- Email: [email protected]
Endogenous respiration of yeast. I. The endogenous substrate
ARCHIVES
OF
BIOCHEMISTRY
Endogenous
AND
Respiration
BIOPHYSICS
88, 17-25
of Yeast. NORMAN
From
the Radioisotope Microbiology
(1960)
I. The
Endogenous
R. EATON
Service, Veterans Administration Hospital and and Medicine, University of Washington, Seattle, Received
Substrate’
August
the Departments Washington
of
10, 1959
1. Kinetic and chemical data suggest the presence of three endogenous substrates in a strain of Saccharomyces cerevisiae. These consist of two metabolically distinct glycogen pools and the disaccharide, trehalose. Lipides do not serve as a substrate for endogenous respiration. 2. Utilization of one glycogen pool requires the presence of oxygen, while the other can be metabolized either aerobically or anaerobically. Trehalose is used only anaerobically. 3. Anaerobically ethanol accumulates in amounts equal to the CO, produced. In addition, there is a slow anaerobic disappearance of the “oxidizable” glycogen pool with an equivalent accumulation of a TCA-soluble carbohydrate that is utilizable only aerobically. 4. It is suggested that the rapid oxygen uptake observed after periods of anaerobiosis is due to oxidation of ethanol and the TCA-soluble carbohydrate and that the low R.Q.‘s reported previously are the result of ethanol oxidation.
petted from lipide rather than carbohydrate oxidation. On the basis of these observations Stickland concluded that carbohydrate does not serve as the endogenous substrate in yeast. It must be noted, however, that the methods used by Stickland would probably not have detected a disappearance of acidsoluble carbohydrate such as trehalose. While there appears to have been no unequivocal demonstration of glycogen disappearance in amounts equivalent to the COZ produced during aerobic utilization of endogenous reserves, several well-documented cases of glycogen disappearance under anaerobic conditions have been reported. These latter studies have been performed with both normal resting suspensions and in the presence of dinitrophenol. In the former case, the metabolic rates, as measured by carbon dioxide production, have been found to be low in comparison with the aerobic release of carbon dioxide (4). It is interesting to note, however, that evidence exists for the anaerobic breakdown of reserves without evolution of CO2 (3), leading to the accumulahion of
INTRODUCTION
The nature of the substance (or substances) stored and subsequently degraded during endogenous respiration of yeast is still the subject of some controversy, although most investigators have assumed that this endogenous substrate is a carbohydrate, “probably glycogen” (1). This assumption is based, for the most part, on the respiratory quotient (R.Q.), which is usually found to be equal to about one (1,2), and on t,he disappearance of iodine-staining material (I), hydrolyzable polysaccharide (3), or anthrone-reactive material (4). Recently, however, t’he role of carbohydrate has been questioned by Stickland (5) who was unable to find a disappearance of hydrolyzable polysaccharide during endogenous respiration. In addition, the R.Q.‘s reported by Stickland were of the order of 0.85, a value which approximat’es that exr Supported, in part, by a grant (RG-5875) the Department of Health, Education, and fare, U. S. Public Health Service.
from Wel17
EATON
some intermediate that can subsequently be oxidized rapidly (6, 7). Dinitrophenol-induced fermentation leads to a disappearance of both glycogen and trehalose (8). It is by no means certain, however, that, in either of these cases, the substrates or the metabolic pathways are the same as those of aerobic endogenous metabolism. In addition to glycogen, trehalose is sometimes mentioned as a possible endogenous substrate (9), although Brandt (10) has concluded that the oxidation of stored trehalose by resting suspensions of yeast is extremely feeble. Moreover, while the results obtained by Stickland (5) and certain observations by Lindegren (11) would seem to indicate that lipides might play a role in endogenous respiration, this possibility has not been tested. The idea that endogenous CO2 is derived from a single substrate pool appears to be based upon the results of kinetic studies of Stier and Stannard (1) and of Spiegelman and Nozawa (2). Both groups were led to the conclusion that the rate of endogenous respiration is limited by the concentration of a single substrate, which was equated to the glycogen content of the cell. Recently, however, a re-examination of the kinetics of yeast endogenous respiration (12) has shown that there are, in fact, at least two substrate pools metabolized aerobically. The work reported below describes more exactly the nature of these endogenous reserves. METHODS
AND
MATERIALS
A strain of Saccharomyces cerevisiae, LK2G12, used in previous studies (13), was grown in a medium of the following composition: glucose (containing O-14 pc. CY/lOO ml. medium), 2%; yeast extract (Difco), 0.1%; NHdCl, 0.1%; KHzPO4 , 0.1 M. Inoculation was made from a stock culture slant of the same composition plus 2% agar. The cultures were incubated at 30°C. either anaerobically (standing) for 40-48 hr. or aerobically (shaken) for 20-24 hr. The cells were harvested by centrifugation, washed twice with distilled water and resuspended in 0.1 M KHzPOd to a cell density of 6-7 mg. dry weight/ml. as measured by optical density of the suspension and reference to a previously prepared standard curve. For most of the respiration studies, 60-70 min. elapsed between the beginning of the harvesting procedure and the first manometer reading. Standard manometric techniques were used; each Warburg vessel contained a total volume of 2.2 ml.
FRACTIONATION PROCEDURE Growth of the yeast in the above medium containing glucose randomly labeled with 04 yielded cells in which all of the carbon-containing compounds were randomly labeled. Such cells were allowed to dissimilate their stored reserves for varying periods of time, and cell components were separated by a fractionation procedure based primarily on that of Trevelyan and Harrison (14). Disappearance of Cl4 from the various fractions was compared to the appearance of Cl4 in the carbon dioxide as a measure of the source of endogenous CO* .
Carbon Dioxide CO2 was absorbed in 0.2 ml. of 3 N NaOH the center well of the Warburg vessel.
Trichloroacetic
Acid (TCA)
in
Fraction
Cells were killed by tipping 0.2 ml. of 100% TCA from a side arm into the main compartment of the Warburg vessel. The contents of the vessel were poured into a 15-ml. conical centrifuge tube, and the vessel was rinsed with 1 ml. of 10% TCA which was also added to the centrifuge tube. Cells were removed by centrifugation and were resuspended in about 0.1 ml. water. The extraction procedure was repeated twice with l-ml. portions of 10% TCA, and the combined extracts were brought to 10 ml. with distilled water. Aliquots of 1.0 ml. were taken for Cl4 and carbohydrate determinations. Treatment of the TCA extract with ion-exchange resins [Dowex 50(H) followed by IR4B(HCOI)] removed about 10% of the total carbohydrate (anthrone-reactive material). About 4% of the carbohydrate in the deionized solution was reducing sugar, and hydrolysis resulted in the liberation of one reducing group per hexose unit, after correction for the small amount of reducing sugar initially present. Chromatography in propanol-ethyl acetate-water (15) resulted in only one detectable sugar spot, with an Rf identical to that of a known sample of trehalose. It is concluded, therefore, that the major carbohydrate of this fraction is the nonreducing disaccharide, trehalose [cf. (14)].
Lipide Fraction The trehalose-free cell residue was resuspended in 0.1 ml. water. Two milliliters of ethanol was added, followed by 4 ml. ethyl ether, and the suspension was centrifuged. This procedure was repeated twice, and the combined extracts were dried in wet combustion flasks for subsequent oxidation and assay of C’4.
