In the Laboratory
An in Vivo 13C NMR Analysis of the Anaerobic Yeast Metabolism of 1-13C-Glucose Brent J. Giles, Zenziwe Matsche, Ryan D. Egeland, Ryan A. Reed, Scott S. Morioka, and Richard L. Taber* Department of Chemistry, The Colorado College, Colorado Springs, CO 80903; *
[email protected] A discussion of intermediary metabolism is a large portion of the first-semester course in biochemistry. Yet few biochemistry laboratory experiments have been published that study metabolic pathways. Recently, Mega et al. reported an experiment that uses 13C NMR to follow glycolysis (1). With variations from this experiment, we also have been using NMR in undergraduate biochemistry to monitor, in real time, the anaerobic metabolism of 1-13C-glucose by Saccharomyces cerevisiae (Fleischmann’s Active Dry yeast). We wish to describe the application of this experiment to a study of how yeast maintains internal osmolarity when subjected to osmotic shock by saline solutions. The experiment uses readily available yeast, 1- 13C-labeled glucose, and a standard NMR spectrometer, and students can complete the exercise in one laboratory period. Two reviews of the application of 13C NMR to metabolic studies are listed in the references (2, 3). The experiment can be done in real time by incubating Fleischmann’s Active Dry yeast in an NMR tube containing a buffer. A small quantity of 1-13C-glucose is added to the yeast suspension and the tube is placed in the NMR spectrometer. Ethanol is the principle end product of yeast anaerobic metabolism of glucose. However, yeast also produces glycerol—initially, faster than ethanol (4 ). As the metabolism proceeds, ethanol production predominates until all the glucose is gone. Glycerol production is important to the yeast, since sending some of the carbon of glucose to glycerol rather than ethanol restores the NAD+/NADH coenzyme balance for the early stages of glycolysis. All cells must be able to control their volume in changing osmotic environments. They sometimes do this by increasing the concentration of a metabolite or by changing the permeability of the cell membrane (5). In our experiment, we increased the salinity of the buffer in which we incubated the yeast, and the cells responded by increasing the ratio of glycerol to ethanol. The experiment can be done quickly. There is a onehour incubation time to hydrate the yeast, and, depending on the activity of the yeast, the NMR data are collected over about a 30-minute period. Nevertheless, the single NMR spectrometer that is usually available to a class is a limiting factor in the experiment. As an alternative to the real-time studies, we have used a procedure developed by den Hollander et al. (4) in which we conducted the glucose metabolism in a microcentrifuge tube rather than the NMR tube. We prepared cell extracts after different time intervals by adding about 15% by volume of precooled 70% (w/w) perchloric acid, then freezing the samples in an ethanol–dry ice mixture. We repeated the freeze–thaw three times, centrifuged the samples, and kept them cold until the NMR spectrometer was available to obtain spectra.
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Experimental Section
Real-Time Procedure The following procedure for real-time experiments is modified from den Hollander et al. (4). A buffer was prepared containing, per liter, 0.15 g of K 2HPO 4, 0.5 g of MgSO4, 0.85 g of KH2PO4, and 22.3 g of Na4P2O7, brought to pH 6. To 20 mL of this buffer was added 1.7–2.5 g of Fleischmann’s Active Dry yeast, and the flask was stoppered and shaken until the suspension was homogeneous. Oxygen was removed from the suspension by bubbling with nitrogen for two minutes and immediately re-capping. The suspension was stirred with a magnetic stirrer for one hour and vented by slightly loosening the stopper every five minutes. A solution consisting of 10 mg of 1-13C-glucose (Sigma 29,704-6) in 50 µL of water was prepared and added to 400 µL of the yeast suspension in a 5-mm NMR tube. Another 400 µ L of yeast suspension was then added. This was mixed on a vortex mixer for 30 s and immediately placed into the NMR spectrometer, and the analysis was performed. If the cost of the labeled glucose is a concern, successful analyses can be obtained with half the amount described. The analysis was performed using a Varian Gemini-200 NMR spectrometer set for 13C determinations. Sixteen acquisitions were taken, with a 3-min delay after the first and subsequent acquisitions. Since anaerobic glycolysis produces CO2, the NMR tube was ejected and vented immediately after each acquisition. There was no autolock, and chemical shifts drifted slightly during the experiment. The NMR signals for β-1-13C-glucose and the α anomer were 97.7 