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Mechanism of carbon dioxide fixation by Saccharomyces cerevisiae
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u p t a k e a t o n e h o u r w a s t h e s a m e w h e n t h e ratio of e q u i v a l e n t s of a s c o r b a t e t o iron w a s v a r i e d f r o m 2 to io. A t a ratio of 4 o, t h e r e w a s a 5 o % inhibition. G a s s i n g t h e s o l u t i o n s w i t h N s before i n c u b a t i o n to deplete t h e m i x t u r e s of O s i n h i b i t e d t h i s s y s t e m as well as t h e a p o f e r r i t i n - f e r r o u s s y s t e m . I n c r e a s i n g t h e iron c o n c e n t r a t i o n as m u c h as 8-fold (holding a s c o r b a t e c o n s t a n t a t i. 5 e q u i v a l e n t s p e r g r a m a t o m of apoferritin N) h a d no effect o n t h e reaction. As s h o w n in t h e lower p o r t i o n of T a b l e I, t h e e x t e n t of t h e r e a c t i o n increased w i t h t e m p e r a t u r e u p t o 65 ° C. (At h i g h e r t e m p e r a t u r e s c o a g u l a t i o n of t h e p r o t e i n b e g a n to be significant.) T h i s is in accord w i t h t h e findings of SALTMAN et al. 7 on iron u p t a k e b y r a t liver slices, w h i c h also h a d a h i g h o p t i m u m t e m p e r a t u r e . I n t h a t r e s p e c t it s u p p o r t s t h e i r s u g g e s t i o n t h a t t h e u p t a k e w a s t h e r e s u l t of intracellular ironb i n d i n g b y apoferritin. T h e initial r a t e of t h e iron u p t a k e in o u r s y s t e m a t v a r i o u s t e m p e r a t u r e s f r o m o to 7 3 ° C i n d i c a t e d a n a c t i v a t i o n e n e r g y of 8 kcal/mole, c a l c u l a t e d f r o m t h e A r r h e n i u s equation. T h e u p t a k e of ferrous iron w a s inhibited b y ascorbic acid (at a c o n c e n t r a t i o n 5 t i m e s t h e e q u i v a l e n t s of iron present) to a n a m o u n t c o m p a r a b l e to t h e a s c o r b a t e - m e d i a t e d u p t a k e of /erric iron. MAZUR el al. s a n d BIELIG AND BAYERs h a v e d e m o n s t r a t e d t h e release of s o m e of t h e ferric iron f r o m ferritin in t h e presence of ascorbic acid, g l u t a t h i o n e , c y s t e i n e or a n a e r o b i c liver tissue, a n d its s u b s e q u e n t t r a n s f e r to t h e p l a s m a iron-binding protein, in w h i c h iron aLso occurs in t h e ferric s t a t e . T h e evidence p r e s e n t e d here i n d i c a t e s t h a t t h e reverse r e a c t i o n c a n occur. U n d e r aerobic c o n d i t i o n s a n d in t h e presence of ascorbic acid or boiled liver e x t r a c t , ferric iron c a n be reduced, a b s o r b e d o n t o apoferritin a n d reoxidized b y a t m o s p h e r i c o x y g e n t o a f o r m w i t h optical a n d a n t i g e n i c p r o p e r t i e s of ferritin. M A R Y W . LOEWUS Department o/Physiological Chemistry, RICHARD A. FINEBERG
University o/Cali/ornia School o/Medicine, Berkeley, Calf/. ( U.S.A .) 1 S. G R A N I C K A N D L. MICHAELIS, J. Biol. Chem., 147 (I943) 91. t A. MAZUR, S. BAEZ AND E. SHORR, J. Biol. Chem., 2I 3 (1955) 147. a S. GRANICK AND P. F. HAHN, J. Biol. Chem., 155 (1944) 661. ~J. BIELIG AND E. BAYER, Naturwissenschatten, 42 (1955) I25. 6 M. V. LAUFSERGER, Bull. soc. chim. biol., 19 (1937) I575. • H . BORSOOK, A. ABRAMS AND P. H. LowY, J. Biol. Chem., 215 (I955) IZI. T p. SALTMAN, R. D. FISKIN AND S. B. BELLINGER, J. Biol. Chem., 220 (1956) 741. s j. BIELIG AND E. BAYER, Naturwissenscha/ten, 42 (1955) 466. R e c e i v e d A u g u s t 4th, 1957
Preliminary Notes