RESPIRATION OF YEAST. I Polysaccharide
Fraction
The residue remaining aft.er extraction of lipides was suspended in 0.1 ml. water. Two milliliters of 0.25 M Na&03 was added (16), and the suspension was heated at 100°C. for 30 min. Four milliliters of ethanol was added, the precipitate was removed by centrifugation, and the supernatant liquid was discarded. The precipitate was resuspended in 2 ml. water and reprecipitated with 2 vol. ethanol, and the supernatant liquid was again discarded. The residue was taken up in 2 ml. of 2 N HISO, and heat,ed at 100°C. for 15 min. The extraction with H,SO, was repeated once, and the combined supernatant liquids, containing glycogen and mannan (lb), were brought to 10 ml. with distilled water. illiquots of 0.5 ml. were taken for determination of carbohydrate and 0”. Using preparations of glycogen and mannan isolated by the method of Jeanloz (I?), it was found that this procedure resulted in partial hydrolysis but no detectable destruction of the polysaccharide as measured by the anthrone reaction. Estimation of the glycogen component of the polysaccharide mixture isolated from randomly Cl”-labeled cells was based on the following considerat,ions: Since mannose gives only 557% of the color of an equivalent amount of glucose with anthrone (18), ,1 = G + 0.55 M and 6” = G + M where il = carbon estimated by anthrone, G = carbon in the glycogen component, X = carbon in the mannan component, and C = carbon estimated by 04 assav. Therefore, G = A -
0.55 C 0.45
It should be noted that this is based upon the assumption that only glycogen and mannan are present in the fraction, and any contaminating radioactive material would tend to decrease the value for G. The glycogen content calculated in this way should therefore probably be considered a minimum estimate. Carbon-l d-Assay All samples were converted to CO2 by wet oxidation. Lipides were oxidized with Van Slyke reagent (19). More reproducible results for wet oxidation of water-soluble materials however, were obtained with the persulfate method of Katz el al. (20). The resultant CO? was collected in XaOH, converted to BaC03, and plated as previously described (13). The samples were counted with an ultrathin window Geiger tube, and the observed activities were correct,ed for self-absorption and
19
efficiency by reference to a standard curve. All activities were calculated as disintegrations/min. and converted to equivalent microliters carbon.
Chemical Determinations Total carbohydrate was determined by the anthrone method (14) and reducing sugar by the method of Park and Johnson (21). Glucose was used as a standard in both cases. Ethanol was determined with alcohol dehydrogenase as previously reported (13). RESULTS
AEROBIC METABOLISM It has been suggested earlier (12) that, under aerobic conditions, the endogenous carbon dioxide produced by a resting suspension of yeast is derived simultaneously from two substrate pools. This conclusion was based upon the result’s of experiments designed to test the apparent reaction order of the process. The amount of endogenous substrate available to the cell (as carbon dioxide) was estimated from the amount of carbon dioxide evolved as CO% evolut’ion approached an asymptotic value, A. The logarithm of A minus X, the amount of carbon dioxide evolved after various time intervals, was plotted as a function of time. If the process has a first-order character, i.e., if carbon dioxide is derived from a single substrate, bhe concentration of which is limiting, log (A - X) will be directly proportional to t,ime, and a straight’ line should result. Such plots have shown, however, that carbon dioxide evolut,ion assumes first-order kinetics only after about 4 hr. [(12) ; cf. 13g. lA, curve 11. Extrapolation of the logarithmic portion of the curve to zero time (Fig. lA, curve 2) and replotting the difference between curves 1 and 2 results in another logarithmic curve (lcig. I A, curve 3). Thus the evolution of carbon dioxide from endogenous reserves is characterized by two “firstorder” processes occurring simultaneously. In order to explain t,hese results it is necessary to assume the presence of two endogenous substrate pools. The amount,s of material in each of these pools is given by the intercept’s of the logarithmic curves with t’he ordinate. The nat,ure of the two substrat’e pools was
20
EAT01
800 -I
i
4
6 4
lb 1’2
HOURS
I’
2
I’,‘,‘,‘,
4
6
8
IO
12
HOURS
FIG. 1. Graphic analysis of endogenous CO2 evolution. Curve 1: total available substrate remaining (as C0.J. Curve 2: extrapolation of linear (logarithmic) portion of Curve 1 to zero time. Curve 3: replot of the difference between Curves 1 and 2. A. Aerobic. B. Anaerobic. Each flask contained 11.9 mg. (dry wt.) of cells grown anaerobically. [A = total endogenous substrate (as COz) ; z = COz evolved; ordinate = log (A - 2) = log (remaining substrate).]
investigated with cells randomly labeled with C4. After growth in a medium containing glucose-Cl4 as the sole carbon source, cells were washed, resuspended in phosphate buffer, and allowed to oxidize their endogenous reserves for varying periods of time. The process was stopped by addition of trichloroacetic acid, the cell components were separated according to the schemepresented above, and the amount of Cl4 and carbohydrate remaining in each of these fractions was determined. The results of several representative experiments using this procedure are shown in Table I. The amount of aeration of the culture during growth appeared to have no effect on the kind of material oxidized. In all casesthe carbon present in the lipide and TCA fractions remained constant throughout, within the limits of experimental error. The only fraction showing significant disappearance of carbon was that containing the polysaccharides, glycogen and mannan (14). Furthermore, this
disappearance could be accounted for by a decrease in the calculated glycogen content of the fraction, and it was essentially equivalent to the amount of carbon appearing as” carbon dioxide. Thus, glycogen appears to be the only substrate oxidized, and one is forced to conclude that, under aerobic conditions, the endogenous stores consist of two metabolically distinct glycogen pools. ANAEROBIC
METABOLISM
Although Stier and Stannard (1) and Spiegelman and Nozawa (2) have reported that the fermentation of endogenous reserves is negligible, the strain of yeast used in these experiments produced significant amounts of carbon dioxide anaerobically. Fales (4) and, more recently, Chester (22) have also reported fermentation of the stored materials. In Table II the carbon dioxide produced anaerobically over an extended period of time is compared to the ethanol
RESPIRATION
OF
TABLE DISAPPEARANCE I
I
OF CELL
CARBON
21
I
I DURING
AEROBIC
RESPIRATION’ T-
I
Fraction
Cells
toe
if2
TCA
I
I
MkWl0metric
.-
YEAST.
C”
Polysaccharides
C”
Anthrone
I-
Anthrone
nzg.
min.
