Yeast Metabolism of D-[U-14C]-Glucose: A Student Study of the Early stages of Mycolysis Richard L. Taber a n d Betty G. Harwood Colorado College, Colorado Springs, CO 80903 Modern undereraduate courses in hiochemistrv spend a great deal of t i m i o n understanding metabolism &dLon t h e methods for elucidating metabolic pathways. Thus, students are introduced t o the usefulness of radioisotopes as tools in tracing metabolic pathways in most lecture courses. Unfortunately, i t is less common t h a t the student encounters t h e use of radioisotopes in the undergraduate biochemical laboratory. Since radioisotopes are u s i d everywhere in biochemistry, a n introduction t o their use should be a standard part of t h e laboratory program. We would like to offer a n experiment which can be accomplished with a minimum of equipment in a reasonable time t h a t our students have found t o be interesting and informative. In this experiment, the student studies the early stages of glycolysis, incubating yeast extract with D-[U-'4C]glucose, isolatineu the earlv " elvcolvtic -. " intermediates and seoaratiner them by thin layer chromatography. T h e student then determines the distribution of radiolaheled intermediates along t h e thin layer plate b y two methods: liquid scintillation counting and fluorography. Comparison of t h e distribution of radioactivity with the migration distances for known c o m ~ o u n d allows s the student to make tentative identification of the metabolic products. This experiment can be accomplished in two t o three laboratory periods and gives the student experience with (1)the uncertainties encountered in studying metabolic pathways, (2) the handling and use of radioisotopes, (3) the application of thin layer chromatography to a difficult separation problem, and (4) liquid scintillation counting and fluorography a s methods of detecting radioactivity. There are a large number of possible radioactive metabolites t h a t could be produced from the metabolism of glucose, and i t would be virtually impossible t o separate all the radioactive glycolytic intermediates from each other by a one dimensional thin layer chromatography. Thus, in order t o simplify t h e problem of thin layer separation, in this experiment the yeast glycolytic enzymes are exposed to the radioactive glucose for a very short period of time. I n this way, perhaps only t h e earliest intermediates of glycolysis will acquire the radiolabel. A second approach used by students t o limit glycolysis in this experiment was to add A T P t o the yeast extract in hope of inhibiting phosphofructokinase. T h e students are briefed thoronrhlv on radiochemical principles and techniques, general safety rules for working with radioisoto~es,and the uotential biolorical hazards t h a t soft beta emitters present before they are alrowed to begin the experiment. It is essential that students recognize potential hazards in working with radioisotopes and t h a t there also are common sense rules t h a t enable radioactive materials to be used safely.
-
Experimental Procedure The procedure is adapted from same of the early investigations of glyrolysa using isotopic carbon (1-3). In a mortar and pestle, thoroughly grind about 1 g of baker's yeast m 2 ml of 0.06 M disodium phosphate buffer (pH 8.5 and 0.001M in EDTA). Add another 2 ml of this buffer to the suspension and centrifuge at 25,000 X g for 15 min.