Mechanism of carbon dioxide fixation by SaccharomycescerevisMe B a k e r ' s y e a s t (Saccharomyces cerevisiae) a s s i m i l a t e s c a r b o n dioxidet, S, s, 4, p r e s u m a b l y w i t h f o r m a t i o n of a Ct-dicarboxylic acid (KLEINZELLER a, STOPPANI $ AND DAVm eta/.) t. Several m e c h a n i s m s m a y be p r o p o s e d to e x p l a i n t h e i n c o r p o r a t i o n of CO s, e.g., (i), s y n t h e s i s of o x a l o a c e t a t e b y c o n d e n s a t i o n of CO s a n d p h o s p h o e n o l p y r u v a t e (UTTER eta/. s, BANDURSKI AND I~PMANHT); (2), r e d u c t i v e c a r b o x y l a t i o n of p y r u v a t e w i t h f o r m a t i o n of m a l a t e (OCHOA eta/, s) ; (3), c a r b o x y l a t i o n o f p r o p i o n a t e w i t h f o r m a t i o n of s u c c i n a t e (LARDY AND ADLRRS). If t h e y e a s t w e r e allowed t o oxidize s u b s t r a t e s (acetaldehyde, acetate, p y r u v a t e or glucose ) in t h e p r e s e n c e o f 1~COs, t h e kinetics of t h e d i s t r i b u t i o n of t h e r a d i o a c t i v i t y fixed in t h e m e t a b o l i c pools c o u l d e l u c i d a t e t h e initial s t e p of c a r b o n dioxide assimilation, a n d f u r t h e r m o r e , t h e n a t u r e of t h e ' m a i n p a t h o f s u b s t r a t e o x i d a t i o n , w h i c h in S. cerevisiae is still a m a t t e r of c o n t r o v e r s y 1°. T h e p r o c e d u r e w a s s i m i l a r t o t h a t applied b y CALVIN et a/. n to s t u d y t h e p a t h of c a r b o n i n p h o t o s y n t h e s i s . S u s p e n s i o n s of s t a r v e d y e a s t were i n c u b a t e d w i t h r a d i o a c t i v e b i c a r b o n a t e a n d s u b s t r a t e s in a closed flask c o n n e c t e d to a v o l u m e c o m p e n s a t o r (c/. STOPPANI et a/.lS), t h e O t u p t a k e of t h e y e a s t b e i n g m e a s u r e d i n d e p e n d e n t l y u n d e r s i m i l a r e x p e r i m e n t a l c o n d i t i o n s . Controls w i t h o u t s u b s t r a t e were r u n s i m u l t a n e o u s l y . W i t h acetic a n d p y r u v i c acids, t h e initial p H of t h e m e d i u m w a s 2.5, in o r d e r t o increase t h e y e a s t p e r m e a b i l i t y . T h e s t r o n g a c i d i t y did n o t affect t h e y e a s t m e t a b o l i c p a t t e r n s , as w i t h glucose or a c e t a l d e h y d e as s u b s t r a t e s , e x p e r i m e n t s c a r r i e d o u t a t p H 2.5 a n d 7-4 g a v e n e a r l y t h e s a m e results. A f t e r i n c u b a t i o n (from x 5 sec to 3 k
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according to t h e e x p e r i m e n t ) , a l i q u o t s of t h e y e a s t s u s p e n s i o n s were a d d e d to a n d t h o r o u g h l y m i x e d w i t h 9 vol. m e t h a n o l . T h e r a d i o a c t i v i t y of t h e m e t h a n o l s u s p e n s i o n s a n d t h a t of t h e clear s u p e r n a t a n t fluids o b t a i n e d b y c e n t r i f u g a t i o n were m e a s u r e d . T h e r a d i o a c t i v e c o m p o u n d s dissolved in t h e m e t h a n o l - w a t e r m i x t u r e were c o n c e n t r a t e d to a s m a l l v o l u m e , s e p a r a t e d b y p a p e r c h r o m a t o g r a p h y (BENSON et al.13), a u t o r a d i o g r a p h e d a n d t h e a c t i v i t y of e a c h c o m p o u n d c o u n t e d on t h e c h r o m a t o g r a m . T h e a s s i m i l a t i o n of CO S w a s g r e a t e r in t h e p r e s e n t of s u b s t r a t e . T h u s , w i t h o.o2 M acetate, t h e Q o , (/zl O 2 u p t a k e / m g d r y cells/h) w a s I3.8 a n d t h e r a d i o a c t i v i t y fixed 87o c t s / m i n / m g d r y cells/h, w h e r e a s in t h e a b s e n c e of acetate, t h e respective values were 1.1 a n d lO 3. Similar r e s u l t s were o b t a i n e d w i t h glucose, p y r u v a t e a n d a c e t a l d e h y d e , w h i c h confirms a n d e x t e n d s t h e r e s u l t s o b t a i n e d b y o t h e r workersl, 2, 3, 4. A t t h e b e g i n n i n g of t h e fixation, all t h e r a d i o a c t i v i t y a p p e a r s in m e t h a n o l - w a t e r soluble c o m p o u n d s . T h e s e were aspartic, g l u t a m i c , malic a n d citric (plus aconitic a n d isocitric) acids. A f t e r 2 - 3 h, t h e d i s t r i b u t i o n (%) of 14C r e a c h e d a s t e a d y state. If t h e r a d i o a c t i v i t y (%) a p p e a r