Volume 17, number 3

MOLECULAR & CELLULARBIOCHEMISTRY

October 7, 1977

ISOCITRATE DEHYDROGENASE OF TETRAHYMENA PYRIFORMIS Pilar VIDAL and Alberto M A C H A D O

Centro de Biologia Molecular, C.S.I.C. Universidad Aut6noma de Madrid. Facultad de Ciencias, Madrid 34, Spain. (Received March 28, 1977)

Summary We have studied the isocitrate dehydrogenase of Tetrahymena pyriformis. This enzyme is able to utilize both NAD and NADP, but kinetic studies suggest that the enzymatic activity with NAD is not of physiological significance. Some of the factors that might regulate the NADP-dependent isocitrate dehydrogenase were also studied. This enzyme has an absolute requirement for divalent cations; Mg 2÷ and Mn 2÷ will serve as cofactors but the latter is more effective than the former. It is known that this enzyme is subject to a concerted inhibition by oxaloacetate and glyoxylate. Either glyoxylate or oxaloacetate alone also are capable of inhibiting the enzyme although higher concentrations are required. We have found concerted inhibition also for the NAD-dependent isocitrate dehydrogenase from rat liver and yeast. The activity of the Tetrahymena pyriformis enzyme is inhibited by NADPH. This inhibition is competitive with NADP. The Ki and Km values are, respectively, 2 3 / ~ i and 18 p,i.

Introduction All eucaryotic organisms so far examined possess two enzymes which catalyse the oxidation of isocitrate to 2-oxoglutarate; the NAD-linked isocitrate dehydrogenase (EC 1.1.1.4.1) and the NADP-linked isocitrate dehydrogenase (EC 1.1.1.4.2). Procaryotic organisms have only one

isocitrate dehydrogenase. Most of them have an NADP specific enzyme, but some have an NAD specific enzyme 1-4. The ciliate protozoon Tetrahymena pyriformis is an eucaryote with certain procaryotic characteristics, such as a "bacterial" type cytochrome C-5535'6. MULLER et al.7 have investigated the presence and localization of the enzymes of the tricarboxylic acid cycle and glyoxalate cycle enzymes in Tetrahymena. They did not find NAD-linked isocitrate dehydrogenase. ALLEN8 however claimed to have detected the existence of isozymes of the NAD-linked enzyme by polyacrilamide gel electrophoresis. We can detect only a single NAD-linked isocitrate dehydrogenase in Tetrahymena. Its properties indicate that it is identical with the NADP-linked enzyme previously described 7. The difference between the Km values for NAD and NADP suggest that the activity with NAD is not of physiological significance. The regulation of the NADP-linked isocitrate dehydrogenase in Tetrahymena is important for the regulation of the glyoxalate cycle. Our data confirm the results of LEVy9;NADP-dependent isocitrate dehydrogenase from Tetrahymena is subject to concerted feedback inhibition by glyoxalate and oxaloacetate. We do not believe that either the inhibition of isocitrate dehydrogenase by oxalomalate (the product of the condensation of glyoxalate and oxaloacetate, RtJFFO et al~.°) or the NAD-dependent activity of the enzyme are of physiological significance. We suggest that the inhibition of isocitrate dehydrogenase by N A D P H results in the diversion of material into the glyoxalate bypass in

Dr. W. Junk b.v. Publishers- The Hague, The Netherlands

151

Tetrahymena. Such a control system would resemble the control of NADPH production by glucose 6-phosphate dehydrogenase in organisms which possess this enzyme.

Glucose-6-phosphate dehydrogenase activity was measured by the method of KuBY et al. 15. Protein was assayed on trichloroacetic acid precipitates by the Lowry procedures as described

by LAYNE16. Materials and Methods

Chemicals NAD, NADP and DL-isocitrate were Obtained from Sigma, glucose-6-phosphate dehydrogenase from Boehringer and Sephadex G-75 and G-200 from Pharmacia Fine Chemicals. Other reagents were of the highest purity available from commercial sources. All preparations were made in glass distilled water.