a microliter syringe fitted through a rubber septum in the center neck, and tubing, leading to a 4 ml test tube, is fitted through a cork placed
in the other side neck of the flask. The large syringe is loaded with 5 ml of absolute ethanol, the small syringe is loaded with about 1 microcurie of D-[U-14C]-glucose,and 1 ml of 10 per cent sodium hydroxide is placed in the test tube. A micro stirring bar is placed in the flask and the whole apparatus is positioned over a magnetic stirrer. The metabolism of radioactive elueose can lead to the formation
that involve radioactive material. Transfer 0.5 ml of the centrifuged yeast extract to the reaction vessel, inject the 14C-glucoseinto the gently stirred extract, wait 2 to 3 sec, and inject the 5 rnl of absolute ethanol into the reaction mixture. Gently flush the system withnitrogen to force any radioactiveearhon dioxide that might he formed into the sodium hydroxide trap. The addition of the absolute ethanol causes protein and the phosphorylated glycolytic sugars to precipitate, leaving most of the unreacted radioactiveglucose in solution. The next step is to extract the phosphorylated sugars from the protein with water and concentrate to a small volume for thin layer chromatography. Carefully pour the reaction mixture into a small centrifuge tube and centrifuge for 5 min with a table top eentrifuge. Carefully pour off the supernatant from this centrifugation into a small tapered centrifuge tube and save for concentration and analysis far radioactive glucose. Rinse the reaction vessel with 1 ml of distilled water. Add the washing to the precipitate and mix thoroughly. Centrifuge this suspension for 5 min, and pour off the supernatant into a second small, tapered centrifuge tube. Extract the precipitate with a second 1 ml of water, centrifuge for 5 min, and add this to the supernatant from the first aqueous extract. If time is a problem, this second extraction can he omitted. Concentrate the supernatants from the original ethanol preeipitation and the aqueous extractions to a final volume of about 0.1 ml by placing the centrifugetubes under a heat lamp and passing a gentle stream of air into the tuhes. Thin Layer chromatography The thin layer chromatography of the phosphorylated sugar extracts is accomplished in one dimension on commercially prepared cell~doseplates (Analtech, Ine., Avicel plates). The developing solvent found to be most effective in separating the henose monophosphates is ethyl acetate:acetir aeid:water:ammonia (6:fi:Z:I) and should be reasonably freshly prepared. Spot 1microliter of the concentrated, aqueous extract near the left and right edges of the plate. Alsci spot 1 microliter samples of 1 per cent solutions of glucose, glueose-6phosphate, fructose-G-phosphate,and fructose-1,6-diphosphate as knows Place the plate in the thin layer chromatography tank and allow the solvent front to migrate about 15 cm, which takes about 3 hr. Analysis by Liquid Scintillation Counting Starting from the origin of the spotting on one edge of the TLC plate, carefully mark off rectangles every 0.25 cm up to about 10 cm from the oriein. Scram each rectanele of absorbant into a scintillation
and count to estimate the amount of unreacted radioactive glucose.
If any radinactive carhon dioxide is produced from the metabolism of the radiolabeled glucose, it will he absorbed hy the sodium hydroxide tram Since the tra~oine .. solution is stronelv " " basic. addition of the scintillation fluid creates a chemiluminescencethat &es very large backgmund counts. The chemiluminescence decreases with Volume 60
Number 3
March 1983
249
2500
:1000 FDP
1
2
3
4
5
6
7
8
Distance, Crn
A plot of counts per minute against migration distance on cellulose plates of intermediates produced from a 3 second exposure of yeast extracts to D-[U-"CI-glucose.
time, and the sample should he ccmnted until a constant count rate is obtained. Visualization of the Knowns After scraping samples from the plate for liquid scintillation analysis, and protecting the remaining radioactive portion from the spray, visualize the known sugars by spraying with aniline-oxalate reagent. The sugars appear as brown spots on a white background after heatingfor 15 to45 mininan oven at 110'. Fructop6-phosphate and fructose-1.6-di~hosohate are often faint. The aniline-oxalate reagent is prepared-by adding 0.9 g o d i c acid to 200 ml water, and when dissolved, add 1.8 ml of freshly distilled aniline. Analysis by Fluorography Not all undergraduate biochemistry laboratories have access to liquid scintillation counters. However, the radioactive glycolytie intermediates can be located very easily on the thin layer plate by fluorography (4-6). Prepare a 7 per cent solution (wiv) of 2,5-diphenyloxazole (PPO) in diethyl ether. Spray the dry chromatogram with the PPO solution as quickly