i n g in each c o m p o u n d a f t e r t h e b e g i n n i n g of fixation is p l o t t e d as a f u n c t i o n of time, t h e d i s t r i b u t i o n c u r v e s allow a decision b e t w e e n t h e several possible p a t h s of c a r b o n i n c o r p o r a t i o n 14, e v e n if oxaloacetic (on a c c o u n t of instability) is n o t p r e s e n t in t h e c h r o m a t o g r a m s . T h u s , if t h e first c o m p o u n d in w h i c h t h e C O , is fixed is oxaloacetic acid, t h e c u r v e s s h o w i n g t h e d i s t r i b u t i o n of r a d i o a c t i v i t y in its i m m e d i a t e d e r i v a t i v e s (aspartic, malic a n d citric acids) will initially h a v e a positive slope. However, if t h e t r a n s a m i n a t i o n of oxaloacetic acid is a m u c h f a s t e r r e a c t i o n t h a n t h o s e l e a d i n g to malic a n d citric acids, it is possible t h a t t h e positive slope of t h e a s p a r t i c acid w o u l d be finished before m e a s u r e m e n t s are begun, a n d t h e plot of a s p a r t i c acid would t h e n a p p e a r w i t h a n e g a t i v e slope. O n t h e o t h e r h a n d , if t h e c a r b o n is i n c o r p o r a t e d t h r o u g h t h e " m a l i c r e a c t i o n " , t h e c u r v e for malic acid w o u l d h a v e to be t h e first one s h o w i n g a n e g a t i v e slope. Fig. I s h o w s t h a t a s p a r t i c acid is t h e o n l y c o m p o u n d to h a v e a d i s t r i b u t i o n c u r v e w i t h a n e g a t i v e slope, w h i c h leads to t h e conclusion t h a t oxaloacetic is t h e first p r o d u c t of fixation. T h e e s s e n t i a l role of oxaloacetic is s u p p o r t e d b y t h e presence of "oxaloacetic" c a r b o x y l a s e in cell-free e x t r a c t s of b a k e r ' s yeast, w h e r e a s in t h e s a m e e x t r a c t s , no " m a l i c e n z y m e " c a n be d e t e c t e d ( o b s e r v a t i o n s w i t h J. CANNATA, to be published). Therefore t h e " m a l i c r e a c t i o n " as well as o t h e r less likely m e c h a n i s m s c a n be ruled o u t as a n e x p l a n a t i o n of CO 2 fixation b y yeast.
u.
10C 9G
~8C
¢o ~70 .,~.k
~C. 6° Fig. I. R e l a t i v e d i s t r i b u t i o n of t4C a f t e r f i x a t i o n of 14CO~ b y S. cerevisiae in t h e p r e s e n c e of glucose. 285 m g y e a s t ; 5 ° /*moles glucose; 4.2 /*moles NaH14CO3 (2.8-IO s c t s / m i n ) ; p h o s p h a t e buffer p H 7.I, o.~ m M . T o t a l v o l u m e ; Io ml. Air in t h e g a s p h a s e . T e m p . : 23 °. A, a s p a r t i c ; G, g l u t a m i c ; M, malic a n d C, citric (plus cis-aconitic a n d isocitric) acids.
ua~ 50
~ (.j 40 3G Q. ?0 t0 15
30
45
TIME (M~A,.)
60
T h e d e g r a d a t i o n of r a d i o a c t i v e a s p a r t i c a n d g l u t a m i c acids o b t a i n e d f r o m y e a s t w h i c h w a s allowed to oxidize p y r u v i c acid in t h e presence of 14CO 2, s h o w s t h a t a t first all t h e 14C fixed is c o n c e n t r a t e d in t h e 4 - c a r b o n of a s p a r t i c a n d in t h e 1-carbon of g l u t a m i c acids respectively, b u t later t h e r a d i o a c t i v i t y a p p e a r s also in t h e 1-carbon of a s p a r t i c acid. T h i s m a y be e x p l a i n e d b y equilibration of oxaloacetic w i t h malic, a n d t h r o u g h t h e l a t t e r w i t h t h e a p p a r e n t l y s y m m e t r i c a l l y labelled f u m a r i c acid. T h e r e s u l t s s u m m a r i z e d a b o v e are c o n s i s t e n t w i t h t h e o p e r a t i o n of t h e t r i c a r b o x y l i c acid cycle as t h e m a i n o x i d a t i v e p a t h w a y in b a k e r ' s yeast. A l t h o u g h y e a s t cells c o n t a i n isocitritase 15, t h e r e c e n t l y described is g l y o x a l a t e cycle does n o t s e e m to be of m u c h i m p o r t a n c e , as is s h o w n b y t h e s t r o n g labelling of g l u t a m i c acid. A full a c c o u n t of t h i s w o r k will be given in a n o t h e r journal.