Anaerobic
10.8
15 240 X-240
58 550 +492
59h.6 561f51 +502*39
1373f231 1376zt98 +3*193
89&k34 854f76 -44f64
295f15 350f133 t55*92
1706f67 1441f57 - 265f68
Anaerobic
10.3
15 105 210 G-210
28 213 324 296
28f9 215&24 324f34 +-296zk29
1324f33 1365zt62 1342f37 +I&41
711fll 706f19 691f14 -2Of15
244f18 250&25 2783t30 i-54&28
2252f65 2016&11: 1876185 -376*86
Aerobic
14.6
15 105 240 15-240
22 150 284 f262
21f5 157f29 277&19 f256f52
1375f50 1423f33 1387f40 flOf53
198f6 205f3 199f15 Slf14
724*143 787~1~82 741f90 f17f128
1902f67 1789f64 1680&67 -228f71
values
pl.
a All
Calculated glycogen
1426f73 1123f29 -303f60
1084f89 734f60 350f83
1484&20 1264zt27 1141f30 -343f30
545f61 339*212 243*95 -302f94
Ii
C f
95cj0 confidence
interval.
found in the supernatant medium. The amount of ethanol formed corresponded closely to the carbon dioxide evolved. These results are consistent with the idea that the anaerobic degradation of endogenous reserves proceeds via the glycolytic sequence of reactions. A graphic analysis of the kinetics of the anaerobic process in terms of carbon dioxide produced is shown in Fig. 1B. The amount of endogenous subst,rate available was estimated from manometric data, and the logarithm of the difference between this value (A) and the CO2 produced (z) was plotted as a function of time. The resulting curve was analyzed as previously described (12). For comparison, a similar analysis of cells respiring aerobically is shown in Fig. 1A. It is clear that anaerobically, as well as aerobically, two logarithmic (‘Yirst-order”) processes occur simultaneously, suggesting the presence of two endogenous substrate pools. The amount of substrate present in each of these pools, as CO2 equivalents, was determined by extrapolation of the logarithmic portions of the curves to zero time. In the case of anaerobic carbon dioxide production this value must be multiplied by three for
TABLE
II
ANAEROBIC FORMATION OF CO2 AND ETHANOL Each flask contained 11.9 mg. (dry wt.) anaerobically grown cells. Gas phase, NZ . Temp., 30°C. Hours 2 4 12 22
co2
Ethanol
pmoles
&l?noles
3.56 4.39 6.29 7.65
3.60 4.68 6.12 7.56
comparison with aerobic experiments, since only one-third of the hexose carbon appears as carbon dioxide, the remainder accumulating as ethanol (cf. Table II). Reference to Fig. 1 shows that t,he rapidly metabolized components (curves 3), but not the slowly metabolized components (curves 2), are present in equivalent amounts. This suggests that there are three substrate pools, one of which can be metabolized either aerobically or anaerobically. The identit,y of these two rapidly metabolized components is further shown by the following experiment: Cells were allowed to oxidize their reserves for several hours and were subsequently placed under anaerobic conditions. It was to be expected that, if
22
EATON
The chemical nature of the anaerobic endogenous substrates was investigated as described above: Cells labeled with Cl4 were shaken under nitrogen, and samples were removed periodically for determination of the carbon content of the various cell fractions. The results of these studies are shown in Table III. In addition to a disappearance x 60of glycogen, carbohydrate disappeared from ’ 604 577 the TCA fraction as well, during fermenta40tion of endogenous stores. The decrease in glycogen aft.er ext.ended fermentat,ion was equivalent in amount to the rapidly metabolized anaerobic component,, and, as shown above, the lat’ter appears to be identical to I .\, , , , , the rapidly metabolized aerobic component. 2 4 6 S IO This suggests that, the slowly metabolized HOURS anaerobic component might, in fact, be FIG. 2. Graphic analysis of fermentative COZ production after oxidation. Curves 1, 2, and 3: as trehalose. If this is true, one can calculate, constant based on manin Fig. 1; before oxidation. Curve 4: available ana- using a “first-order” ometric data, the amount of carbon expected erobic substrate remaining (as CO,) after 9 hr. aerobiosis. Each flask contained 9.4 mg. (dry wt.) to disappear from the TCA fraction. In the anaerobically grown cells. Temp. 30°C. Gas phase, case of the anaerobically grown cells shown N2 . in Table III, this amount is 405 ~1. C, a value considerably higher than the 197 11. these two components are identical, graphic found. analysis of the fermentative carbon dioxide This inconsistency was resolved by a study after an extended period of oxidation would of the behavior of the TCA fraction during yield only a single logarithmic curve, the oxidation after periods of anaerobiosis. more rapidly metabolized anaerobic comWhen disappearance of carbohydrate from ponent having been oxidized. The results t’his fraction was measured (Fig. 4), it was (Fig. 2) show that after oxidation for 9 hr. found after anaerobiosis that a portion of the rapidly metabolized anaerobic compothe TCA-soluble carbohydrat’e was oxidiznent was, indeed, reduced to negligible able and that the amount utilized was a amounts [from 3(202 - 145) = 171, to function of the time the cells were main3(132 - 120) = 361. The amount of matetained under anaerobic conditions. Since no rial in the slowly metabolized anaerobic carbon disappears from the TCA fract,ion of cells not previously kept anaerobic (i.e., component, however, was decreased little, since there is no aerobic ut’ilization of treif at all, by oxidation. The converse experiment, i.e., oxidation halose, cf. Table I), anaerobiosis results in of aerobically utilizable of reserves after a period of fermentation, is the accumulation carbohydrate. Moreover, the amount of this shown in Fig. 3. Cells shaken under Nz were accumulating (equivalent to tested after 9 and 22 hr. After removal of carbohydrate 107 ~1. C after a 22-hr. period of anaerobithe supernatant liquid, the cells were resuspended in buffer for determination of aerobic osis; cf. Fig. 4) is about equal to the decrease in the slowly met’abolized aerobic glycogen COZ production. Again, the rapidly metabpool (125 in the present example; cf. Fig. 3). olized aerobic component virtually disappeared during anaerobic incubation (decreasFurther evidence that the more slowly metabolized anaerobic substrat,e is trehalose ing from 203 to 25 in 22 hr.). In addition, is shown by the equivalence of this pool, as however, there was a decrease in the amount of the slowly metabolized aerobic pool determined graphically, with t’he total car(from 470 to 420 to 345 over’ a period of 22 bohydrate content of the TCA fraction. In addit,ion, the rat,e of disappearance of trehr.). 400-
RESPIRATION
OF
YEAST.
23
I
600 673.
600
470, 420,
400 345203,
200 X
I a
100 60 60 40,
20.
.
h
i
’
6
.
6
i.
HOURS 3. Graphic analysis of aerobic CO* production after fermentation. Fig. 1; before fermentation. Curve 4: available aerobic substrate (as COJ Curve 5: after 22 hr. anaerobiosis. Each flask contained 11.9 mg. anaerobically Gas phase, air. FIG.
TABLE DISAPPEARANCE
OF CELL
Curves 1, 2, and 3: as in after 8 hr. anaerobiosis. grown cells. Temp. 30°C.
III
CARBON
DURING
FERMENTATION”
-
Fraction Cells (dry wt.)
Growth conditions
7-
Time
-
Lipide
TCA Anthrone
(C”)
Calculated glycogen
Polysaccharide AMhr0ne
C’4
1373f231 1480flOO t 107&193b
1485f73 1244f32 -241f61
1765&67 1510f123 - 255f 108
953f25 828f22 - 125+32
967f15 823f18 -144f21
1363f57 1174f81 - 189f76
C’”
-~
%=.
hr.
Anaerobic
10.8 27
0 27 O-27
295f15 294f39 -1f35
898f34 701f67 - 197f62
Aerobic
10.0
0 22 (f22
501f146 550f180 f49f194
127f8 96f4 -3lf8
-
-
a All values pl. C f 95y0 confidence interval. b It should be noted that this does not show a decrease corresponding the supernatant liquid was not removed prior to fractionation and this tween accumulated ethanol and trehalose disappearance.
to the anthrone figure represents
1143+x84 92Ort86 - 223*95 475*75 398f62 -77f-75
value because a balance be-
24
EATON
0
900 890
I
60
r
NOURS
I
120
’
ANAEROBIC .