as possible at about a 5-in. distance so as to get an even and thorough wetting of the plate. Immediately take the sprayed chromatogram to a darkroom, where all subsequent work is to be done under a photographic safelight. A strip of X-ray film is placed over the edge of the thin layer plate where the distribution of separated radioactive intermediates should be located. Place the chromatagram and film between two glass plates, secure with rubher hands, wrap in black plastic, and place in a freezer. After 48 hr, take the chromatoaram - and film hack to the darkroom and develoo the film according to manufacturer's instructions. Dark spots appear on the X-ray film wherever radioactive materials were located on the plate. The distances from the origin on the X-ray film can be compared with the &values for the knowna. Results and Discussion Typical student results for the metaholism of D-[U-14C]glucose are presented as a zonal profile in the figure. Students
usuallv obtain four main peaks of radioactivity, which correspond'to fi values for fr"ctose-1,6-diphosph&, glucose-6v h o s.~ h a t e .fructose-6-phosphate and glucose. T h e relative . sizes of the peaks varieb from student t o student, and some of them also found small unidentified peaks. Students who do not scrape the thin layer plate carefully, taking narrow sections of the adsorbant for analysis, lose the resolution hetween the two hexose monophosphates. Likewise, as the thin layer developing solvent ages, resolution between t h e sugars diminishes. Students who do not concentrate the aqueous extracts sufficiently before spotting them on the plates have peaks of radioactivity t h a t are very small. In our investigations, n o student found radioactivity i n t h e carbon dioxide t r a t~h a t was avvreciablv above the background. gome studen;; atteml;ted t o inhibit ph&phofructokinase by adding ATP to the yeast extract. In a few instances, addition of ATP t o t h e extract did seem t o inhibit t h e formation of fructose-1,6-diphosphateas was shown by a disappearance of the first peak in the figure. When the glycolytic study was first tried by students, they chromatographed the ethanol extract concentrate as well as t h e aqueous extracts. They found that glucose was virtually the only substance containing radioactivity in the ethanol. T h e students need t o be reminded t h a t althouah a good correlation between peaks of radioactivity and Rrvalues for the known sugars are a good indication of which compounds those peaks represent, they still have not actually isolated and identified them. T h e student can be referred t o some of the original studies on t h e metaholism of glucose b y yeast, where the glycolytic intermediates were actually separated and chemicallv identified 11-31. A lack hf a liquid scintillation counter need not prevent teaching laboratories from doing this investigation. Our stndents had good success analyzing the distribution of radioactivitv on the thin laver . d.a t e hv fluoromavhv. - . .Most students were able to identify the main areas of radioactivity by exposing the X-ray film for 24 hr, h u t for some, a 48-hr exposure of the film was necessary. I t was easier for the students t o make correlations with RF values from knowns using the ~
solid scintzlant on the plate, but a 48-hi exposure of the x-ray film produced no spots.
Literature Cited (1) Kosh1md.D. E., and We8theimer.F. H., J. Amer Chem Soc.72.3383(1950). (2) Blumenthd, H.J., L-E, X. P. and Weinhnuie, S.. J. Amer Chrm. Soc., 76,6W3
,.""-,. ,,OZ",
(31 Benson,A.A.,Barshnm,LA.,Cslvin,M.,Goodde,T.C..Haas,V.A.,andStepka. W., . IAmtr.Chrm.Snc .72,1710(1950). ( 4 ) Touchd."ne, J. C.. mdDohhins. M. F.,"PracticeofThinhyer Chromatography,"John WBw and Sons, New York, 19% D. 280. (51 Randerath X., Anal. Binchem.,34.188 (1970). (6) Prydr, S.. Melo,T.B..and Koren, J.F.,Anal Chem ,dS, 2106 (19731.
ACS Information Pamphlet on Acid Rain Available
I I 250
In an effort to promote better understanding of the chemistry behind science-related regulatory issues, the Office of Federal Regulatory Programs of the Society's Department of Public Affairs has published the "Acid Rain Infmmation Pamohlet."
knowledge and comprehension of this controversial topic. Aimed at the educated and informed non-scientists, the information pamphlet explores the phenomenon of acid rain by addressingthe causes of acidic deposition,long~termtrends, atmospheric processes, techniques for assessment,modeling, and monitoring networks. Written in clear, concise language, educators will find it useful in the classroonl for high school and colleee " students alike. T o obtam a copy of the pamphlet, write to Ms. Jean A. P a r , American Chemical Society, Department of Public Affairs, 1155 16th Street, NW Washington, D.C. 20036, or call 2021812-4395.
Journal of Chemical Education
