C O N T R I B U T I O N OF T H E P E N T O S E - P H O S P H A T E P A T H W A Y T O G L U C O S E M E T A B O L I S M IN S A C C H A R O M Y C E S C E R E V I S I A E : A C R I T I C A L A N A L Y S I S ON T H E USE OF L A B E L L E D G L U C O S E
JUANA M. GANCEDO and ROSARIO LAGUNAS
Instituto de Enzimolog(a del Consejo Superior de Investigaciones Cient[ficas, Facultad de Medicina de la Universidad Autdnoma, l141adrid-34 (Spain)
(Received December 4th, 1972) (Revision received January 15th, 1973)
SUMMARY
The val id i ty of the p r o c e d u r e o f Ka tz and Wood ~ for the d e t e r m i n a t i o n of the c o n t r i b u t i o n o f the p e n t o s e - p h o s p h a t e p a t h w a y (PPP) to glucose me- t abo l i sm in yeas t is es tabl ished, and it is shown tha t a n u m b e r of m e t h o d s are l iable to yield e r r o n e o u s results. In yeas t growing on glucose wi th NH~ as n i t rogen source , the p e n t o s e - p h o s p h a t e p a t h w a y was f o u n d to a c c o u n t for 2.5% of the to t a l m e t a b o l i s m o f glucose, while in yeas t g rowing on an en- r iched m e d i u m where NH~ had been rep laced b y Difco yeas t e x t r a c t the con- t r i bu t ion was lowered to 0.9%. These resul ts c o n f i r m tha t a ma jo r role for this p a t h w a y is to supp ly N A D P H for b io syn the t i c react ions .
INTRODUCTION
The use of label led glucose for de t e rmin ing the c o n t r i b u t i o n o f the PPP to glucose m e t a b o l i s m requires a n u m b e r of s impl i fy ing a s sumpt ions . I f these a s s u m p t i o n s are no t expl ic i t ly s ta ted and their val idi ty assessed, p rocedu re s using rad ioac t ive subs t ra tes are liable to yie ld e r r o n e o u s results.
Quan t i t a t i ve i n f o r m a t i o n on me tabo l i c pa t t e rns for glucose u t i l iza t ion by yeas t is scarce 2 -4 ; m o r e o v e r the p rocedures used to gain this i n f o r m a t i o n were inadequa te , as it will be shown later. I t t h e r e f o r e appea red useful to examine the val id i ty of d i f f e ren t p rocedu re s in o rder to a p p l y the m o r e con- ven ien t to growing yeas t . Since the c o n t r i b u t i o n of the PPP seems to be re-
Abbreviations: EM, Embden-Meyerhof; GICO2, CO2 specific yield from C-1 of glucose -- ratio of radioactive CO 2 formed to [ 1 -I 4C]glucose utilized; G6CO2, CO2 specific yield from C-6 of glucose -- ratio of radioactive CO2 formed to [6 -14 C] glucose utilized; NTP, non-triose-P pathway; PPP, pentose-phosphate pathway.
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lated with biosynthetic requirements of the cell s,~ measurements have been carried out in yeast growing on different nitrogen sources.
MATERIAL AND METHODS
Saccharomyces cerevisiae {strain 13 gal ) was grown with vigorous shaking at 30 ° in a synthetic medium 7 or in an enriched medium where 1% Difco yeast extract was substituted for (NH4)2 HPO4. Glucose 2% was added as carbon and energy source.
Actively growing yeast was filtered through Millipore and resuspended in fresh medium of the same composition containing 0.5% glucose. From the resulting suspension, which contained about 2 mg wet yeast/ml, 12-ml ali- quots were transferred to 125-ml Erlenmeyer flasks containing 0.2 pCi of [ 1 -~4 C] glucose or [6 -~4 C] glucose (The Radiochemical Centre, Amersham). The flasks were stoppered with rubber caps fitted with a windowed plastic test tube and vigorously shaken at 30 ° for 70 min. After this time 0.5 ml of 1 M hyamine was injected into the plastic tube and 3 ml of 5.4 N HC1 was added to the medium; shaking was resumed for 1 h to allow total absorption of the CO2 formed. The hyamine was transferred to a counter vial containing 2 ml of scintillation liquid by washing with 0.5 ml methanol, and radioactivi- ty was determined in a Packard 3320 Tri-Carb scintillation spectrometer. An aliquot of the acidified yeast suspension was centrifuged and glucose assayed in the supernatant, using glucose oxidase s for the synthetic medium and the Dische procedure 9 for the enriched medium; this latter method was used since the enriched medium produced interference with the glucose oxidase system. However, since glucose standards are run in parallel both procedures yield comparable results.