I 180
’
I
240
MINUTES
FIG. 4. Aerobic utilization of TCA-soluble carbohydrate before and after anaerobiosis. Cells: as in Fig. 3. Cell suspensions were shaken under NP for the indicated times, then exposed to air. Residual TCA-soluble carbohydrate was determined with anthrone and converted to equivalent ~1. C. Temp. 30°C. TABLE COMPARISON
OF
POOLS
THE
IV
NONGLYCOGEN
ANAEROBIC
DISCUSSION
DETERMINED KINETICALLY AND CHEMICALLY Slow logarithmic component
Trehaloseb
3.42 x 10-S 630
Kf c, d.
4.53 x 10-s 608
a First-order constant (min.?/mg. cells). b Anthrone-positive material in TCA fraction. TABLE RESPIRATORY
QUOTIENTS QUENT
V OF CELLS
SUBSE-
TO ANAEROBIOSIS
Cells grown anaerobically in a medium containing inactive glucose (Expt. 1) or Cl4 glucose (Expt. 2). Gas phase, air. Temp. 30°C. , I I
__-~ 1 2
~___ hr.
mg.
pl.
0 2 8 0 9
11.9 213 318 328 10.4 287 288
Pl.
208 264 269 267 238
0.98 0.83 0.82 0.93 0.83
with those of Stickland (5), it was of some interest to calculate the respiratory quotients of cells after various periods of anaerobiosis. This has been done in Table V for the first 60 min. of oxidation subsequent to anaerobiosis [the time period used by Stickland (5) and by Spiegelman and Nozawa (2)]. It may be seen that a period of anaerobiosis as short as 2 hr. results in an R.Q. considerably lower than that of the freshly harvested cells, as might be expected from the oxidation of accumulated ethanol. Furthermore, during this short tJme interval the amount of polysaccharide in the cell shows little decrease (Table V), since most of the oxidation is at the expense of carbon contained in the TCA fraction, presumably ethanol and the TCAextractable carbohydrate that accumulate anaerobically.
A/L
Apl.
-47
-176
o After 60 min. oxidation subsequent indicated period of anaerobiosis. b Disappearance of Cl4 after 60 min.
to the
halose is essentially the same as that of the slowly metabolized anaerobic pool. These results are summarized in Table IV. Because of the variance of these results
Kinetic analysis of endogenous respiration has shown that yeasts respiring aerobically draw upon two endogenous substrate pools. A chemical fractionation of the respiring cells, however, shows that only glycogen disappears during respiration, and it, is concluded that there are two metabolically distinct glycogen pools in the yeast cell, Two alternatives appear possible: The two glycogens may, indeed, comprise species of distinct molecular configuration, or the glycogen may be of only one chemical or con-figurational type but spatially separated in the cell. At the present time, there is no evidence available to distinguish between these possibilities. Alkali-extractable and acid-extractable glycogens have been described by Trevelyan et al. (14) and others. In the present case, however, the quantities of these two fractions do not correspond closely to the amounts of the two glycogen pools described kinetically. On the other hand, Northcote (23) has succeeded in separating electrophoretically two glycogen components from a yeast preparation. It would appear from the method of preparation that these components also do not correspond to the acidand alkali-soluble glycogens. Whether they are equivalent to the metabolically differentiated glycogens described here is, as yet, unknown.
RESPIRATION
Anaerobically, kinetic analysis also indicates the presence of two substrate pools. Under these conditions, however, in addition to glycogen disappearance there is a disappearance of carbohydrate from the TCA fraction. Since trehalose represents the only major carbohydrate in t’his fraction, it is assumed that it is, in fact, trehalose that is fermented. The fermentable glycogen corresponds in amount to the rapidly metabolized aerobic and anaerobic pools, and it is concluded that these two pools are identical. The more slowly metabolized aerobic (“oxidizable”) glycogen also disappears slowly under anaerobic conditions. This is accompanied by an accumulation of an equivalent amount of TCA-ext’ractable carbohydrate that can be oxidized only. This suggests that “oxidizable” glycogen can be degraded to a relatively low-molecularweight carbohydrate and t’hat it is the further utilization of the latter that requires the presence of oxygen. In addition to the TCA-soluble carbohydrate, ethanol is formed in an amount equal t,o the carbon dioxide produced. It seems probable, therefore, that the material accumulating anaerobically and subject to rapid oxidation, as reported by several workers (6, 7), is ethanol (if trehalose and fermentable glycogen are available to the cell) or the carbohydrate derived from “oxidizable” glycogen or, as in the present case, both. As would be expected from the oxidation of accumulated ethanol, t’he R.Q.‘s of cells respiring after periods of anaerobiosis are considerably lower than one. This observation may help to explain the results obtained by Stickland (5): If his suspensions were allowed to stand (anaerobic) for any length of time before testing, a low R.Q. would
OF
YEAST.
25
I
likely obtain over the short interval used, and little or no disappearance of polysaccharide (cf. Table V), would be observed. REFERENCES 1. STIER, T. J. B., AND STANNARD, J. N., J. Gen. Physiol. 19, 461 (1936). 2. SPIEGELMAN, S., AND NOZAWA, M., Arch. Biothem. 6, 303 (1945). 3. SCOTT, G. T., JACOBSON, M. A., AND RICE, M. E., Arch. Biochem. 30, 282 (1951). 4. FALES, F. W., J. Biol. Chem. 193, 113 (1951). L. H., Biochem. J. 64, 498 (1956). 5. STICKLAND, 6. WINZLER, R. J., AND BAUMBERGER, J. P., J. Cellular Comp. Physiol. 12. 183 (1938). 7. BRADY, T. G., MCGANN, C., AND TULLY, E., Biochem. J. 64, 44p (1956). A., Arch. Bio8. BERKE, H. L., AND ROTHSTEIN, them. Biophys. 72, 380 (1957). 9. TREVELYAN, W. E., in “The Chemistry and Biology of Yeasts” (A. H. Cook, ed.), p. 406. Academic Press, New York, 1958. 10. BRANDT, K. M., Biochem. 2. 309, 190 (1941). 11. LINDEGREN, C. C., Arch. Biochem. 8, 119 (1945). 12. EATON, N. R., Biochim. et Biophys. Ada 36, 259 (1959). 13. EATON, N. R., AND KLEIN, H. P., Biochem. J. 67, 373 (1957). W. E., AND HARRISON, J. S., Bio14. TREVELYAN, them. J. 60, 298 (1952). 15. LAMBOU, M. G., ilnal. Chem. 29, 1449 (1957). W. E., AND HARRISON, J. S., Bio16. TREVELYAN, them. J. 63, 23 (1956). 17. JEANLOZ, R., Helv. Chim. Acta 27, 1501 (1944). 18. DIMLER, R. J., SCHAEFER, W. C., WISE, C. S., AND RIST, C. E., ,4nal. Chem. 24,141l (1952). 19. VAN S,LYKE, II. II., PLAZIN, J. P., AND WEISIGER, J. R., J. Biol. Chem. 191, 299 (1951). 20. KATZ, J., ABRAHAM, S., AND BAKER, N., Anal. Chem. 26, 1503 (1954). 21. PARK, J. T., AND JOHNSON, M. J., J. Biol. Chem. 161, 149 (1949). 22. CHESTER, V. E., iliature 183, 902 (1959). 23. NORTHCOTE, D. H., Biochem. J. 68, 353 (1951).