and 93.8 ppm, respectively, with an impurity in the sample that did not metabolize appearing at 94.9 ppm. The 2- 13C-ethanol peak appeared at 18.7 ppm, and the 1-13C-glycerol signal was at 63.7 ppm. Each NMR spectrum was plotted, and peak heights were measured. In preparing the samples in the NMR tube, we found that the yeast suspension could not be too viscous. Considerable bubbling occurs as a result of carbon dioxide production, and in viscous suspensions this bubbling would cause the yeast to be pushed to the top of the NMR tube, resulting in variable NMR intensities. Yeast Extract Procedure If laboratory time does not permit that both the incubation and NMR analysis be done in the same period, the two operations can be separated. The yeast can be incubated in a microcentrifuge tube and the reaction stopped at specific times, with the NMR analysis done when the instrument is available. In this experiment, the yeast cells were incubated as in the real-time experiment. After incubation, 800 µL of
Journal of Chemical Education • Vol. 76 No. 11 November 1999 • JChemEd.chem.wisc.edu
In the Laboratory
yeast suspension was added to each of 4 microcentrifuge tubes. Subsequently, 25 µ L of a 20 mg/100 µL solution of 1-13C-glucose was rapidly added to each tube and a stopwatch was started. Each tube was briefly mixed on a vortex mixer. After 7.5 min one batch of yeast cells were killed with 125 µL of precooled 70% (w/w) perchloric acid. The yeast in the other tubes were killed at 15, 22.5, and 30 min. The suspensions were freeze–thawed three times using dry ice in ethanol, centrifuged, neutralized with 10% KOH, centrifuged again, and stored at {20 °C until analyzed. The NMR analysis was done as before except that D2O was added to achieve a 10% concentration for locking, and 256 acquisitions were taken.
Osmotic Shock Procedure To study the effect of increased external salinity on the ratio of glycerol to ethanol produced, we repeated the previous perchlorate-killed yeast experiment by adding, before the yeast was introduced, sodium chloride to the 20 mL of suspension buffer to give salt concentrations over a range of 0.1 to 0.8 M. A 1.6 M sodium chloride concentration killed the yeast. Results and Discussion One advantage of a real time, in vivo NMR analysis of metabolism is that the student can see the changes in metabolite concentration occur as they happen. Figure 1 is a NMR spectrum showing the α - and β-anomers of 1-13C-glucose, 1-13C-glycerol, and 2-13C-ethanol obtained from the yeast fermentation. The students obtain spectra over time to monitor the disappearance of the glucose and the appearance of the ethanol and glycerol. Typical results are shown in Figure 2. We found, as did Mega et al. (1), that the α-1-13Cglucose disappeared faster than the β-anomer. With larger numbers of students, we prefer to use the perchlorate-killed yeast method. Doing the experiment this way, the students do not get the immediate visualization of the events of metabolism as they do in the real-time experiments. However, it is sometimes more convenient logistically to separate the metabolic events from the running of the NMR spectra. The perchorate-killed experiments do not have the problem of carbon dioxide production in the capped NMR tube. As we used the perchlorate-killed method in our lab, we assigned one pair of students to analyze the extract that was stopped at 7.5 min, and each of the other pairs to analyze 15-, 22.5-, and 30-min extracts. In this way, 8 students can be involved in taking four NMR spectra and pooling their information to plot the data shown in Figure 2. The students need to be reminded that using NMR peak heights to measure the quantities of ethanol and glycerol does not give a true indication of the molar ratios of the two substances. den Hollander et al. did experiments that showed the molar glycerol/ethanol ratio to be about 0.09 (4). They also found that there was a 2–3 min delay for the appearance of ethanol, but no delay for glycerol. In our real-time experiments, the first NMR data were typically obtained at about 4 min, and by then ethanol formation was already occurring. In the presence of external osmotic stress, cells must adjust to maintain their volume. In response to altered extracellular
Figure 1. A real-time 13 C NMR spectrum of yeast taken 21 min after the introduction of 1-13C- glucose. The added glucose is nearly depleted at this point in time. The identity of the peaks was determined by running known samples of glucose, ethanol, and glycerol. This spectrum was run without locking, and the chemical shifts are slightly downfield from the standards. The peak at 94.9 ppm is unknown; it represents a small impurity in the added glucose that was not metabolized.