For the determination of growth yields, aliquots of the cultures were filtered through Millipore, dried at 105 ° for 24 h and weighed. Aliquots of the cul- tures were also centrifuged for 5 min at 5000 × g and glycerol and glucose were measured in the supernatants. Glycerol was determined enzymatically 10, and glucose, as indicated above.
RESULTS AND DISCUSSION
The most elaborated model for calculating the contribution of the PPP to glucose metabolism has been developed by Wood and associates ',~,~2. We will examine here the salient features of this model in order to ascertain if it is applicable to S. cerevisiae growing on glucose.
(1) Equilibration of the hexose phosphates is assumed to be complete. In yeast a very active phosphoglucose isomerase maintains glucose-6-P and fruc- tose-6-P near equilibrium ~3. This equilibrium between hexose phosphates results in the recycling of a part of the fructose-6-P formed by the PPP, so calculations made without considering recycling 3 are invalidated.
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(2) Complete equilibration of the triose phosphates is also assumed. This assumption is difficult to test, but it has been established ,2 that if the CO2 yields from C-1 of glucose are twice or more those from C-6, a fair estima- tion of the PPP will be obtained even when the equilibration of the triose phosphates is limited and provided that glycerol synthesis does not exceed 20%. These conditions are fulfilled by S. cerevisiae growing on glucose since glycerol production is less than 10% (Table I) and the CO2 yield from C-1 of glucose is 2 to 7 times greater than the CO: yield from C-6 {Table II).
(3) It is considered that the reaction: fructose-6-P -~ fructose-l ,6-diP is ir- reversible. In the case of yeast growing on glucose, no significant amounts of fructose-6-P can be formed from fructose diphosphate since the synthesis of fructose-l ,6-diphosphatase is strongly repressed 14
(4) The original calculations ' were developed for a case where only the EM pathway and the PPP were operative. In growing yeast this is obviously not the case, since polysaccharides, nucleic acids, etc. are being synthesized. How- ever, since only about 10% of the glucose utilized is assimilated in cellular components (Table I), the error introduced by neglecting the NTP ' would be less than 5%.
(5) A substantial recycling of COs would introduce serious error in the estimation of the PPP using CO2 yields. In growing yeast some recycling may occur since the biosynthesis of purines involves a carboxylation, and draining of intermediates of the citric acid cycle for the synthesis of aspartate and glutamate makes the operation of pyruvate carboxylase necessary. However, from the yeast content in purines, aspartate and glutamate, it can be calcu- lated that the CO2 involved in these reactions represents less than 1% of the total COs formed and this amount would not affect the results.
(6) Reactions of transaldolase exchange could affect the distribution of the radioactivity in the metabolic products and invalidate the results ,s. However.
T A B L E I
G L U C O S E A S S I M I L A T I O N A N D G L Y C E R O L P R O D U C T I O N BY S. cerevisiae
Cul tures o f S. cerevisiae grown as ind ica ted in M A T E R I A L A N D M E T H O D S were f i l tered at d i f f e ren t phases of growth . G r o w t h yields, glycerol and glucose were e s t ima ted as descr ibed in M A T E R I A L AND METHODS. T he resul ts are refer red to 1 g o f yeast (d ry we igh t ) pro- duced. Figures are m e a n values o f 3 e x p e r i m e n t s fo l lowed by t he s t anda rd devia t ion .