OF
BIOCHEMISTRY
Endogenous
AND
Respiration
BIOPHYSICS
88, 17-25
of Yeast. NORMAN
From
the Radioisotope Microbiology
(1960)
I. The
Endogenous
R. EATON
Service, Veterans Administration Hospital and and Medicine, University of Washington, Seattle, Received
Substrate’
August
the Departments Washington
of
10, 1959
1. Kinetic and chemical data suggest the presence of three endogenous substrates in a strain of Saccharomyces cerevisiae. These consist of two metabolically distinct glycogen pools and the disaccharide, trehalose. Lipides do not serve as a substrate for endogenous respiration. 2. Utilization of one glycogen pool requires the presence of oxygen, while the other can be metabolized either aerobically or anaerobically. Trehalose is used only anaerobically. 3. Anaerobically ethanol accumulates in amounts equal to the CO, produced. In addition, there is a slow anaerobic disappearance of the “oxidizable” glycogen pool with an equivalent accumulation of a TCA-soluble carbohydrate that is utilizable only aerobically. 4. It is suggested that the rapid oxygen uptake observed after periods of anaerobiosis is due to oxidation of ethanol and the TCA-soluble carbohydrate and that the low R.Q.‘s reported previously are the result of ethanol oxidation.
petted from lipide rather than carbohydrate oxidation. On the basis of these observations Stickland concluded that carbohydrate does not serve as the endogenous substrate in yeast. It must be noted, however, that the methods used by Stickland would probably not have detected a disappearance of acidsoluble carbohydrate such as trehalose. While there appears to have been no unequivocal demonstration of glycogen disappearance in amounts equivalent to the COZ produced during aerobic utilization of endogenous reserves, several well-documented cases of glycogen disappearance under anaerobic conditions have been reported. These latter studies have been performed with both normal resting suspensions and in the presence of dinitrophenol. In the former case, the metabolic rates, as measured by carbon dioxide production, have been found to be low in comparison with the aerobic release of carbon dioxide (4). It is interesting to note, however, that evidence exists for the anaerobic breakdown of reserves without evolution of CO2 (3), leading to the accumulahion of
INTRODUCTION
The nature of the substance (or substances) stored and subsequently degraded during endogenous respiration of yeast is still the subject of some controversy, although most investigators have assumed that this endogenous substrate is a carbohydrate, “probably glycogen” (1). This assumption is based, for the most part, on the respiratory quotient (R.Q.), which is usually found to be equal to about one (1,2), and on t,he disappearance of iodine-staining material (I), hydrolyzable polysaccharide (3), or anthrone-reactive material (4). Recently, however, t’he role of carbohydrate has been questioned by Stickland (5) who was unable to find a disappearance of hydrolyzable polysaccharide during endogenous respiration. In addition, the R.Q.‘s reported by Stickland were of the order of 0.85, a value which approximat’es that exr Supported, in part, by a grant (RG-5875) the Department of Health, Education, and fare, U. S. Public Health Service.
from Wel17
EATON
some intermediate that can subsequently be oxidized rapidly (6, 7). Dinitrophenol-induced fermentation leads to a disappearance of both glycogen and trehalose (8). It is by no means certain, however, that, in either of these cases, the substrates or the metabolic pathways are the same as those of aerobic endogenous metabolism. In addition to glycogen, trehalose is sometimes mentioned as a possible endogenous substrate (9), although Brandt (10) has concluded that the oxidation of stored trehalose by resting suspensions of yeast is extremely feeble. Moreover, while the results obtained by Stickland (5) and certain observations by Lindegren (11) would seem to indicate that lipides might play a role in endogenous respiration, this possibility has not been tested. The idea that endogenous CO2 is derived from a single substrate pool appears to be based upon the results of kinetic studies of Stier and Stannard (1) and of Spiegelman and Nozawa (2). Both groups were led to the conclusion that the rate of endogenous respiration is limited by the concentration of a single substrate, which was equated to the glycogen content of the cell. Recently, however, a re-examination of the kinetics of yeast endogenous respiration (12) has shown that there are, in fact, at least two substrate pools metabolized aerobically. The work reported below describes more exactly the nature of these endogenous reserves. METHODS
AND
MATERIALS
A strain of Saccharomyces cerevisiae, LK2G12, used in previous studies (13), was grown in a medium of the following composition: glucose (containing O-14 pc. CY/lOO ml. medium), 2%; yeast extract (Difco), 0.1%; NHdCl, 0.1%; KHzPO4 , 0.1 M. Inoculation was made from a stock culture slant of the same composition plus 2% agar. The cultures were incubated at 30°C. either anaerobically (standing) for 40-48 hr. or aerobically (shaken) for 20-24 hr. The cells were harvested by centrifugation, washed twice with distilled water and resuspended in 0.1 M KHzPOd to a cell density of 6-7 mg. dry weight/ml. as measured by optical density of the suspension and reference to a previously prepared standard curve. For most of the respiration studies, 60-70 min. elapsed between the beginning of the harvesting procedure and the first manometer reading. Standard manometric techniques were used; each Warburg vessel contained a total volume of 2.2 ml.
FRACTIONATION PROCEDURE Growth of the yeast in the above medium containing glucose randomly labeled with 04 yielded cells in which all of the carbon-containing compounds were randomly labeled. Such cells were allowed to dissimilate their stored reserves for varying periods of time, and cell components were separated by a fractionation procedure based primarily on that of Trevelyan and Harrison (14). Disappearance of Cl4 from the various fractions was compared to the appearance of Cl4 in the carbon dioxide as a measure of the source of endogenous CO* .
Carbon Dioxide CO2 was absorbed in 0.2 ml. of 3 N NaOH the center well of the Warburg vessel.
Trichloroacetic
Acid (TCA)
in
Fraction
Cells were killed by tipping 0.2 ml. of 100% TCA from a side arm into the main compartment of the Warburg vessel. The contents of the vessel were poured into a 15-ml. conical centrifuge tube, and the vessel was rinsed with 1 ml. of 10% TCA which was also added to the centrifuge tube. Cells were removed by centrifugation and were resuspended in about 0.1 ml. water. The extraction procedure was repeated twice with l-ml. portions of 10% TCA, and the combined extracts were brought to 10 ml. with distilled water. Aliquots of 1.0 ml. were taken for Cl4 and carbohydrate determinations. Treatment of the TCA extract with ion-exchange resins [Dowex 50(H) followed by IR4B(HCOI)] removed about 10% of the total carbohydrate (anthrone-reactive material). About 4% of the carbohydrate in the deionized solution was reducing sugar, and hydrolysis resulted in the liberation of one reducing group per hexose unit, after correction for the small amount of reducing sugar initially present. Chromatography in propanol-ethyl acetate-water (15) resulted in only one detectable sugar spot, with an Rf identical to that of a known sample of trehalose. It is concluded, therefore, that the major carbohydrate of this fraction is the nonreducing disaccharide, trehalose [cf. (14)].
Lipide Fraction The trehalose-free cell residue was resuspended in 0.1 ml. water. Two milliliters of ethanol was added, followed by 4 ml. ethyl ether, and the suspension was centrifuged. This procedure was repeated twice, and the combined extracts were dried in wet combustion flasks for subsequent oxidation and assay of C’4.