Figure 2. A plot of the NMR signal versus time showing the disappearing of the α- and β-anomers of glucose and the appearance of labeled glycerol and ethanol for a real-time incubation of yeast with 1-13C- glucose.
osmolarity, many cells respond to intracellular volume changes by modulating metabolic pathways or membrane transport (6 ). Yeast responds to osmotic shock by increasing the internal concentration of polyols, including glycerol, and by altering glycerol transport across the cell membrane. Blomberg and Alder found that in the presence of 0.7 M NaCl, Saccharomyces cerevisiae increased its rate of glycerol production by about 3-fold (6 ). Glycerol is produced in two steps. The first is the glycerol phosphate dehydrogenase–NADH reduction of dihydroxyacetone phosphate to glycerol phosphate. This is followed by a dephosphorylation by glycerol-3-phosphatase to glycerol. The exposure of yeast to osmotic stress increases levels of
JChemEd.chem.wisc.edu • Vol. 76 No. 11 November 1999 • Journal of Chemical Education
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Figure 3. A plot of the ratio of the 1-13C-glycerol–to–2-13 C-ethanol NMR peak heights against the concentration of sodium chloride in the incubation medium. Data were collected using the perchloratekilled yeast method after incubation with labeled glucose and sodium chloride for 15 min.
glycerol phosphate dehydrogenase (7–9) and glycerol-3phosphatase (10) and an increased glycerol retention occurs. Norbeck and coworkers found two isomers of glycerol-3phosphatase in yeast and identified the genes, GPP1 and GPP2, for them (10). Their studies indicated that GPP2 is the target for the osmosensing signal, which results in an increase in one of the isomers of the phosphatase during osmotic stress. The use of the 13C NMR to study how yeast adjusts glycolysis in response to osmotic stress was more exciting to students than simply observing that glycerol and ethanol were the end products in anaerobic glycolysis. We had students monitor the production of 1-13C-glycerol and 2-13C-ethanol when the yeast was incubated with concentrations of NaCl ranging from 0 to 0.8 molar. We used the perchlorate-killed method, letting the incubation occur for 15 min. We divided the work among groups of students so that one pair used extract with no NaCl, another with 0.1 M NaCl, and so forth
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up to 0.8 M salt concentration. They pooled their data and plotted the ratio of the glycerol-to-ethanol NMR peak heights against the sodium chloride concentration. Figure 3 shows that when the external NaCl concentration was raised to 0.8 M, the glycerol/ethanol ratio of NMR peak heights increased from 0.29 to 0.48. The NMR peak height for the 13C-label in glycerol and ethanol is not a quantitative measure of the molar ration of glycerol to ethanol. However, it does demonstrate the dynamics of glucose metabolism during osmotic shock, as was observed by Blomberg and Alder (6 ). The cost of the experiment need not be excessive. Only 5 mg of 1-13C-glucose is required for one experiment, and if the students work in pairs and at current prices for the labeled glucose, the cost is about $0.65 per student. Acknowledgments We wish to thank Harold Jones for assistance with the NMR spectrometer. The Margaret T. and Otis A. Barnes Trust provided a summer stipend for Brent Giles to develop the experiment. The Hughes Undergraduate Research Program, funded by grants to HHMI to Colorado College, provided money to purchase supplies. Literature Cited 1. Mega, T. L.; Carlson, C. B.; Cleary, D. A. J. Chem. Educ. 1997, 74, 1474. 2. Shulman, R. G.; Brown, T. R.; Ugurbil, K.; Ogawa, S.; Cohen, S. M.; den Hollander, J. A. Science 1979, 205, 160. 3. Cohen, S. M. Methods in Enzymology, Vol. 177; Academic: New York, 1989; p 417. 4. den Hollander, J. A.; Brown, T. R.; Ugurbil, K.; Shulman, R. G. Proc. Natl. Acad. Sci. USA 1979, 76, 6096. 5. Chamberlin, M. E.; Strange, K. Am. J. Physiol. 1989, 257, C159. 6. Blomberg, A.; Adler, L. J. Bacteriol. 1988, 171, 1087. 7. Andre, L.; Hemming, A.; Adler, L. FEBS Lett. 1991, 286, 13. 8. Blomberg, A.; Adler, L. Adv. Microb. Physiol. 1992, 33, 145. 9. Albertyn, J.; Hohmann, S.; Thevelin, J. M.; Prior, B. A. Mol. Cell. Biol. 1994, 14, 4135. 10. Norbeck, J.; Pahlman, A. K.; Akhtar, N.; Bomberg, A.; Adler, L. J. Biol. Chem. 1996, 271, 13875.
Journal of Chemical Education • Vol. 76 No. 11 November 1999 • JChemEd.chem.wisc.edu