Cell growth and harvesting conditions Cells of Tetrahymena pyriformis strain W (culture obtained from Dr. Mot~oz, Department of Biochemistry, Faculty of Science, Complutense University, Madrid) were grown at 28°C, on autoclaved medium containing proteose peptone 0.5% w/v, bacto tryptone 0.5% w/v, K2HPO4. 3H20 0.026% w/v, sodium acetate 0.1% w/v and glucose 0.25% w/v. The cultures were aerated by shaking in 600 ml portions in 11. Erlenmeyer flasks. The cells were harvested at the beginning of the stationary phase (5 days of growth) by centrifugation at 15380 × g, 4°C, and washed in cold Ringer-phosphate solution ~. Crude extracts were obtained by cell disruption in a MSK (B. Braun) cell homogenizer at 2000 r.p.m, for 4 minutes and 4000 r.p.m, for 1 minute and centrifuged at 3000 × g for 10 minutes. The supernatants were used for enzyme assays. Yeast cells (Saccharomyces cerevisiae) were grown aerobically at 30 °C on Lindegren medium ~2 supplemented with 1% glucose. The cells were harvested in the stationary phase (24 hours of growth). Crude extracts were obtained as described previously 13. Rat liver mitochondria were isolated by the method of CARVALHO14.

Assay methods The activity of isocitrate dehydrogenase was measured by the method of MACHADO et al. ~3, except that AMP and ADP were omitted for the assay of the NAD-dependent activity of Tetrahymena extracts. ADP was added to the incubation mixtures with rat liver extract. 152

Results

Heat inactivation isocitrate dehydrogenase activities toward N A D and N A D P Table 1 shows the effect of heating the crude extract to different temperatures for periods of 5 minutes. In this experiment, the isocitrate dehydrogenase activities towards NAD and NADP were lost concomitantly. The same resuits were obtained after heating extract to 50 °C for varying periods of time, which suggests that both reactions are catalyzed by the same protein.

Enzyme purification Table 2 summarizes the results of enzyme purification which was carried out at 4 °C unless otherwise indicated. Step 1. Enzyme from Tetrahymena pyriformis. A washed cell pellet weighing about 60 g was suspended in 120 ml of 0.1 M phosphate buffer pH 7 and broken by a MSK (B. Braun) cell homogenizer. The suspension was centrifuged at 3000 x g for 10 minutes. Step 2. Salt fractionations. The supernatant solution from the previous step was precipitated by the slow addition of solid (NH4)2804, with mechanical stirring over a period of 1 hour, to obtain a 50% saturated solution. The precipitate was removed by centrifugation for 15 minutes at 15000 × g and discarded. The protein remaining in the supernatant fluid was precipitated by the slow addition of solid T a b l e i: Effect of 5 minutes of exposure to dilIcrent temperatures o n activity o f

Tetrahy~na isociuate dehydrogenase.

Enzyme activity is expressed as percent o f the controls. Results u e mean values ± S.E. o f 4 determinations.

TEMPERATURE

NAD activity NADP activity

3O *C

4O oC

45 °C

5O °C

55 °C

6O °C

100 ± 0 100 ± 0

94 ± 5 9l * 8

81 ± 4 87 * 5

79 ± 8 79 ± 3

01 1±

0 0

T a b l e 2. Purification of isocit~ate d e h y d r o g e n a s ¢ of

Spe¢. a c t Fraction Homegenate (NH,), $O, pr~ipitate Sephadex G- 75 eluate Scphadex G- 2O0 eluate

NAD n motes x rain t x rag-' 9.9 39.1 77 398

Tetrahyme~.

Spee. act. NADP n moles x rain I x mg -I 464 1438 3100 22400

Purifi~tion (-fold) [DH-NAD I 3.9 7.7 43.7

IDH-NADP 1 3.2 6.7 48.2

Ratio NADP Activity: NAD Activity 46 40 40 56

(NH4)2SO4 with mechanical stirring over 90 minutes to obtain a 70% saturated solution. The precipitate was removed by centrifugation for 15 minutes at 15000 x g and was redissolved in 12 ml 0.1 M phosphate buffer pH 7. This solution was dialyzed overnight against 0.005 M phosphate buffer, containing 10 -4 M DTE (dithioerythritol) and 10 -4 MDLisocitrate. Step 3. Gel filtration on Sephadex. Two ml of the dialyzed solution were placed on the top of a column (1.5 cm x 76 cm) of Sephadex G-75 previously equilibrated with 0.1 M buffer phosphate pH 7. The column was developed with the same buffer at a flow rate of 0.2 ml per minute; 5 ml samples were collected and assayed for activity. The fractions containing the enzyme were passed through a Sephadex G200 column ( 3 c m x 7 1 cm). The flow rate was adjusted to 12-18 ml/h. The active fractions were pooled and stored at 4 °C. The NAD- and NADP-dependent isocitrate dehydrogenase activities were eluted in the same fractions and, as summarized in Table 2, at all steps during purification of the enzyme, the ratio of NADP dependent activity to NAD dependent activity was constant. These results support the original idea: that both reactions were catalyzed by the same protein because they have the same properties after heating and after chromatography on Sephadex G-75 and G-200. Furthermore, the ratio of activities during the processes of purification is constant.