RESPIRATION OF YEAST. I Polysaccharide
Fraction
The residue remaining aft.er extraction of lipides was suspended in 0.1 ml. water. Two milliliters of 0.25 M Na&03 was added (16), and the suspension was heated at 100°C. for 30 min. Four milliliters of ethanol was added, the precipitate was removed by centrifugation, and the supernatant liquid was discarded. The precipitate was resuspended in 2 ml. water and reprecipitated with 2 vol. ethanol, and the supernatant liquid was again discarded. The residue was taken up in 2 ml. of 2 N HISO, and heat,ed at 100°C. for 15 min. The extraction with H,SO, was repeated once, and the combined supernatant liquids, containing glycogen and mannan (lb), were brought to 10 ml. with distilled water. illiquots of 0.5 ml. were taken for determination of carbohydrate and 0”. Using preparations of glycogen and mannan isolated by the method of Jeanloz (I?), it was found that this procedure resulted in partial hydrolysis but no detectable destruction of the polysaccharide as measured by the anthrone reaction. Estimation of the glycogen component of the polysaccharide mixture isolated from randomly Cl”-labeled cells was based on the following considerat,ions: Since mannose gives only 557% of the color of an equivalent amount of glucose with anthrone (18), ,1 = G + 0.55 M and 6” = G + M where il = carbon estimated by anthrone, G = carbon in the glycogen component, X = carbon in the mannan component, and C = carbon estimated by 04 assav. Therefore, G = A -
0.55 C 0.45
It should be noted that this is based upon the assumption that only glycogen and mannan are present in the fraction, and any contaminating radioactive material would tend to decrease the value for G. The glycogen content calculated in this way should therefore probably be considered a minimum estimate. Carbon-l d-Assay All samples were converted to CO2 by wet oxidation. Lipides were oxidized with Van Slyke reagent (19). More reproducible results for wet oxidation of water-soluble materials however, were obtained with the persulfate method of Katz el al. (20). The resultant CO? was collected in XaOH, converted to BaC03, and plated as previously described (13). The samples were counted with an ultrathin window Geiger tube, and the observed activities were correct,ed for self-absorption and
19
efficiency by reference to a standard curve. All activities were calculated as disintegrations/min. and converted to equivalent microliters carbon.
Chemical Determinations Total carbohydrate was determined by the anthrone method (14) and reducing sugar by the method of Park and Johnson (21). Glucose was used as a standard in both cases. Ethanol was determined with alcohol dehydrogenase as previously reported (13). RESULTS
AEROBIC METABOLISM It has been suggested earlier (12) that, under aerobic conditions, the endogenous carbon dioxide produced by a resting suspension of yeast is derived simultaneously from two substrate pools. This conclusion was based upon the result’s of experiments designed to test the apparent reaction order of the process. The amount of endogenous substrate available to the cell (as carbon dioxide) was estimated from the amount of carbon dioxide evolved as CO% evolut’ion approached an asymptotic value, A. The logarithm of A minus X, the amount of carbon dioxide evolved after various time intervals, was plotted as a function of time. If the process has a first-order character, i.e., if carbon dioxide is derived from a single substrate, bhe concentration of which is limiting, log (A - X) will be directly proportional to t,ime, and a straight’ line should result. Such plots have shown, however, that carbon dioxide evolut,ion assumes first-order kinetics only after about 4 hr. [(12) ; cf. 13g. lA, curve 11. Extrapolation of the logarithmic portion of the curve to zero time (Fig. lA, curve 2) and replotting the difference between curves 1 and 2 results in another logarithmic curve (lcig. I A, curve 3). Thus the evolution of carbon dioxide from endogenous reserves is characterized by two “firstorder” processes occurring simultaneously. In order to explain t,hese results it is necessary to assume the presence of two endogenous substrate pools. The amount,s of material in each of these pools is given by the intercept’s of the logarithmic curves with t’he ordinate. The nat,ure of the two substrat’e pools was
20
EAT01
800 -I
i
4
6 4
lb 1’2
HOURS
I’
2
I’,‘,‘,‘,
4
6
8
IO
12
HOURS
FIG. 1. Graphic analysis of endogenous CO2 evolution. Curve 1: total available substrate remaining (as C0.J. Curve 2: extrapolation of linear (logarithmic) portion of Curve 1 to zero time. Curve 3: replot of the difference between Curves 1 and 2. A. Aerobic. B. Anaerobic. Each flask contained 11.9 mg. (dry wt.) of cells grown anaerobically. [A = total endogenous substrate (as COz) ; z = COz evolved; ordinate = log (A - 2) = log (remaining substrate).]
investigated with cells randomly labeled with C4. After growth in a medium containing glucose-Cl4 as the sole carbon source, cells were washed, resuspended in phosphate buffer, and allowed to oxidize their endogenous reserves for varying periods of time. The process was stopped by addition of trichloroacetic acid, the cell components were separated according to the schemepresented above, and the amount of Cl4 and carbohydrate remaining in each of these fractions was determined. The results of several representative experiments using this procedure are shown in Table I. The amount of aeration of the culture during growth appeared to have no effect on the kind of material oxidized. In all casesthe carbon present in the lipide and TCA fractions remained constant throughout, within the limits of experimental error. The only fraction showing significant disappearance of carbon was that containing the polysaccharides, glycogen and mannan (14). Furthermore, this
disappearance could be accounted for by a decrease in the calculated glycogen content of the fraction, and it was essentially equivalent to the amount of carbon appearing as” carbon dioxide. Thus, glycogen appears to be the only substrate oxidized, and one is forced to conclude that, under aerobic conditions, the endogenous stores consist of two metabolically distinct glycogen pools. ANAEROBIC
METABOLISM
Although Stier and Stannard (1) and Spiegelman and Nozawa (2) have reported that the fermentation of endogenous reserves is negligible, the strain of yeast used in these experiments produced significant amounts of carbon dioxide anaerobically. Fales (4) and, more recently, Chester (22) have also reported fermentation of the stored materials. In Table II the carbon dioxide produced anaerobically over an extended period of time is compared to the ethanol
RESPIRATION
OF
TABLE DISAPPEARANCE I
I
OF CELL
CARBON
21
I
I DURING
AEROBIC
RESPIRATION’ T-
I
Fraction
Cells
toe
if2
TCA
I
I
MkWl0metric
.-
YEAST.
C”
Polysaccharides
C”
Anthrone
I-
Anthrone
nzg.
min.
