Experiment I, Part III - Tripeptide and Protein Modeling 3

Experimental In this experiment you will study the energy-dependent uptake (active transport) of radioactive methyl thiogalactoside (14C-TMG), a nonmetabolizable substrate. Since you will be attempting to produce accumulation of TMG against a concentration gradient, it is clear that an energy source is needed to drive the uptake. Yet the anaerobe, S. lactis 7962 cannot undertake oxidative phosphorylation. And the sample of cells that we prepare for you will have been washed free of all metabolizable substrates by several centrifugations in either distilled water or tris buffer. Thus the only way you can establish the protonmotive force necessary to drive uptake of TMG is by (1) changing the pH of the medium containing the cells, (2) artificially inducing a membrane potential (e.g., a K+-diffusion potential), or (3) adding a metabolizable substrate such as glucose, arginine, or galactose, which provide energy for anaerobic metabolism leading to the synthesis of ATP and the pH gradient and the membrane potential that result from ATP hydrolysis. You will measure the active transport of 14C-TMG by S. lactis cells driven by energy sources (1), (2), and (3). Therefore, in one experiment you will artificially generate a pH gradient across the cell membrane by lowering the pH of the medium containing the cells. In another experiment, you will artificially induce a membrane potential by using the K+-selective ionophore, valinomycin. Glucose will provide the energy source in the third experiment. The second experiment is based on the following considerations. Cells that were grown in a high potassium concentration medium can be considered to be K+-rich inside (even after washing, since the cell membrane is relatively impermeable to K+). If the cells are suspended in a K+-free medium (sodium phosphate buffer) there is a large K+gradient across the membrane, but the K+ diffusion potential is still zero because no K+ can move across the membrane. If valinomycin is added to the medium, a diffusion potential is immediately established that has a magnitude given approximately by the Nernst potential. VM=59mV log (K+in / K+out) Since K+out~0 in your experiment, you will be generating the maximum VM possible when you add valinomycin. Preparation, Filtration, and Timing Have 10 filters ready for each experiment and a place to put each of them after the sample has been taken. Before taking a sample, place a filter on the suction apparatus, fasten the chimney, and pass 5 ml of the appropriate buffer through the filter under suction. Keep the vacuum on until the sample has been taken and washed. When applying the cell sample to the filter, put it dead-center on the filter and then rapidly wash with 5 ml buffer. Cut the vacuum, remove the filter, and set up for another sample. The wet filters containing the cell samples should be placed at a labeled and 36 PROTECTED location to dry for at least half an hour (a gust of wind as a student walks by will scatter and mix the dried filters). After drying the filters, place each in a separate scintillation counting vial which has been appropriately labeled with 1/4-inch letters just below the threads for the lid. (Remember scintillations of light must be able to get out of the counting vial to reach the photomultiplier tube during the counting process.) the label on the scintillation vial should include your lab day, group symbol, experiment number, and sample number. A short time is required for the TMG concentration to come to equilibrium once the cells have been added to the TMG-containing buffer. If t = 0 is defined as the time at which you add the valinomycin, glucose, or acid to begin the uptake reaction, i.e., to energize the cells, then you should take samples at approximately -5, -3, and -1 minute in order to determine the equilibrium TMG distribution before cell energization. After energization, the first 3 samples should be taken as rapidly as possible. The last 4 samples can then be stretched out so that the last one is taken at about 10 minutes after energization. Before you begin each experiment I suggest making a table that has the sample number, anticipated time of sampling, and blanks for the actual time of sampling. In a day or two we will have counted your samples. The results will be posted in the course web site. In addition we will post the concentration of the stock cell suspension (mg dry cell weight per ml stock suspension). From this value you may calculate the total internal cell volume in your “uptake-reaction” mixture, since it has been determined that the internal volume of S. lactis is 1.83 l cell water per mg dry cell weight. We will also provide you with the specific activity of the 14C-TMG and with the counts per minute of the TMG-containing buffer solution to which you added cells to begin an uptake experiment. pH Experiment You will be provided with a “pH” buffer tube. this tube contains 14C-TMG and 0.1 M KCl in 1 ml of dilute (5 mM) KPO4 buffer at pH 