G r o w t h Glucose Glucose Glycerol Glucose c o n d i t i o n s c o n s u m e d ass imi la ted p roduced t r a n s f o r m e d
in to glycerol (g) (%) (g) (%)
Minimal m e d i u m glucose 10.5 + 0.3 9 .5 0.77 -+ 0 .09 7.4 Enr i ched m e d i u m glucose 7.8 + 0.3 13 0 .33 -+ 0 .03 4.2
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TABLE II
CONTRIBUTION OF THE PPP TO GLUCOSE METABOLISM IN S. cerevisiae
S. cerivisiae grown as indicated was resuspended in fresh medium containing radioactive glucose labelled either in C-1 or C-6. After incubation at 30 ° for 70 rain radioactivity in- corporated in CO2 was measured. For details see MATERIAL AND METHODS. Contribu- tion of the PPP was estimated as described by Katz and Wood I. Figures are mean values of 8 determinations, followed by the standard deviations.
these exchange r eac t ions , even extensive, do n o t a f fec t the calculat ions made using specific yields f rom [1-14C] glucose and [6-14C] glucose ~
The above cons idera t ions show tha t the model fo r glucose metabo l i sm pro- posed by Katz and Wood ~ is valid for yeas t growing on glucose. Th e re fo r e it is possible to app ly the p rocedures t hey have deve loped for calculat ing the c o n t r i b u t i o n o f the PPP.
The c o n t r i b u t i o n o f the PPP to glucose metabo l i sm will be expressed as the f rac t ion (glucose t r ans fo rmed in to g lycera ldehyde-3-P b y the PPP)/(ca- t abo l ized glucose)
G1CO2 -- G6CO2 ppp = 3 -- 2G1CO2 -- G6CO2
The results should then no t be d i rec t ly c o m p a r e d with those ob ta ined using o the r def in i t ions for PPP.
Radioac t iv i ty i nco rpo ra t ed in some t r iose -phospha te derivat ive (i.e. lipids) m a y also be used to calculate the PPP con t r ibu t ion . However , this m e t h o d can be subjec t to a cons iderable e r ror w h e n the c o n t r i b u t i o n o f the PPP is low.
The ra t io ~4CO2 f rom [1-~4C] glucose/14CO2 f rom [6-~4C] glucose is still widely used as an index for the p r o p o r t i o n of glucose ca tabol ized t h ro u g h the PPP J6,17. This is misleading, since variat ions in the p r o p o r t i o n of glucose ox id ized t h r ough the citric acid cycle marked ly a f fec t this rat io. Only in par- t icular cases where the con t r i bu t i on of the citric cycle remains cons t an t would a change in this ra t io ref lec t a change in the p ro p o r t i o n o f glucose channel led
th rough the PPP. Ano the r m e t h o d for es t imat ing the glucose catabol ism pa t te rn , based on
the p r o p o r t i o n of the to ta l CO2 p roduced v ia PPP, py ruva te d e c a r b o x y l a t i o n or citric acid cycle has also been p roposed 4. However , this m e t h o d can be also subjec t to cons iderable e r ror since the CO: p r o d u c t i o n is no t p ropor - t ional to the glucose me tabo l i zed bu t is i tself a func t ion of the ca tabol i sm
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pa t t e rn . Fo r ins tance , the f rac t ion o f CO2 f o r m e d in the PPP did n o t change s ignif icant ly wi th ae ra t ion levels, bu t if the da ta are p rocessed as descr ibed above it appea r s tha t , as ae r a t i on decreases , the c o n t r i b u t i o n o f the PPP to the ca t abo l i sm o f glucose is l owered f r o m 7.4% to 2.3%.
Most m e a s u r e m e n t s of the PPP have been carr ied ou t in non-g rowing cells 2,3 and in some cases wha t was d e t e r m i n e d cou ld have been a mixed pa t t e rn o f m e t a b o l i s m since the e x p e r i m e n t was e x t e n d e d unt i l the glucose p resen t was e x h a u s t e d 3. In our case, a d i lu ted yeas t suspens ion in g rowth m e d i u m was used, the c o n c e n t r a t i o n o f glucose (0.5%) was such t ha t it would n o t be re- duced to less t han 50% at the end of the e x p e r i m e n t , and the 70-min incuba- t ion t i m e was cons ide rab ly longer than the 15-min lag fo l lowing f i l t ra t ion and y e t shor t enough fo r the m e t a b o l i c cond i t ions to r ema in r easonab ly con- s tant .