Anaerobic
10.8
15 240 X-240
58 550 +492
59h.6 561f51 +502*39
1373f231 1376zt98 +3*193
89&k34 854f76 -44f64
295f15 350f133 t55*92
1706f67 1441f57 - 265f68
Anaerobic
10.3
15 105 210 G-210
28 213 324 296
28f9 215&24 324f34 +-296zk29
1324f33 1365zt62 1342f37 +I&41
711fll 706f19 691f14 -2Of15
244f18 250&25 2783t30 i-54&28
2252f65 2016&11: 1876185 -376*86
Aerobic
14.6
15 105 240 15-240
22 150 284 f262
21f5 157f29 277&19 f256f52
1375f50 1423f33 1387f40 flOf53
198f6 205f3 199f15 Slf14
724*143 787~1~82 741f90 f17f128
1902f67 1789f64 1680&67 -228f71
values
pl.
a All
Calculated glycogen
1426f73 1123f29 -303f60
1084f89 734f60 350f83
1484&20 1264zt27 1141f30 -343f30
545f61 339*212 243*95 -302f94
Ii
C f
95cj0 confidence
interval.
found in the supernatant medium. The amount of ethanol formed corresponded closely to the carbon dioxide evolved. These results are consistent with the idea that the anaerobic degradation of endogenous reserves proceeds via the glycolytic sequence of reactions. A graphic analysis of the kinetics of the anaerobic process in terms of carbon dioxide produced is shown in Fig. 1B. The amount of endogenous subst,rate available was estimated from manometric data, and the logarithm of the difference between this value (A) and the CO2 produced (z) was plotted as a function of time. The resulting curve was analyzed as previously described (12). For comparison, a similar analysis of cells respiring aerobically is shown in Fig. 1A. It is clear that anaerobically, as well as aerobically, two logarithmic (‘Yirst-order”) processes occur simultaneously, suggesting the presence of two endogenous substrate pools. The amount of substrate present in each of these pools, as CO2 equivalents, was determined by extrapolation of the logarithmic portions of the curves to zero time. In the case of anaerobic carbon dioxide production this value must be multiplied by three for
TABLE
II
ANAEROBIC FORMATION OF CO2 AND ETHANOL Each flask contained 11.9 mg. (dry wt.) anaerobically grown cells. Gas phase, NZ . Temp., 30°C. Hours 2 4 12 22
co2
Ethanol
pmoles
&l?noles
3.56 4.39 6.29 7.65
3.60 4.68 6.12 7.56
comparison with aerobic experiments, since only one-third of the hexose carbon appears as carbon dioxide, the remainder accumulating as ethanol (cf. Table II). Reference to Fig. 1 shows that t,he rapidly metabolized components (curves 3), but not the slowly metabolized components (curves 2), are present in equivalent amounts. This suggests that there are three substrate pools, one of which can be metabolized either aerobically or anaerobically. The identit,y of these two rapidly metabolized components is further shown by the following experiment: Cells were allowed to oxidize their reserves for several hours and were subsequently placed under anaerobic conditions. It was to be expected that, if
22
EATON
The chemical nature of the anaerobic endogenous substrates was investigated as described above: Cells labeled with Cl4 were shaken under nitrogen, and samples were removed periodically for determination of the carbon content of the various cell fractions. The results of these studies are shown in Table III. In addition to a disappearance x 60of glycogen, carbohydrate disappeared from ’ 604 577 the TCA fraction as well, during fermenta40tion of endogenous stores. The decrease in glycogen aft.er ext.ended fermentat,ion was equivalent in amount to the rapidly metabolized anaerobic component,, and, as shown above, the lat’ter appears to be identical to I .\, , , , , the rapidly metabolized aerobic component. 2 4 6 S IO This suggests that, the slowly metabolized HOURS anaerobic component might, in fact, be FIG. 2. Graphic analysis of fermentative COZ production after oxidation. Curves 1, 2, and 3: as trehalose. If this is true, one can calculate, constant based on manin Fig. 1; before oxidation. Curve 4: available ana- using a “first-order” ometric data, the amount of carbon expected erobic substrate remaining (as CO,) after 9 hr. aerobiosis. Each flask contained 9.4 mg. (dry wt.) to disappear from the TCA fraction. In the anaerobically grown cells. Temp. 30°C. Gas phase, case of the anaerobically grown cells shown N2 . in Table III, this amount is 405 ~1. C, a value considerably higher than the 197 11. these two components are identical, graphic found. analysis of the fermentative carbon dioxide This inconsistency was resolved by a study after an extended period of oxidation would of the behavior of the TCA fraction during yield only a single logarithmic curve, the oxidation after periods of anaerobiosis. more rapidly metabolized anaerobic comWhen disappearance of carbohydrate from ponent having been oxidized. The results t’his fraction was measured (Fig. 4), it was (Fig. 2) show that after oxidation for 9 hr. found after anaerobiosis that a portion of the rapidly metabolized anaerobic compothe TCA-soluble carbohydrat’e was oxidiznent was, indeed, reduced to negligible able and that the amount utilized was a amounts [from 3(202 - 145) = 171, to function of the time the cells were main3(132 - 120) = 361. The amount of matetained under anaerobic conditions. Since no rial in the slowly metabolized anaerobic carbon disappears from the TCA fract,ion of cells not previously kept anaerobic (i.e., component, however, was decreased little, since there is no aerobic ut’ilization of treif at all, by oxidation. The converse experiment, i.e., oxidation halose, cf. Table I), anaerobiosis results in of aerobically utilizable of reserves after a period of fermentation, is the accumulation carbohydrate. Moreover, the amount of this shown in Fig. 3. Cells shaken under Nz were accumulating (equivalent to tested after 9 and 22 hr. After removal of carbohydrate 107 ~1. C after a 22-hr. period of anaerobithe supernatant liquid, the cells were resuspended in buffer for determination of aerobic osis; cf. Fig. 4) is about equal to the decrease in the slowly met’abolized aerobic glycogen COZ production. Again, the rapidly metabpool (125 in the present example; cf. Fig. 3). olized aerobic component virtually disappeared during anaerobic incubation (decreasFurther evidence that the more slowly metabolized anaerobic substrat,e is trehalose ing from 203 to 25 in 22 hr.). In addition, is shown by the equivalence of this pool, as however, there was a decrease in the amount of the slowly metabolized aerobic pool determined graphically, with t’he total car(from 470 to 420 to 345 over’ a period of 22 bohydrate content of the TCA fraction. In addit,ion, the rat,e of disappearance of trehr.). 400-
RESPIRATION
OF
YEAST.
23
I
600 673.
600
470, 420,
400 345203,
200 X
I a
100 60 60 40,
20.
.
h
i
’
6
.
6
i.
HOURS 3. Graphic analysis of aerobic CO* production after fermentation. Fig. 1; before fermentation. Curve 4: available aerobic substrate (as COJ Curve 5: after 22 hr. anaerobiosis. Each flask contained 11.9 mg. anaerobically Gas phase, air. FIG.
TABLE DISAPPEARANCE
OF CELL
Curves 1, 2, and 3: as in after 8 hr. anaerobiosis. grown cells. Temp. 30°C.
III
CARBON
DURING
FERMENTATION”
-
Fraction Cells (dry wt.)
Growth conditions
7-
Time
-
Lipide
TCA Anthrone
(C”)
Calculated glycogen
Polysaccharide AMhr0ne
C’4
1373f231 1480flOO t 107&193b
1485f73 1244f32 -241f61
1765&67 1510f123 - 255f 108
953f25 828f22 - 125+32
967f15 823f18 -144f21
1363f57 1174f81 - 189f76
C’”
-~
%=.
hr.
Anaerobic
10.8 27
0 27 O-27
295f15 294f39 -1f35
898f34 701f67 - 197f62
Aerobic
10.0
0 22 (f22
501f146 550f180 f49f194
127f8 96f4 -3lf8
-
-
a All values pl. C f 95y0 confidence interval. b It should be noted that this does not show a decrease corresponding the supernatant liquid was not removed prior to fractionation and this tween accumulated ethanol and trehalose disappearance.
to the anthrone figure represents
1143+x84 92Ort86 - 223*95 475*75 398f62 -77f-75
value because a balance be-
24
EATON
0
900 890
I
60
r
NOURS
I
120
’
ANAEROBIC .