Effect of concentrations of isocitrate, N A D and DADP The effect of substrate, NAD and NADP concentrations on the initial velocity of the enzyme-catalyzed reaction with the partial purified enzyme was investigated. Michaelis constants were obtained by the method of LINEWEAWR and BURK. Km values of NADP isocitrate dehydrogenase for DL-isocritrate and NADP were 22/~M and 1 8 / x i respectively. However, with NAD as the electron acceptor, the Km values for DL-isocitrate and NAD were 2 4 / ~ i and 22 mM respectively. The values for both isocitrate and NADP are in good agreement with those of the NADP-linked isocitrate dehydrogenase from Azotobacter vinelandii strain 0, which are 36 ~M for DL-isocitrate and 1 8 / x i for NADP ~7. The high Km for NAD

E

:

!

2

a_ v >

~/[M,,2÷] (~o -~ ) .

Fig. 1. Lineweaver-Burk plot for the determination of Km value for Me 2+. O, Mn2+; Mg 2÷.

was not due to traces of contaminating NADP since the NAD preparation showed no activity when incubated with glucose-6-phosphate and glucose-6-phosphate dehydrogenase. Isocitrate dehydrogenase from Tetrahymena has therefore some activity with NAD which is relatively greater than that found for the enzyme from Es-

cherichia coli ~8. Metal co-factor requirements The isocitrate dehydrogenase required Mg 2÷ or Mn 2÷ for maximal activity. Only 10% of maximum activity was found in the absence of metal ion. This low degree of activity could have been due to trace amounts of cations present in the assay reagents. The enzyme showed no activity upon the addition of 0.33 m i EDTA. Maximal activity of enzyme was found with 4× 10 -5 M Mn 2+ which is twice the maximal activity with Mg 2÷ at 8 x 10 -5 M. The apparent Km for Mg 2+ is 83/zM and for Mn 2÷ 14/ZM (Fig. 1). The requirement for the divalent ions and the maximum effect of Mn 2÷ are in agreement with nearly all isocitrate dehydrogenase activity studies 17-24 but is in contrast to the report by CHUNG and FRAZEN25 that the enzyme from A z otobacter vinelandii (ATCC 9104) is better stimulated by Mg 2÷. This supports the belief that the active substrate for the dehydrogenase reaction is an isocitrate-Mn 2÷ (Mg 2÷) complex26,27"

Effect of pH on enzyme activity The enzyme activity was determined as a function of pH with 30 mM phosphate buffer for 153

p H 6 to 8 a n d 30 mM T r i s - H C l for 8.5 to 9. T h e e n z y m e activity i n c r e a s e d slightly with p H . F o r t h e r o u t i n e assays we u s e d p H 7 as we c o n s i d e r e d it to b e m o r e physiological.

Effect of nucleotides on enzyme activity T h e n u c l e o t i d e s t e s t e d at different c o n c e n t r a tions c a u s e d , in g e n e r a l , v e r y little effect o n t h e i s o c i t r a t e d e h y d r o g e n a s e activity. M a x i m u m inh i b i t i o n is p r o d u c e d b y A T P ; at 2 mM c o n c e n t r a t i o n s t h e n u c l e o t i d e causes 5 0 % i n h i b i t i o n . This i n h i b i t i o n was n o t r e v e r s e d by t h e inc r e a s e of M g 2÷ c o n c e n t r a t i o n . S i m i l a r effects (40% i n h i b i t i o n ) w e r e p r o d u c e d b y A D P , G T P a n d G M P at 5 m s . I n all cases, t h e e x t e n t of t h e i n h i b i t i o n was p r o p o r t i o n a l to the c o n c e n t r a t i o n of t h e n u c l e o t i d e a d d e d . A M P a n d G D P at 5 mM c a u s e d t h e least i n h i b i t i o n ( 2 0 % ) .