The resul ts shown in Table II indica te t ha t in S. cerev i s iae growing on glu- cose the c o n t r i b u t i o n o f the p e n t o s e - p h o s p h a t e p a t h w a y to the m e t a b o l i s m of glucose is on ly 2.5%. When the yeas t is growing on a m e d i u m where in- organic n i t rogen has been rep laced b y yeas t ex t r ac t the c o n t r i b u t i o n o f the PPP is l owered to 0.9%. In the p resence o f yeas t ex t r ac t the b i o s y n t h e t i c pa th- ways for a m i n o acids, pur ines and pyr imid ines should be repressed, and the r e q u i r e m e n t s for N A D P H accord ing ly lowered . T h e r e f o r e the resul ts ob t a ined show tha t there is a co r re la t ion b e t w e e n N A D P H r e q u i r e m e n t s and ope ra t i on of the PPP. Al though the mo lecu l a r m e c h a n i s m o f con t ro l is no t es tab l i shed it appea r s t ha t the c o n t r i b u t i o n of the PPP could be regula ted by the N A D P supply .
ACKNOWLEDGEMENTS
We are i n d e b t e d to Drs. Patr ic ia McLean, A. Sols and H.G. Wood for criti- cal read ing of the m a n u s c r i p t and to Miss Eulalia Moreno for able t echnica l assistance. R.L. was a Research Fe l low of the F u n d a c i 6 n Juan March.
REFERENCES
1 J. Katz and H.G. Wood, J. Biol. Chem., 238 (1963) 517. 2 H.J. Blumenthal, K.F. Lewis and S.J. Weinhouse, J. Am. Chem. Soc., 76 (1954) 6093. 3 C.H. Wang, I. Stern, C.M. Gilmour, S. Klungsoyr, D.J. Reed, J.J. Bialy, B.E. Christensen
and V.H. Cheldelin, J. Bacteriol., 76 (1958) 207. 4 S.L. Chen, Biochim. Biophys. Acta, 32 (1959) 470. 5 H. Holzer and I. Witt, Biochim. Biophys. Acta, 38 (1960) 163. 6 C.B. Osmond and T.Ap. Rees, Biochim. Biophys. Acta, 184 (1969) 35. 7 C. Gancedo, M.L. Salas, A. Giner and A. Sols, Biochem. Biophys. Res. Commun., 20
(1965) 15. 8 A. Sols and G. DelaFuente, Biochim. Biophys. Acta, 24 (1957) 206. 9 G. Ashwell, in S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. 3,
Academic Press, New York, 1957, p. 73. 10 O. Wieland, in H.U. Bergmeyer (Ed.), Methods in Enzymatic Analysis, Academic Press,
New York and London, 1963, p. 211.
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11 B.R. Landau, G.E. Bartsch, J. Katz and H.G. Wood, J. Biol. Chem., 239 (1964) 686. 12 J. Katz, B.R. Landau and G.E. Bartsch, J. Biol. Chem., 241 (1966) 727. 13 Y~ Nagakawa and E.A. Noltmann, J. Biol. Chem., 240 (1965) 1877. 14 C. Gancedo, J.M. Gancedo and A. Sols, Biochem. Biophys. Res. Commun., 26 (1967)
923. 16 R. Ela, W. Chefurka and J.R. Robinson, J. Insect Physiol., 16 (1970) 2137. 17 P.K. Maitra, J. Bacteriol., 107 (1971) 759.
A P P E N D I X
Calculation o f the error in t roduced in the de terminat ion o f PPP when non- triose-P p a t h w a y s are operative
In a general case glucose is me tabo l i zed th rough a var ie ty of pa thways , EM, PPP and NTP; f rom the glucose t r ans fo rmed into t r iose phospha t e a f rac t ion (K) is c o m p l e t e l y ox id ized to CO: in the t r icarboxyl ic acid cycle.