Department of Biochemistry, Faculty o/ Medicine, University of Buenos Aires and Laboratory for Cell Metabolism ( C . N . E . A .), Buenos Aires (Argentina)
A. O. M. STOPPANI SUSANA L. S. DE FAVELUKES L u c f A CONCHES F . L . SACERDOTE
1 j . RUNNSTROM, K. BRANDT AND R. MARCUSE, Arkiv Kemi Mineral. Geol., I7A (I943) no. 3z S. RUBEN AND M. KAMEN', Proc. Natl. Acad. Sci. U.S., 26 (I94o) 418. * A. KLEINZELLER, Biochem. J., 35 (1941) 495-
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* j . W. DAvIs, V. H. CHELDELIN, B. E. CHRISTENSEN AND C. H. WANG, Biochim. Biophys. ,4 cta, 21 (1956) 1oi. s A. O. M. STOPPANI, Nature, 167 (1951) 653. s M. F. UTTER AND K. KURAHASHI,J. Biol. Chem. 207 (1954) 821. 7 R. S. BANDURSKI AND F. LIPMANN, J. Biol. Chem., 219 (1956) 741. s S. OCHOA, A. H. MEHLER AND k. KOENEERG,J. Biol. Chem., 174 (1948) 979. 9 H. A. LARDY AND J. ADLER, J . Biol. Chem., 219 (1956) 9331 0 H. A. KREBS, S. GURIN AND L. V. EGGLESTON, Biochem. J., 51 (I952) 614. 11 M. CALVIN AND P. MASSINI, Experientia, 8 (1952) 445. 12 A. O. M. STOPPANI, R. C. FULLER AND M. CALVIN,J. Bacteriol., 69 (1955) 491. xs A. A. BENSON, J. A. BASSHAM, M. CALVIN,T. C. GOODALE,V. A. HAAS AND W. STEPKA,J. Am. Chem. Soc., 72 (195 o) 171o. 14 W. J. WHITEHOUSE AND J. L. PUTMAN, Radioactive Isotopes, Oxford Univ. Press, London, 1953, p. 292. 15 j . A. OLSON, Nature, 174 (1954) 695. le H. L. KORNBERG AND H. A. KREBS, Nature, 179 (1957) 988. Received July 6th, I957
Polyribonucleotide synthesis with highly purified polynucleotide phosphorylase* Earlier preparations of polynucleotide phosphorylase of Azotobaaer vinelandii z, z, s did not permit one to determine whether, in analogy with muscle phosphorylase, a primer was required for polyribonucleotide synthesis. Highly purified preparations of this enzyme have now been obtained with which a requirement for priming has been established. These have been obtained by use of steps previously reported4, followed by other steps involving protamine, ammonium sulfate, and gels. The specific activity of the enzyme, expressed as in a previous paper 1, increases from about I.O in the initial extract to 25o-3oo in the final preparation for which a purity of 7o-8o% is suggested by sedimentation studies**. At this stage the enzyme contains about 3% nucleic acid (or nucleotide) and has a turnover number of about 35oo (moles of ADP***/min] i o5 g protein at 3o °) for polyadenylic acid synthesis from o.o6 M ADP. When tested at various purity levels by the radioactive phosphate "exchange" assay previously described1, the specific activity of the enzyme, up to the purest fractions, increases approximately to the same extent for each of the five nucleoside diphosphates ADP, GDP, UDP, CDP, and IDP, strongly suggesting a single enzyme. As shown in Fig. IA, there is no lag in the synthesis of poly U by the gel eluate fraction (specific activity, 45), which contains up to 4 ° % nucleic acid, but there is a definite lag phase when an equivalent amount of enzyme of specific activity 15o is used. It may be seen t h a t the lag is partially eliminated by addition of a boiled extract of the gel eluate. Fig. IB shows the priming effect of yeast RNA, poly AGUC, or Azotobacter RNA on poly U synthesis. The experiments of Fig. 2 were carried out with enzyme of specific activity 200. Fig. 2A shows "self" priming of the synthesis of poly A, poly U, and poly C, while Fig. 2B shows the priming of poly AGUC synthesis by yeast RNA, poly AGUC, or Azotobacter RNA. Further investigation has shown t h a t different polynucleotides may prime, have no effect, or even inhibit the synthesis of certain polynucleotides. Thus, the synthesis of poly C is primed only by poly C but is strongly inhibited by poly A, poly U, or RNA, natural or synthetic. On the other hand, poly C primes the synthesis of all the polynucleotides so far tested including poly * Aided by grants from the National Institute of Arthritis and Metabolic Diseases (Grant A-529) and the National Cancer Institute (Grant C-2784) of the National Institutes of Health, United States Public Health Service, the American Cancer Society (recommended by the Committee on Growth, National Research Council), the Rockefeller Foundation, and by a cofvtract (N6 onr 279, T.O. 6) between the Office of Naval Research and New York University College of Medicine. ** We are indebted to Dr. R. C. WARNER for the ultracentrifuge runs. *** Abbreviations: Synthetic polynucleotides of adenylic, uridylic, or cytidylic acid, poly A, poly U, or poly C; natural ribonucleic acid, RNA; synthetic ribonucleic acid, poly AGUC; 5'-diphosphates (pyrophosphates) of adenosine, guanosine, uridine, cytidine, or inosine, ADP, GDP, UDP, CDP, or IDP; adenosine triphosphate, ATP; oxidized and reduced diphosphopyridine nucleotide, DPN + and D P N H ; tris(hydroxymethyl)aminomethane, Tris; ethylenediamLue tetracetate, versene.