7.5. Allow this tube to come to room temperature, add 0.2 ml of the stock cell suspension and begin taking samples. Wash these samples with the dilute pH 7.5 buffer (labeled “Dilute pH-wash”). Energize the cells at t = 0 by adding 0.2 ml of the 0.5 M KPO4 acidic (pH 4.7) buffer. The addition of this acidic buffer should bring the cell incubation medium pH down to about 5.5. Wash cell samples taken after energization with the pH 4.5 buffer (labeled “Acid pH wash”). Valinomycin Experiment You will be given a “VAL” buffer tube. This tube contains 14C-TMG in 1 ml 0.1 M NaPO4 buffer at pH 7.5. Allow this tube to come to room temperature, add 0.2 ml of the stock cell suspension, and begin taking samples. Wash the samples with NaPO4 pH 7.5 buffer (labeled “Val Wash”). Have the lab instructor add a few l of valinomycin solution when you want to energize the cells (make sure the instructor is nearby and has VAL before you begin the experiment). The same wash buffer can be used after energization. 37 Glucose Experiment This experiment will be run exactly as the VAL experiment, except that you will energize with 40 uL of 1 M glucose rather than with valinomycin. Write-Up 1. 2. 3. 4. 38 Plot counts/min vs. time for the three experiments. On the right hand vertical axis plot [TMG]in/[TMG]out. Discuss whether your results support the Mitchell Theory for the active uptake of nutrients by bacterial cells. Speculate about why some of your curves for 14C-TMG uptake rapidly decrease after several minutes. Why isn't the membrane potential VM =  when K+-containing cells are suspended in K+-free medium in the presence of valinomycin? MITOCHONDRIA: OXIDATION AND PHOSPHORYLATION Introduction In this experiment we will measure mitochondrial electron transport and the mechanism and efficiency of its coupling to the synthesis of ATP. Mitochondrial electron transport will be measured by measuring the consumption of O2 using the Clark polarographic electrode (whose operation is described in the Appendix). The tools for analyzing the system will be various substrates and inhibitors; the method for application of these tools will be measurement of their effect on the rate of ADT-dependent oxygen consumption. You are not provided with specific protocols for these experiments. Rather, you are in the position of those scientists who first developed the schemes for oxidative phosphorylation that we have studied and discussed outside the laboratory. To illuminate your path, you should consult the text and lecture notes for basic information about the mechanism of electron transport and oxidative phosphorylation. Biochemicals The solutions of substrates and inhibitors which will be provided in the laboratory are detailed in TABLE I Reagents. Many of these are expensive and should not be wasted. The amounts to be added can be calculated from the suggested dilutions from stock and a measurement of the volume of your incubation chamber. All acids have been adjusted to pH 7.2 with NaOH. These solutions may be added to the reaction chambers with Pipetmen micropipets. When making these additions, exercise care to minimize the introduction of bubbles. ADP (50 l) should be addeded accurately. Interpretation of your experiments will require knowing how much ADP has been added. Every effort will be made to provide an ADP solution whose A260 (1 cm) is 0.502 when diluted 1000-fold. The Merck Index gives 260 for ADP as 15.4 x 103 liters/mole cm. If the actual value for the A260 is different, you will be informed at the beginning of the laboratory. Mitochondrial Preparations Rat-liver mitochondria will be prepared by the instructors before you arrive, by the following procedure. The liver is homogenized in a medium containing 0.25 M sucrose, 0.003 M EDTA, and 0.005 M Tris-Cl, pH 7.4. A teflon-pestle Potter-Elvenhjem homogenizer (clearance, about 0.006 inches) operating at about 300 RPM is used and care is taken to keep the tube immersed in an ice bath during homogenization. After removal of nuclei and unbroken cells by centrifugation at 600 g for 10 minutes, the mitochondria are pelleted at 10,000 g for 15 minutes. In order to remove endogenous substrates, the mitochondria are washed twice with 0.25 M sucrose, 0.005 M Tris Cl, pH 7.4 by centrifugation at 10,000 g for 15 minutes. After the final wash, the 39 mitochondrial preparation is suspended in the sucrose-Tris solution to give about 25 mg protein/ml and stored on ice. Addition of about 0.3 ml of this suspension to incubation medium in the reaction chamber should result in a satisfactory rate of O2 consumption. If not, the amount of mitochondrial suspension added should be adjusted to give a rate (in the presence of substrate alone) which will result in the exhaustion of the O2 in the chamber in no more than 5 minutes. The incubation medium contains: Sucrose 0.10 M KCl 0.020 M Potassium Phosphate 0.003 M Tris-Cl 0.005 M pH 7.5 The function of the sucrose in all of the above solutions is to maintain isotonicity without increasing ionic strength; Tris-Cl is a buffer, and