I 180
’
I
240
MINUTES
FIG. 4. Aerobic utilization of TCA-soluble carbohydrate before and after anaerobiosis. Cells: as in Fig. 3. Cell suspensions were shaken under NP for the indicated times, then exposed to air. Residual TCA-soluble carbohydrate was determined with anthrone and converted to equivalent ~1. C. Temp. 30°C. TABLE COMPARISON
OF
POOLS
THE
IV
NONGLYCOGEN
ANAEROBIC
DISCUSSION
DETERMINED KINETICALLY AND CHEMICALLY Slow logarithmic component
Trehaloseb
3.42 x 10-S 630
Kf c, d.
4.53 x 10-s 608
a First-order constant (min.?/mg. cells). b Anthrone-positive material in TCA fraction. TABLE RESPIRATORY
QUOTIENTS QUENT
V OF CELLS
SUBSE-
TO ANAEROBIOSIS
Cells grown anaerobically in a medium containing inactive glucose (Expt. 1) or Cl4 glucose (Expt. 2). Gas phase, air. Temp. 30°C. , I I
__-~ 1 2
~___ hr.
mg.
pl.
0 2 8 0 9
11.9 213 318 328 10.4 287 288
Pl.
208 264 269 267 238
0.98 0.83 0.82 0.93 0.83
with those of Stickland (5), it was of some interest to calculate the respiratory quotients of cells after various periods of anaerobiosis. This has been done in Table V for the first 60 min. of oxidation subsequent to anaerobiosis [the time period used by Stickland (5) and by Spiegelman and Nozawa (2)]. It may be seen that a period of anaerobiosis as short as 2 hr. results in an R.Q. considerably lower than that of the freshly harvested cells, as might be expected from the oxidation of accumulated ethanol. Furthermore, during this short tJme interval the amount of polysaccharide in the cell shows little decrease (Table V), since most of the oxidation is at the expense of carbon contained in the TCA fraction, presumably ethanol and the TCAextractable carbohydrate that accumulate anaerobically.
A/L
Apl.
-47
-176
o After 60 min. oxidation subsequent indicated period of anaerobiosis. b Disappearance of Cl4 after 60 min.
to the
halose is essentially the same as that of the slowly metabolized anaerobic pool. These results are summarized in Table IV. Because of the variance of these results
Kinetic analysis of endogenous respiration has shown that yeasts respiring aerobically draw upon two endogenous substrate pools. A chemical fractionation of the respiring cells, however, shows that only glycogen disappears during respiration, and it, is concluded that there are two metabolically distinct glycogen pools in the yeast cell, Two alternatives appear possible: The two glycogens may, indeed, comprise species of distinct molecular configuration, or the glycogen may be of only one chemical or con-figurational type but spatially separated in the cell. At the present time, there is no evidence available to distinguish between these possibilities. Alkali-extractable and acid-extractable glycogens have been described by Trevelyan et al. (14) and others. In the present case, however, the quantities of these two fractions do not correspond closely to the amounts of the two glycogen pools described kinetically. On the other hand, Northcote (23) has succeeded in separating electrophoretically two glycogen components from a yeast preparation. It would appear from the method of preparation that these components also do not correspond to the acidand alkali-soluble glycogens. Whether they are equivalent to the metabolically differentiated glycogens described here is, as yet, unknown.
RESPIRATION
Anaerobically, kinetic analysis also indicates the presence of two substrate pools. Under these conditions, however, in addition to glycogen disappearance there is a disappearance of carbohydrate from the TCA fraction. Since trehalose represents the only major carbohydrate in t’his fraction, it is assumed that it is, in fact, trehalose that is fermented. The fermentable glycogen corresponds in amount to the rapidly metabolized aerobic and anaerobic pools, and it is concluded that these two pools are identical. The more slowly metabolized aerobic (“oxidizable”) glycogen also disappears slowly under anaerobic conditions. This is accompanied by an accumulation of an equivalent amount of TCA-ext’ractable carbohydrate that can be oxidized only. This suggests that “oxidizable” glycogen can be degraded to a relatively low-molecularweight carbohydrate and t’hat it is the further utilization of the latter that requires the presence of oxygen. In addition to the TCA-soluble carbohydrate, ethanol is formed in an amount equal t,o the carbon dioxide produced. It seems probable, therefore, that the material accumulating anaerobically and subject to rapid oxidation, as reported by several workers (6, 7), is ethanol (if trehalose and fermentable glycogen are available to the cell) or the carbohydrate derived from “oxidizable” glycogen or, as in the present case, both. As would be expected from the oxidation of accumulated ethanol, t’he R.Q.‘s of cells respiring after periods of anaerobiosis are considerably lower than one. This observation may help to explain the results obtained by Stickland (5): If his suspensions were allowed to stand (anaerobic) for any length of time before testing, a low R.Q. would
OF
YEAST.
25
I
likely obtain over the short interval used, and little or no disappearance of polysaccharide (cf. Table V), would be observed. REFERENCES 1. STIER, T. J. B., AND STANNARD, J. N., J. Gen. Physiol. 19, 461 (1936). 2. SPIEGELMAN, S., AND NOZAWA, M., Arch. Biothem. 6, 303 (1945). 3. SCOTT, G. T., JACOBSON, M. A., AND RICE, M. E., Arch. Biochem. 30, 282 (1951). 4. FALES, F. W., J. Biol. Chem. 193, 113 (1951). L. H., Biochem. J. 64, 498 (1956). 5. STICKLAND, 6. WINZLER, R. J., AND BAUMBERGER, J. P., J. Cellular Comp. Physiol. 12. 183 (1938). 7. BRADY, T. G., MCGANN, C., AND TULLY, E., Biochem. J. 64, 44p (1956). A., Arch. Bio8. BERKE, H. L., AND ROTHSTEIN, them. Biophys. 72, 380 (1957). 9. TREVELYAN, W. E., in “The Chemistry and Biology of Yeasts” (A. H. Cook, ed.), p. 406. Academic Press, New York, 1958. 10. BRANDT, K. M., Biochem. 2. 309, 190 (1941). 11. LINDEGREN, C. C., Arch. Biochem. 8, 119 (1945). 12. EATON, N. R., Biochim. et Biophys. Ada 36, 259 (1959). 13. EATON, N. R., AND KLEIN, H. P., Biochem. J. 67, 373 (1957). W. E., AND HARRISON, J. S., Bio14. TREVELYAN, them. J. 60, 298 (1952). 15. LAMBOU, M. G., ilnal. Chem. 29, 1449 (1957). W. E., AND HARRISON, J. S., Bio16. TREVELYAN, them. J. 63, 23 (1956). 17. JEANLOZ, R., Helv. Chim. Acta 27, 1501 (1944). 18. DIMLER, R. J., SCHAEFER, W. C., WISE, C. S., AND RIST, C. E., ,4nal. Chem. 24,141l (1952). 19. VAN S,LYKE, II. II., PLAZIN, J. P., AND WEISIGER, J. R., J. Biol. Chem. 191, 299 (1951). 20. KATZ, J., ABRAHAM, S., AND BAKER, N., Anal. Chem. 26, 1503 (1954). 21. PARK, J. T., AND JOHNSON, M. J., J. Biol. Chem. 161, 149 (1949). 22. CHESTER, V. E., iliature 183, 902 (1959). 23. NORTHCOTE, D. H., Biochem. J. 68, 353 (1951).