For each set of values o f EM, PPP, NTP and K it is possible to calculate G1CO: and G6CO~ (ref. 1)
G1CO2 = [(EM X K) + 3 PPP] Q
G6CO: = (EM + PPP) K
1 Q - 1 + 2 PPP
is a co r rec t ion fac to r which takes in to accoun t d i lu t ion o f the specific act ivi ty of glucose-6-P f rom [ 1-'4 C] glucose by recycling.
F rom the values o f G1CO: and G6CO2 ob ta ined PPP can be ca lcula ted using
ppp = G1CO: -- G6CO: 3 -- 2 G I C O : -- G6CO: "
The results of such a ca lcula t ion are given in Table I. As can be seen the fig- ures are near ident ical to the original values, even when NTP is as high as 0.2, tha t is when 20% of the glucose ut i l ized is me tabo l i zed t h ro u g h NTP. The error i n t roduc e d exceeds 5% on ly when the p r o p o r t i o n o f glucose comple te - ly ox id ized to CO: exceeds 50% (K ~> 0.5).
There fore , in the case o f yeas t growing on glucose where K is of the order of 0.02, no significant e r ror is i n t roduced by neglect ing the NTP.
Calculation o f the error in troduced in the de terminat ion o f PPP by neglecting recycling o f CO:
Since 1 g yeas t (dry weight) contains , on the average, 125 pmoles purines and 725 t~moles aspar ta te + g lu tamate ~ ( for the synthesis of 1 g yeas t 850 pmoles CO: would be f ixed.
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TABLE I (Appendix)
EFFECT OF NEGLECTING METABOLISM BY NTP IN ESTIMATING THE PPP
Contribution of the different pathways to glucose metabolism
In yeast grown on minimal medium, 10.5 g glucose are consumed in the synthesis of 1 g yeast, so that 0.015 pmole CO2 is fixed per ~mole glucose utilized. In yeast grown on enriched medium 0.020/~mole CO2 is fixed per ~mole glucose utilized.
In yeast growing on glucose each pmole of glucose yields about 1.6 pmole of CO2, mostly through decarboxylat ion of pyruvate (Lagunas and Gancedo, unpublished}, so the CO2 fixed represents about 1% of the total CO2 formed.
Several possibilities appear for the fixation of CO2. (1) The CO2 formed in the decarboxylat ion of pyruvate, in the PPP and
in the tr icarboxylic acid is randomly equilibrated within the cell, and used by the carboxylases. In this case the amounts of G1CO2 and G6CO2 actually evolved would be 1% higher than the values observed. Therefore, in the for- mula
p p p = G I C O 2 - - G 6 C O 2
3 - - 2 G I C O 2 - - G 6 C O 2 '
the numera tor should be 1% higher. Since the denominator would remain vir- tually unchanged, the error in the determinat ion would not exceed 1--2%.
(2) The pyruvate carboxylase (plausibly mitochondrial) utilizes preferen- tially the CO2 formed in the tr icarboxylic acid cycle. This CO2 is originated from C-1, -2, -5 and -6 of glucose in equimolecular proportions. From 0.015-- 0.020 pmole CO2 f ixed/pmole glucose one-fourth would be derived from C-1 and the same amount f rom C-6. Therefore, although the actual values of G1CO2 and G6CO2 would be higher than those observed, the difference GICO2 -- G6CO2 would remain unchanged. Since the expression 3 -- 2G1CO2
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- - G6CO2 would bare ly change (less than 1% decrease) no s ignif icant e r ror wou ld be i n t r o d u c e d in the d e t e r m i n a t i o n o f PPP.
(3) The ca rboxy la ses do n o t use m e t a b o l i c COs bu t ma in ly the COs f r o m air. In this case no recyc l ing wou ld occur and t h e r e f o r e no er ror could be in- t r o d u c e d for this cause.
REFERENCES
] J. Katz and H.G. Wood, J. Biol. Chem., 238 (1963) 517. 2 A.A. Eddy, in A.H. Cook (Ed.), The Chemistry and Biology of Yeasts, Academic Press,