phosphate a requirement for oxidative phosphorylation. The EDTA in the homogenization medium serves to remove Ca++, which can act as an uncoupling agent. It is of the utmost importance that the mitochondria be kept cold until they are used; deterioration and uncoupling can result if they are warmed. Experimental: Oxygen Uptake An example of a typical experiment is attached, to give you an idea of how the data from your experiments might look. Consider this diagram carefully, so that you understand what each of the parts measures, and how each of the parts might vary given the presence of different substrates and/or inhibitors. Your expectations in this regard are the hypotheses which your experiments should test. Note that the graph contains a critical number: the concentration of O2 which is present at the beginning of the experiment. This number incorporates a conversion factor between voltage (what your recorder actually measures) and O2 concentration. This factor is only valid if the buffer is O2-saturated at the beginning of the experiment (before addition of mitochondria). To be sure that this conversion factor applies, it is essential that your buffer be well aerated, and temperature equilibrated (at room temperature) before beginning the experiment. Conversion of this concentration to moles of oxygen requires, in turn, that you know (and record) the total volume in the reaction chamber. Other hints: Once mitochondria have been added to the chamber, carry out the experiment without delay. After making an addition, it is necessary to observe the recorder tracing until the full effects of the addition have been displayed, after which further additions should be made quickly. For example, after addition of substrate, allow the reaction to proceed far enough to establish a straight 40 line on the recorder before making the next addition. After adding ADP, observe the stimulation of O2 uptake (if any); when the rate returns to a steady state at or near its pre-ADP slope, a further addition may be made if desired. Be sure to wash the chamber and electrode thoroughly after using an inhibitor. The first run with any new mitochondrial preparation should include a test for stimulation by ADP and note should be made of the respiratory control ratio (see below). A preparation which is not stimulated by ADP to give a control ratio of at least 3 is damaged and uncoupled and useless for many of the experiments described below. Suggested Experiments You should devise and carry out any experiments desired. Some suggestions: 1. Determine P:O ratios for several substrates and thereby deduce the relative contribution to phosphorylation within the electron transport chain. NOTE: It is very difficult to determine P:O ratios for ascorbate-TMPD, as the latter can act as an uncoupling agent. Addition of Mg++ (5-10 mM) has been reported to prevent this action. 2. Using inhibitors, establish the order of entry of various substrates into the electron transport chain. Where does the endogenous substrate enter? 3. Investigate whether the different uncouplers such as dinitrophenol, valinomycin, Ca++, or the electron shunt TMPD, all operate in the same way. References Packer, L. (1967). Experiments in Cell Physiology, Academic Press, NY. See experiments 14, 15, and 19. Estabrook, R. W. and M. E. Pullman (eds.). (1967). Methods in Enzymology, Vol. X, academic Press, NY. See articles 6 and 8. 41 TABLE I: Reagents Stock Concentration Suggested Final Concentration for Trial Item M Solvent M Sodium Succinate 1 H2O 5x10-3 Sodium Ketoglutarate 1 H2O 5x10-3 Sodium Hydroxybutyrate 1 H2O 5x10-3 Na ADP 0.03 H2O 3x10-4 2,4-Dinitrophenol 0.01 EtOH 10-4* Na Azide 1 H2O 10-2* Na Malonate 1 H2O 2x10-2* Oligomycin 1x10-3M EtOH 2x10-6*, (or 5x more) Na Ascorbate 1 H2O 5x10-3 Rotenone 1x10-4 EtOH 10-6* Antimycin 1x10-3 EtOH 10-6-10-5* TMPD 0.05 H2O 1x10-4, (or less) MgCl2 1 H2O 5 to 10x10-3* CaCl2 1 H2O 5 to 10x10-3* Valinomycin 1x10-4M EtOH 1x10-6* * concentrations should be increased if necessary 42 43 APPENDIX THE CLARK OXYGEN ELECTRODE The oxygen electrode (illustrated below) consists of a platinum cathode and a silver anode immersed in a KCl solution. this assembly is separated from the solution to be tested by a thin teflon membrane which is freely permeable to O2 but not to water, salts, and most other solutes. A potential of 0.6-0.8 volts is maintained across the electrode. In the presence of O2, the following reactions take place: Anode: 2Ag + 2Cl- -> 2AgCl + 2e- Cathode: 2H+ + 1/2 O2 + 2e- -> H2O Because these reactions depend on the availability of oxygen, the current which flows through the electrode is stoichiometrically proportional to the O2 concentration. This current may be carried through a standard resistance and the voltage across the resistance recorded with a strip-chart recorder, or the current may be passed into an operational amplifier current to voltage converter. With either circuit the output voltage (recorder deflection) is proportional to O2 concentration at the electrode membrane. 44