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u p t a k e a t o n e h o u r w a s t h e s a m e w h e n t h e ratio of e q u i v a l e n t s of a s c o r b a t e t o iron w a s v a r i e d f r o m 2 to io. A t a ratio of 4 o, t h e r e w a s a 5 o % inhibition. G a s s i n g t h e s o l u t i o n s w i t h N s before i n c u b a t i o n to deplete t h e m i x t u r e s of O s i n h i b i t e d t h i s s y s t e m as well as t h e a p o f e r r i t i n - f e r r o u s s y s t e m . I n c r e a s i n g t h e iron c o n c e n t r a t i o n as m u c h as 8-fold (holding a s c o r b a t e c o n s t a n t a t i. 5 e q u i v a l e n t s p e r g r a m a t o m of apoferritin N) h a d no effect o n t h e reaction. As s h o w n in t h e lower p o r t i o n of T a b l e I, t h e e x t e n t of t h e r e a c t i o n increased w i t h t e m p e r a t u r e u p t o 65 ° C. (At h i g h e r t e m p e r a t u r e s c o a g u l a t i o n of t h e p r o t e i n b e g a n to be significant.) T h i s is in accord w i t h t h e findings of SALTMAN et al. 7 on iron u p t a k e b y r a t liver slices, w h i c h also h a d a h i g h o p t i m u m t e m p e r a t u r e . I n t h a t r e s p e c t it s u p p o r t s t h e i r s u g g e s t i o n t h a t t h e u p t a k e w a s t h e r e s u l t of intracellular ironb i n d i n g b y apoferritin. T h e initial r a t e of t h e iron u p t a k e in o u r s y s t e m a t v a r i o u s t e m p e r a t u r e s f r o m o to 7 3 ° C i n d i c a t e d a n a c t i v a t i o n e n e r g y of 8 kcal/mole, c a l c u l a t e d f r o m t h e A r r h e n i u s equation. T h e u p t a k e of ferrous iron w a s inhibited b y ascorbic acid (at a c o n c e n t r a t i o n 5 t i m e s t h e e q u i v a l e n t s of iron present) to a n a m o u n t c o m p a r a b l e to t h e a s c o r b a t e - m e d i a t e d u p t a k e of /erric iron. MAZUR el al. s a n d BIELIG AND BAYERs h a v e d e m o n s t r a t e d t h e release of s o m e of t h e ferric iron f r o m ferritin in t h e presence of ascorbic acid, g l u t a t h i o n e , c y s t e i n e or a n a e r o b i c liver tissue, a n d its s u b s e q u e n t t r a n s f e r to t h e p l a s m a iron-binding protein, in w h i c h iron aLso occurs in t h e ferric s t a t e . T h e evidence p r e s e n t e d here i n d i c a t e s t h a t t h e reverse r e a c t i o n c a n occur. U n d e r aerobic c o n d i t i o n s a n d in t h e presence of ascorbic acid or boiled liver e x t r a c t , ferric iron c a n be reduced, a b s o r b e d o n t o apoferritin a n d reoxidized b y a t m o s p h e r i c o x y g e n t o a f o r m w i t h optical a n d a n t i g e n i c p r o p e r t i e s of ferritin. M A R Y W . LOEWUS Department o/Physiological Chemistry, RICHARD A. FINEBERG
University o/Cali/ornia School o/Medicine, Berkeley, Calf/. ( U.S.A .) 1 S. G R A N I C K A N D L. MICHAELIS, J. Biol. Chem., 147 (I943) 91. t A. MAZUR, S. BAEZ AND E. SHORR, J. Biol. Chem., 2I 3 (1955) 147. a S. GRANICK AND P. F. HAHN, J. Biol. Chem., 155 (1944) 661. ~J. BIELIG AND E. BAYER, Naturwissenschatten, 42 (1955) I25. 6 M. V. LAUFSERGER, Bull. soc. chim. biol., 19 (1937) I575. • H . BORSOOK, A. ABRAMS AND P. H. LowY, J. Biol. Chem., 215 (I955) IZI. T p. SALTMAN, R. D. FISKIN AND S. B. BELLINGER, J. Biol. Chem., 220 (1956) 741. s j. BIELIG AND E. BAYER, Naturwissenscha/ten, 42 (1955) 466. R e c e i v e d A u g u s t 4th, 1957
Preliminary Notes