Effect of compounds related to the tricarboxylic acid and glyoxylate cycles M o s t of t h e i n t e r m e d i a t e s of the t r i c a r b o x y l i c acid cycle a n d r e l a t e d c o m p o u n d s t e s t e d h a d no effect o n t h e N A D P - l i n k e d e n z y m e r e a c tion. T h e effects of t h e s e c o m p o u n d s in the p r e s e n c e o r a b s e n c e of g l y o x y l a t e o r o x a l a c e t a t e a r e s h o w n in T a b l e 3. T h e o n l y c o m p o u n d s which c a u s e d i n h i b i t i o n of the e n z y m e w e r e o x a l o a c e t a t e a n d g l y o x y late, s e p a r a t e l y a n d c o m b i n e d . This is in c o m p l e t e a g r e e m e n t with t h e results o b t a i n e d b y LEVY 9 a n d o t h e r s 4'28'32. H o w e v e r the lack of i n h i b i t i o n b y 2 - 0 x o g l u t a r a t e distinguishes t h e Tetrahymena e n z y m e f r o m t h a t of g e r m i n a t i n g c a s t o r b e a n s , which is c o m p e t i t i v e l y i n h i b i t e d b y this c o m p o u n d with a Ki of 0.3 mM a c c o r d ing to NAGAMACHI et al. 33. SATOH 34 also f o u n d this i n h i b i t i o n in m a t u r i n g c a s t o r b e a n s e e d s enzyme.

Inhibition by oxaloacetate and glyoxylate of NAD-linked isocitrate dehydrogenase RUFFO et al. 35 r e p o r t e d the i n h i b i t i o n of rat liver m i t o c h o n d r i a l r e s p i r a t i o n b y a - h y d r o x y - / 3 o x a l o s u c c i n a t e , a c o m p o u n d f o r m e d by n o n e n z y m a t i c c o n d e n s a t i o n of g l y o x y l a t e a n d o x a l o a c e t a t e 1°. Similarly, HAMFTON et al. 4 r e p o r t e d the s a m e i n h i b i t i o n of t h e N A D dependent isocitrate dehydrogenase from ThiobaciUus thiooxidans. W e s t u d i e d the inhibit i o n b y t h e s e c o m p o u n d s of t h e N A D - l i n k e d isocitrate dehydrogenase from yeast and rat 154

Table 3. Effect of organic acids and aminoaeids related to the tricarboxylic acid and glyoxylate cycles on enzyme activity (IDH-NADP) of Tetrahymena in the presence or absence of glyoxylate or oxaloacetate ] Enzyme activity is expressed as percent of that without additions of other compounds. Ketoacids and aminoacids were added to the standard reaction mixture. Compounds added

M x 10-3

Activity %

Pyruvate 3.3 86 Oxaloacetate 3.3 0 Citrate 3.3 96 2-Oxoglutarate 3.3 93 Suecinate 3.3 90 Malate 3.3 95 Glyoxylate 3.3 0 Aspartate 3.3 93 Glutamate 3.3 93 Oxaloacetate + Glyoxylate 0.06 + 0.06 0 Oxaloacetate + Glyoxylate 0.06 + 0.06 0* Pyruvate + glyoxylate 0.06 + 0.06 101" 2-Oxoglutarate + Oxaloacetate 0.06 + 0.06 93* 2-Oxoglutarate + Pyruvate 0.06 + 0.06 100" 2-Oxoglutarate + Glyoxylate 0.06 + 0.06 98* Oxaloaeetate + Pyruvate 0.06 + 0.06 106" Glyoxylate + Malate 0.06. + 0.06 98* Glyoxylate + Suecinate 0.06 + 0.06 98" Oxaloacetate + Glyoxylate 0.06 + 0.06 20** Oxaloacetate + Glyoxylate 0.03 + 0.03 46** *This combination of ketoacids was mixed and kept at room temperature for 20 min before addition to the reaction mixture. **This combination of ketoaeids was mixed and kept at room temperature over-night before addition to the reaction mixture. liver m i t o c h o n d r i a . T h e results a r e s u m m a r i z e d in T a b l e 4. A s can b e seen, o x a l o a c e t a t e p r o d u c e d c o m p l e t e i n h i b i t i o n of b o t h e n z y m e s , b u t the s a m e c o n c e n t r a t i o n of g l y o x y l a t e p r o d u c e s this effect o n l y on the e n z y m e f r o m r a t liver Table 4. Effect of glyoxylate and oxaloacetate on the isocitrate NADlinked isocitrate dehydrogenase of rat liver and yeast. Enzyme activity is expressed as percent of the activity without addition of other compounds. Each value is the mean of 2 determination. Compounds added

