in a hypertonic solution, a solution that has a higher solute concentration than the cytoplasm, will lose water and become plasmolyzed. The three osmotic states are illustrated in Figure 3-3. hypotonic solution isotonic solution hypertonic solution (Cell turgid) (Cell plasmolyzed) Figure 5-3. Osmotic states of the cell A. Diffusion Through an Artificial Membrane In this section you will design an experiment to test the abilities of various molecules to travel across an artificial membrane. Note that the artificial membrane selects only on the basis of size, while natural membranes also select on the basis of characteristics such as polarity or charge. The pores in the membrane will allow molecules smaller than 35000 daltons in molecular weight (approximately equivalent to a protein of 320 amino acids in size) to cross. Select one of the available solutions to put inside your dialysis bag, and predict which molecules you expect to move across the membrane and what results you expect to see as a result of the movement of those molecules. You should be able to note movement of molecules based on changes in the weight of the bag and the results of colorimetric tests for specific compounds. Once you have established your protocol, set up your experiment and let it incubate while you perform the rest of the laboratory. 5-2
Available solutions to place inside the dialysis bag: Albumin solution or 1/10 th strength albumin (protein; record % concentration for each). Albumin has a molecular weight of approximately 68,000 daltons. Glucose solution or 1/10 th strength glucose (sugar; record % concentration for each). Glucose has a molecular weight of 180 daltons. Tests to detect molecules present in your solution: PROCEDURE 1. As a group of four, select one pair of solutions from the above list. 2. Plan how to detect the presence or absence of protein or simple sugars in your chosen solution and in the distilled water from the beaker. Set up the appropriate test tubes, including controls. (Hint: You may wish to review some of the tests you did in Chapter 2) 3. Each pair should obtain a 15 cm piece of dialysis tubing that has been soaked in distilled water. Tie a knot in one end of the tubing, so that you have a leak proof seal. 4. One pair of your group: Fill the tubing about ½ full with the solution of your choice. 4a. Second pair: dilute the solution 1:10 with distilled water (1 ml solution to 9 ml distilled water for a total volume of 10 ml; make sure to rinse the graduated cylinder very well first!) and fill the tubing about ½ full with the diluted solution. 5. Squeeze out as much air as you can and tie the open end of each bag shut with another leak proof knot. Make sure the walls of your bag are not taut. Rinse the bag with distilled water and dry thoroughly with a paper towel. 6. Weigh your bags and record the starting conditions in Table 5.1. 7. Place each bag into a 250 ml beaker containing distilled water. Stir or swirl the beaker at 15 minute intervals for the next two hours. 8. Record your predictions for movement of molecules across the cellulose membrane. 9. After two hours, remove the bag and dry it thoroughly again, then weigh it. Record the ending weight in Table 5.1. 10. Using the same tests you set up in step 2, test the solution from inside the bag and from the beaker again. Record your results in Table 5.1. Carefully wash all your glassware. 11. Pair up with another group that did the experiment with the other molecule. Record their results in Table 5.1 and compare them with your results. 5-3
Table 5.1: Initial Conditions (molecules predicted to be present outside bag, and test results) Full strength albumin (protein) 1/10 th strength albumin (protein) Full strength glucose (sugar) 1/10 th strength glucose (sugar) Initial Conditions (molecules predicted to be present inside bag, and test results) Predicted Movement of Molecules (relative osmotic states) Initial Weight of Bag Ending Weight of Bag Ending Conditions (test results for molecules present outside bag) Ending Conditions (test results for molecules present inside bag) Deduced Movement of Molecules 5-4
QUESTIONS What did you predict would happen to the weight of the bag? Why? What molecules did you think would be able to move across the membrane? Why? Were you able to demonstrate the movement of molecules across the membrane? How? Is the membrane freely permeable to all molecules? Can you make any statements about the selection of which molecules are able to cross? 5-5