Mechanism of carbon dioxide fixation by SaccharomycescerevisMe B a k e r ' s y e a s t (Saccharomyces cerevisiae) a s s i m i l a t e s c a r b o n dioxidet, S, s, 4, p r e s u m a b l y w i t h f o r m a t i o n of a Ct-dicarboxylic acid (KLEINZELLER a, STOPPANI $ AND DAVm eta/.) t. Several m e c h a n i s m s m a y be p r o p o s e d to e x p l a i n t h e i n c o r p o r a t i o n of CO s, e.g., (i), s y n t h e s i s of o x a l o a c e t a t e b y c o n d e n s a t i o n of CO s a n d p h o s p h o e n o l p y r u v a t e (UTTER eta/. s, BANDURSKI AND I~PMANHT); (2), r e d u c t i v e c a r b o x y l a t i o n of p y r u v a t e w i t h f o r m a t i o n of m a l a t e (OCHOA eta/, s) ; (3), c a r b o x y l a t i o n o f p r o p i o n a t e w i t h f o r m a t i o n of s u c c i n a t e (LARDY AND ADLRRS). If t h e y e a s t w e r e allowed t o oxidize s u b s t r a t e s (acetaldehyde, acetate, p y r u v a t e or glucose ) in t h e p r e s e n c e o f 1~COs, t h e kinetics of t h e d i s t r i b u t i o n of t h e r a d i o a c t i v i t y fixed in t h e m e t a b o l i c pools c o u l d e l u c i d a t e t h e initial s t e p of c a r b o n dioxide assimilation, a n d f u r t h e r m o r e , t h e n a t u r e of t h e ' m a i n p a t h o f s u b s t r a t e o x i d a t i o n , w h i c h in S. cerevisiae is still a m a t t e r of c o n t r o v e r s y 1°. T h e p r o c e d u r e w a s s i m i l a r t o t h a t applied b y CALVIN et a/. n to s t u d y t h e p a t h of c a r b o n i n p h o t o s y n t h e s i s . S u s p e n s i o n s of s t a r v e d y e a s t were i n c u b a t e d w i t h r a d i o a c t i v e b i c a r b o n a t e a n d s u b s t r a t e s in a closed flask c o n n e c t e d to a v o l u m e c o m p e n s a t o r (c/. STOPPANI et a/.lS), t h e O t u p t a k e of t h e y e a s t b e i n g m e a s u r e d i n d e p e n d e n t l y u n d e r s i m i l a r e x p e r i m e n t a l c o n d i t i o n s . Controls w i t h o u t s u b s t r a t e were r u n s i m u l t a n e o u s l y . W i t h acetic a n d p y r u v i c acids, t h e initial p H of t h e m e d i u m w a s 2.5, in o r d e r t o increase t h e y e a s t p e r m e a b i l i t y . T h e s t r o n g a c i d i t y did n o t affect t h e y e a s t m e t a b o l i c p a t t e r n s , as w i t h glucose or a c e t a l d e h y d e as s u b s t r a t e s , e x p e r i m e n t s c a r r i e d o u t a t p H 2.5 a n d 7-4 g a v e n e a r l y t h e s a m e results. A f t e r i n c u b a t i o n (from x 5 sec to 3 k
444
PRELIMINARY NOTES
VOL.
26 (1957)
according to t h e e x p e r i m e n t ) , a l i q u o t s of t h e y e a s t s u s p e n s i o n s were a d d e d to a n d t h o r o u g h l y m i x e d w i t h 9 vol. m e t h a n o l . T h e r a d i o a c t i v i t y of t h e m e t h a n o l s u s p e n s i o n s a n d t h a t of t h e clear s u p e r n a t a n t fluids o b t a i n e d b y c e n t r i f u g a t i o n were m e a s u r e d . T h e r a d i o a c t i v e c o m p o u n d s dissolved in t h e m e t h a n o l - w a t e r m i x t u r e were c o n c e n t r a t e d to a s m a l l v o l u m e , s e p a r a t e d b y p a p e r c h r o m a t o g r a p h y (BENSON et al.13), a u t o r a d i o g r a p h e d a n d t h e a c t i v i t y of e a c h c o m p o u n d c o u n t e d on t h e c h r o m a t o g r a m . T h e a s s i m i l a t i o n of CO S w a s g r e a t e r in t h e p r e s e n t of s u b s t r a t e . T h u s , w i t h o.o2 M acetate, t h e Q o , (/zl O 2 u p t a k e / m g d r y cells/h) w a s I3.8 a n d t h e r a d i o a c t i v i t y fixed 87o c t s / m i n / m g d r y cells/h, w h e r e a s in t h e a b s e n c e of acetate, t h e respective values were 1.1 a n d lO 3. Similar r e s u l t s were o b t a i n e d w i t h glucose, p y r u v a t e a n d a c e t a l d e h y d e , w h i c h confirms a n d e x t e n d s t h e r e s u l t s o b t a i n e d b y o t h e r workersl, 2, 3, 4. A t t h e b e g i n n i n g of t h e fixation, all t h e r a d i o a c t i v i t y a p p e a r s in m e t h a n o l - w a t e r soluble c o m p o u n d s . T h e s e were aspartic, g l u t a m i c , malic a n d citric (plus aconitic a n d isocitric) acids. A f t e r 2 - 3 h, t h e d i s t r i b u t i o n (%) of 14C r e a c h e d a s t e a d y state. If t h e r a d i o a c t i v i t y (%) a p p e a r i n g in each c o m p o u n d a f t e r t h