M x 10-3

Activity % rat liver Yeast mitochondria

Oxaloacetate Glyoxylate Oxaloacetate + glyoxylate

3.3

3.3 0.06 + 0.06

0

0

0 35

85 0

Oxaloacetate and glyoxylate were mixed and used immediately or kept at room temperature for 20 min before addition to the reaction mixture. Both manipulations yielded same results.

i 2

Fig. 2. Inhibition of isocitrate dehydrogenase activity by N A D P H . N A D P H was added to a final concentration of: A, None; B, 4.1 x 10 -2 mM; C, 8.2 x 10 -2 raM.

mitochondria. Concerted inhibition is seen with both enzymes but with the enzyme from yeast it decreases with reaction time. This effect is similar to that found by LEVY 9 and by us with the NADP-linked isocitrate dehydrogenase of Tetrahymena with oxaloacetate and glyoxylate.

Inhibition by N A D P H NADPH, a product of the reaction, is a strong inhibitor of isocitrate dehydrogenase from Tetrahymena. When the NADPH/NADP ratio was above 2, inhibition was almost complete. The inhibition is competitive with respect to NADP (Fig. 2) and the Ki for NADPH is 23/zM. KREBS and EGGLESTON 36 reported that for glucose-6-phosphate dehydrogenase from rat liver the Ki for NADPH was 7/zM and the Km f~r NADP 3/zM. SAPA~ HAGAR et at. 37 with different conditions of assays and at different physiological states of the rat liver, 6phosphoglyconate dehydrogenase, gave Ki values of 7 and 3/zM for NADPH and Km values for NADP of 5 and 9/zM for starved and lipogenic diet respectively.

Discussion The results of temperatura resistance (Table 1) and enzyme purification (Table 2) suggest that both NAD- and NADP-dependent isocitrate dehydrogenase activities are brought about by the same protein. One must take into account that Tetrahymena is a ciliate protozoon with some procaryotic characteristics 5'6 and the absence of a specific NAD-linked isocitrate de-

A

hydrogenase would be another procaryotic characteristic. The presence of a NADP-linked isocitrate dehydrogenase that can use NAD might have evolutionary implications. The same enzyme from Trypanosoma cruzi 38 did not reduce NAD, nor did NAD interfere with the reduction of NADP in equimolar amounts. Besides, the high Km for NAD and the big difference between Vmax for NAD (50 times less) suggest that the enzymatic activity of isocitrate dehydrogenase with NAD does not play a physiological role in the oxidation of isocitrate. Concerted inhibition by glyoxylate and oxaloacetate as found in Tetrahymena by LEVY 9 has been detected for all NADP-dependent isocitrate dehydrogenase4'17"28-34'39'4° and for some NAD-dependent enzymes, such as T. thiooxidans 4. Yet, we have shown concerted inhibition for the NAD-dependent enzymes of rat liver and yeast. It is noteworthy that yeast possesses a glyoxylate cycle that is not found in rat liver. There is some doubt about the physiological role of this inhibition9 because of the report by RUFFO et al. 35 indicating that rat liver mitochondrial respiration is also inhibited. Moreover, it is not clear that, with this concerted inhibition, the isocitrate dehydrogenase could ever function, particularly since in Tetrahymena some of the oxaloacetate produced is not made in the peroxysomes7. It is also unclear how the inhibition of the NADPdependent isocitrate dehydrogenase is reversed when glyoxylate and oxaloacetate are present as a result of action of the glyoxylate cycle. We believe that in the case of Tetrahymena the isocitrate dehydrogenase activity is one of the most important devices for obtaining NADPH. We were unable to elicit any glucose-6-phosphate dehydrogenase activity in this organism. There is also a lack of the malic enzyme, another important source of NADPH 41. On the other hand we have found that the Ki for NADPH of isocitrate dehydrogenase of Tetrahymena is 23/.~M and its Km for NADP is 18/xM. This circumstance strongly suggest that the regulation of this enzyme in Tetrahymena may be similar to that of glucose6-phosphate dehydrogenase36 or of 6phosphogluconate dehydrogenase37, of the hexosemonophosphate shunt. Since in Tetrahymena the calculated ratio of free NADPH to free NADP is of the order of 1042, it is possible that this ratio is of paramount importance in 155