ln the above exercise we demonstrated the selective permeability of an artificial cellophane membrane. In the living cell this same type of selective permeability is accomplished by the cell membrane. B. Diffusion Through Living Membranes 1. Plant cells: Elodea Because plant cells have a cell well, they may become turgid as indicated in fig 5.3, but will not burst. The pressure of the cell against the cell wall is a major factor in the ability of plants to stand upright in absence of a solid structural support. When the plants lose water or are subjected to a hypertonic solution, the loss of turgor pressure causes the plant to wilt as the cells become plasmolyzed. In a plasmolyzed cell, the cell membrane pulls away from the cell walls. PROCEDURE 1. Make three wet mount preparations of single Elodea leaves as follows a. Elodea leaf in distilled water b. Elodea leaf in solution from its container c. Elodea leaf in 10% NaCl 2. Observe the slides under the microscope at low and high power. Focus in on a single Elodea leaf cell with the high power objective. 3. Note the distribution of the chloroplasts in the cytoplasm of the cell. Diagram a typical Elodea leaf cell in the solution from it s container in the space below. 4. Observe the leaf cells in distilled water and 10% NaCl. Diagram cells in each of these conditions and answer the questions below. 5. Add a drop of distilled water to the edge of coverslip on the 10%NaCl slide. Pull the solution through by touching a paper towel to the opposite edge of the coverslip. Make sure you have pulled in the entire drop, then allow the slide to sit for a few minutes. Observe the cells. Cell in container solution Cell in Distilled water Cell in 10% NaCl 5-6
QUESTIONS 1. What was the osmotic state of the cell in distilled water? In the salt solution? (Hint: see fig 5.3) 2. What was the condition of the cells in the leaf after being in the salt solution? 3. Describe exactly what happened to the cells in the salt solution. 4. Describe exactly what happened to the cells after the salt solution was replaced with distilled water. 2. Animal cells: Red Blood Cells Animal cells lack a cell wall and thus are less resistant to osmotic lysis. Red blood cells (RBCs) in particular tend to lyse (blow up) in hypotonic solutions, a process called hemolysis. In a hypertonic solution, the cells will shrink, a process called crenation. Compare the effects of these solutions on red blood cells with the effects on plant cells. 5-7
PROCEDURE 1. Dilute one drop of blood in 4-5 drops of 0.9% NaCl. Make four slides as follows: a. Wet mount: one drop of blood b. Smear: one drop of blood (your TA will demonstrate) c. Wet mount: blood mixed 1:2 with distilled water d. Wet mount: blood mixed 1:2 with 10% NaCl 2. Observe the slides of blood only under the microscope at low and high power. Focus in on a single red blood cell with the high power objective. Diagram the normal appearance of a red blood cell. 3. Compare your other slides to the first. Diagram a cell under each condition and answer the questions below. 4. Draw several drops of distilled water under the coverslip of the 10%NaCl slide and observe the cells after a few minutes. Make a note of any changes. Normal RBC RBC in water RBC in 10%NaCl QUESTIONS 1. What happened to the RBCs in distilled water? What is the osmotic condition of the RBC in distilled water? 2. What happened to the RBCs in 10%NaCl? What is the osmotic condition of the RBC in this solution? 5-8
3. What happened (or what would you have expected to happen) to the RBCs after the 10%NaCl was replaced with distilled water? Explain. The differential permeability of cell membranes is a result of their specific physical and chemical structure. When this structure is disrupted, the property of differential permeability is destroyed. The red color of a beet is due to the presence of a water soluble pigment, anthocyanin, which is located within the cells, (enclosed by a cell membrane). The following experiment will analyze the effects of membrane disruption on permeability using anthocyanin as an indicator substance. C. Differential Permeability of Membranes PROCEDURE 1. Label 3 large test tubes A, B, and C. 2. Dice a segment of red beet into small cubes. Rinse the cubes in cold water several times, removing as much anthocyanin as possible. 3. Divide the cubed sections equally among the three test tubes. 4. Cover the beets in tubes A and B with tap water, cover the beets in tube C with absolute ethanol. 5. Leave tubes A and C for 10 minutes at room temperature, heat tube B in an 80 o C water bath for 10 minutes. 6. Shake each tube briefly, and observe the color of the supernatant in each of the tubes. Record your observations in the table on the next page 5-9
Tube Liquid Temperature Initial Color Final Color A Water RT B Water 80 o C C Alcohol RT Table 2: Color Changes of the Liquid Supernatant after Exposing a Beet Sample to Membrane Disrupting Conditions. QUESTIONS 1. Describe the permeability of the membrane to anthocyanin in cool tap water? What is the effect of boiling water? What is the effect of alcohol? 2. Which component(s) of membrane structure is (are) likely to be disrupted by boiling water? Explain. 3. Which component(s) of membrane structure is (are) likely to be disrupted by alcohol? Explain. 5-10