e b e g i n n i n g of fixation is p l o t t e d as a f u n c t i o n of time, t h e d i s t r i b u t i o n c u r v e s allow a decision b e t w e e n t h e several possible p a t h s of c a r b o n i n c o r p o r a t i o n 14, e v e n if oxaloacetic (on a c c o u n t of instability) is n o t p r e s e n t in t h e c h r o m a t o g r a m s . T h u s , if t h e first c o m p o u n d in w h i c h t h e C O , is fixed is oxaloacetic acid, t h e c u r v e s s h o w i n g t h e d i s t r i b u t i o n of r a d i o a c t i v i t y in its i m m e d i a t e d e r i v a t i v e s (aspartic, malic a n d citric acids) will initially h a v e a positive slope. However, if t h e t r a n s a m i n a t i o n of oxaloacetic acid is a m u c h f a s t e r r e a c t i o n t h a n t h o s e l e a d i n g to malic a n d citric acids, it is possible t h a t t h e positive slope of t h e a s p a r t i c acid w o u l d be finished before m e a s u r e m e n t s are begun, a n d t h e plot of a s p a r t i c acid would t h e n a p p e a r w i t h a n e g a t i v e slope. O n t h e o t h e r h a n d , if t h e c a r b o n is i n c o r p o r a t e d t h r o u g h t h e " m a l i c r e a c t i o n " , t h e c u r v e for malic acid w o u l d h a v e to be t h e first one s h o w i n g a n e g a t i v e slope. Fig. I s h o w s t h a t a s p a r t i c acid is t h e o n l y c o m p o u n d to h a v e a d i s t r i b u t i o n c u r v e w i t h a n e g a t i v e slope, w h i c h leads to t h e conclusion t h a t oxaloacetic is t h e first p r o d u c t of fixation. T h e e s s e n t i a l role of oxaloacetic is s u p p o r t e d b y t h e presence of "oxaloacetic" c a r b o x y l a s e in cell-free e x t r a c t s of b a k e r ' s yeast, w h e r e a s in t h e s a m e e x t r a c t s , no " m a l i c e n z y m e " c a n be d e t e c t e d ( o b s e r v a t i o n s w i t h J. CANNATA, to be published). Therefore t h e " m a l i c r e a c t i o n " as well as o t h e r less likely m e c h a n i s m s c a n be ruled o u t as a n e x p l a n a t i o n of CO 2 fixation b y yeast.
u.
10C 9G
~8C
¢o ~70 .,~.k
~C. 6° Fig. I. R e l a t i v e d i s t r i b u t i o n of t4C a f t e r f i x a t i o n of 14CO~ b y S. cerevisiae in t h e p r e s e n c e of glucose. 285 m g y e a s t ; 5 ° /*moles glucose; 4.2 /*moles NaH14CO3 (2.8-IO s c t s / m i n ) ; p h o s p h a t e buffer p H 7.I, o.~ m M . T o t a l v o l u m e ; Io ml. Air in t h e g a s p h a s e . T e m p . : 23 °. A, a s p a r t i c ; G, g l u t a m i c ; M, malic a n d C, citric (plus cis-aconitic a n d isocitric) acids.
ua~ 50
~ (.j 40 3G Q. ?0 t0 15
30
45
TIME (M~A,.)
60
T h e d e g r a d a t i o n of r a d i o a c t i v e a s p a r t i c a n d g l u t a m i c acids o b t a i n e d f r o m y e a s t w h i c h w a s allowed to oxidize p y r u v i c acid in t h e presence of 14CO 2, s h o w s t h a t a t first all t h e 14C fixed is c o n c e n t r a t e d in t h e 4 - c a r b o n of a s p a r t i c a n d in t h e 1-carbon of g l u t a m i c acids respectively, b u t later t h e r a d i o a c t i v i t y a p p e a r s also in t h e 1-carbon of a s p a r t i c acid. T h i s m a y be e x p l a i n e d b y equilibration of oxaloacetic w i t h malic, a n d t h r o u g h t h e l a t t e r w i t h t h e a p p a r e n t l y s y m m e t r i c a l l y labelled f u m a r i c acid. T h e r e s u l t s s u m m a r i z e d a b o v e are c o n s i s t e n t w i t h t h e o p e r a t i o n of t h e t r i c a r b o x y l i c acid cycle as t h e m a i n o x i d a t i v e p a t h w a y in b a k e r ' s yeast. A l t h o u g h y e a s t cells c o n t a i n isocitritase 15, t h e r e c e n t l y described is g l y o x a l a t e cycle does n o t s e e m to be of m u c h i m p o r t a n c e , as is s h o w n b y t h e s t r o n g labelling of g l u t a m i c acid. A full a c c o u n t of t h i s w o r k will be given in a n o t h e r journal.