the r e g u l a t i o n of isocitrate d e h y d r o g e n a s e a n d in the o p e r a t i o n of the glyoxalate cycle. This is in good a g r e e m e n t with the increase of g l u c o n e o g e n e s i s u n d e r c o n d i t i o n s of low oxygen t e n s i o n 6. U n d e r these c o n d i t i o n s there are increases in the N A D H / N A D 13 a n d N A D P H / N A D P ratios.

Acknowledgements T h e a u t h o r s w o u l d like to t h a n k Dr. J. M. JONES MORTIMER of the U n i v e r s i t y of C a m bridge for his suggestions a n d v a l u a b l e criticism a n d Prof. F. MAYOR ZARAGOZA of A u t o n o m o u s U n i v e r s i t y of M a d r i d for revising the m a n u s cript.

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18. Reeves, H. C., Daumy, G. O., Lin, C. C. and Houston, M., 1972. Biochim. Biophys. Acta 258, 27-55. 19. Marr, J. J. and Weber, M. M., 1968. J. Biol. Chem. 243, 4973-4979. 20. Charles, A. M., 1970. Can. J. Biochem. 48, 95-103. 21. Hubbard, J. S. and Miller, A. B., 1969. J. Bacteriol. 99, 161-168. 22. Howard, R. L. and Backer, R. R., 1970. J. Biol. Chem. 245, 3186-3194. 23. Siebert, G., Carsiotis, M. and Plaut, G., 1957. J. Biol. Chem. 226, 977-991. 24. Plaut, G. W., 1970. In Current Topics in Cellular Regulation, vol. 2, ed. Horecker, B. L. and Stadtman, E. R., Academic Press, New York, 1-27. 25. Chung, A. E. and Franzen, J. S., 1969. Biochemistry 8, 3175-3184. 26. Colman, R. F., 1972. J. Biol. Chem. 247, 215-223. 27. Langan, T. A., 1960. Acta Chem. Scand. 14, 936-938. 28. Marr, J. J. and Weber, M. M., 1969. Biochem. Biophys. Res. Commun. 35, 12-19. 29. Shiio, F. and Ozaki, H., 1969. J. Biochem. 64, 45-53. 30. Marr, J. J. and Weber, M. M., 1969. J. Biol. Chem. 244, 5709-5712. 31. Omran, R. G. and David, T. D., 1971. Plant Physiol. 47, 43-47. 32. Little, C. and Holland, P., 1972. Can. J. Biochem. 50, 1109-1113. 33. Nagamachi, K. and Honda, K., 1967. Nippon Nogei Kagaku Kaishi 41, 99-105. 34. Satoh, Y., 1972. Plant and Cell Physiol. 13, 493-503. 35. Ruffo, A., Adinolfi, A., Budillon, G. and Capobiano, G., 1962. Biochem. J. 85, 588-593. 36. Krebs, H. A. and Eggleston, L., 1974. In Advances io Enzyme Regulation, col. 12, ed. Weber, G., Pergamon Press, Oxford, 421-434. 37. Sapag-Hagar, M., Lagunas, R. and Sols, A., 1973. Biochem. Biophys. Res. Commun. 50, 179-182. 38. Agosin, M. and Weinback, E. C., 1956. Biochim. Biophys. Acta 21, 117-126. 39. Ozaki, H. and Shiio, I., 1968. J. Biochem. 64, 355363. 40. Dhillon, D. S. and Silver, M., 1972. Plant Cell Physiol. 13, 261-272. 41. Shrago, E., Brech, W. and Templenton, K., 1967. J. Biol. Chem. 242, 4060-4066. 42. Nishi, A., Scherbaun, O. H., 1962. Biochim. Biophys. Acta 65, 411-418.

Isocitrate dehydrogenase of Tetrahymena pyriformis.

Volume 17, number 3 MOLECULAR & CELLULARBIOCHEMISTRY October 7, 1977 ISOCITRATE DEHYDROGENASE OF TETRAHYMENA PYRIFORMIS Pilar VIDAL and Alberto M A...
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