Department of Biochemistry, Faculty o/ Medicine, University of Buenos Aires and Laboratory for Cell Metabolism ( C . N . E . A .), Buenos Aires (Argentina)
A. O. M. STOPPANI SUSANA L. S. DE FAVELUKES L u c f A CONCHES F . L . SACERDOTE
1 j . RUNNSTROM, K. BRANDT AND R. MARCUSE, Arkiv Kemi Mineral. Geol., I7A (I943) no. 3z S. RUBEN AND M. KAMEN', Proc. Natl. Acad. Sci. U.S., 26 (I94o) 418. * A. KLEINZELLER, Biochem. J., 35 (1941) 495-
VOL. 26 (Z957)
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* j . W. DAvIs, V. H. CHELDELIN, B. E. CHRISTENSEN AND C. H. WANG, Biochim. Biophys. ,4 cta, 21 (1956) 1oi. s A. O. M. STOPPANI, Nature, 167 (1951) 653. s M. F. UTTER AND K. KURAHASHI,J. Biol. Chem. 207 (1954) 821. 7 R. S. BANDURSKI AND F. LIPMANN, J. Biol. Chem., 219 (1956) 741. s S. OCHOA, A. H. MEHLER AND k. KOENEERG,J. Biol. Chem., 174 (1948) 979. 9 H. A. LARDY AND J. ADLER, J . Biol. Chem., 219 (1956) 9331 0 H. A. KREBS, S. GURIN AND L. V. EGGLESTON, Biochem. J., 51 (I952) 614. 11 M. CALVIN AND P. MASSINI, Experientia, 8 (1952) 445. 12 A. O. M. STOPPANI, R. C. FULLER AND M. CALVIN,J. Bacteriol., 69 (1955) 491. xs A. A. BENSON, J. A. BASSHAM, M. CALVIN,T. C. GOODALE,V. A. HAAS AND W. STEPKA,J. Am. Chem. Soc., 72 (195 o) 171o. 14 W. J. WHITEHOUSE AND J. L. PUTMAN, Radioactive Isotopes, Oxford Univ. Press, London, 1953, p. 292. 15 j . A. OLSON, Nature, 174 (1954) 695. le H. L. KORNBERG AND H. A. KREBS, Nature, 179 (1957) 988. Received July 6th, I957
Polyribonucleotide synthesis with highly purified polynucleotide phosphorylase* Earlier preparations of polynucleotide phosphorylase of Azotobaaer vinelandii z, z, s did not permit one to determine whether, in analogy with muscle phosphorylase, a primer was required for polyribonucleotide synthesis. Highly purified preparations of this enzyme have now been obtained with which a requirement for priming has been established. These have been obtained by use of steps previously reported4, followed by other steps involving protamine, ammonium sulfate, and gels. The specific activity of the enzyme, expressed as in a previous paper 1, increases from about I.O in the initial extract to 25o-3oo in the final preparation for which a purity of 7o-8o% is suggested by sedimentation studies**. At this stage the enzyme contains about 3% nucleic acid (or nucleotide) and has a turnover number of about 35oo (moles of ADP***/min] i o5 g protein at 3o °) for polyadenylic acid synthesis from o.o6 M ADP. When tested at various purity levels by the radioactive phosphate "exchange" assay previously described1, the specific activity of the enzyme, up to the purest fractions, increases approximately to the same extent for each of the five nucleoside diphosphates ADP, GDP, UDP, CDP, and IDP, strongly suggesting a single enzyme. As shown in Fig. IA, there is no lag in the synthesis of poly U by the gel eluate fraction (specific activity, 45), which contains up to 4 ° % nucleic acid, but there is a definite lag phase when an equivalent amount of enzyme of specific activity 15o is used. It may be seen t h a t the lag is partially eliminated by addition of a boiled extract of the gel eluate. Fig. IB shows the priming effect of yeast RNA, poly AGUC, or Azotobacter RNA on poly U synthesis. The experiments of Fig. 2 were carried out with enzyme of specific activity 200. Fig. 2A shows "self" priming of the synthesis of poly A, poly U, and poly C, while Fig. 2B shows the priming of poly AGUC synthesis by yeast RNA, poly AGUC, or Azotobacter RNA. Further investigation has shown t h a t different polynucleotides may prime, have no effect, or even inhibit the synthesis of certain polynucleotides. Thus, the synthesis of poly C is primed only by poly C but is strongly inhibited by poly A, poly U, or RNA, natural or synthetic. On the other hand, poly C primes the synthesis of all the polynucleotides so far tested including poly * Aided by grants from the National Institute of Arthritis and Metabolic Diseases (Grant A-529) and the National Cancer Institute (Grant C-2784) of the National Institutes of Health, United States Public Health Service, the American Cancer Society (recommended by the Committee on Growth, National Research Council), the Rockefeller Foundation, and by a cofvtract (N6 onr 279, T.O. 6) between the Office of Naval Research and New York University College of Medicine. ** We are indebted to Dr. R. C. WARNER for the ultracentrifuge runs. *** Abbreviations: Synthetic polynucleotides of adenylic, uridylic, or cytidylic acid, poly A, poly U, or poly C; natural ribonucleic acid, RNA; synthetic ribonucleic acid, poly AGUC; 5'-diphosphates (pyrophosphates) of adenosine, guanosine, uridine, cytidine, or inosine, ADP, GDP, UDP, CDP, or IDP; adenosine triphosphate, ATP; oxidized and reduced diphosphopyridine nucleotide, DPN + and D P N H ; tris(hydroxymethyl)aminomethane, Tris; ethylenediamLue tetracetate, versene.