BIOCHIMIE, 1981, 63, 247-249.

Relationship between glyoxylate cycle activation and NADPH/NADP rise in Tetrahymena pyriformis. Alberto MACHADO and Jorgina SATRUSTEGUI.

(Refue le 17-10-1980, accept( le 23-12-1980 apr~s r(vision)

Departamento de Bioqubnica y Biologia Molecular, Facultad de Ciencias, Centro de Biologla Molecular, Universidad ,4ut6noma de Madrid, Madrid - 34, Spain.

Mots-cl6s : cycle du glyoxylate / Tetrahymena / rap- port NADPH/NADP.

Key-words : g l y o x y l a t e c y c l e / Tetrahymena / NADPH/NADP ratio.

I n t r o d u c t i o n .

The glyoxylic acid cycle is a pathway that leads to the production of dicarboxylic acids required for anaplerotic functions [1]. It is of paramount importance to those organisms that grow on two- carbon substrates and also provides dicarboxylic acids necessary for gluconeogenesis during the germination of fat-storing plant seeds.

The cycle includes several Krebs cycle reac- tions and two specific enzymes: isocitrate lyase (E.C. 4.1.3.1.5) and malate synthase (E.C. 4.1.1.3.2). Thus the glyoxylate bypass can be regarded as an alternative pathway with respect to the enzymes of the Krebs cycle lying between isocitrate and succinate, the branchpoint being located at the level of the common substrate iso- citrate. The cycle is regulated by the levels of the specific enzymes, isocitrate lyase and malate syn- thase, that are activated by the presence of their nutritional inducers in E. coli [1], Tetrahymena pyrilormis [2], Candida tropicalis [3], Saccharo- myces cerevisiae [4] and Coprinus lagopus [5]. Over the Iast years evidence of specific inactivation of isocitrate lyase by endogenous factors has been presented for several plants [6-8]. The enzyme from various parasites [9] and baker's yeast [4] are proteolyzed in vitro or in vivo, respectively. However, regulation by enzyme levels cannot account for the operation of the cycle. The kinetic characteristics of the enzymes competing for the use of isocitrate strongly favour the channelling of this substrate through the tricarboxylic acid cycle: NADP-isocitrate dehydrogenase has both a higher activity and a lower Km for isocitrate than does isocitrate lyase.

We have suggested that the N A D P H / N A D P ratio can actually modulate the activity of the NADP-isocitrate dehydrogenase, an enzyme cata- lyzing an equilibrium reaction [10]. A rise in the N A D P H / N A D P ratio could inhibit the NADP- isocitrate debydrogenase resulting in a stimulation of the glyoxylate bypass. An increase of the N A D P H / N A D P ratio takes place in acetate- grown E. coli [10] and S. cerevisiae (Machado and Bautista submitted for publication) in relation with the values found in glucose-grown organisms. We are now presenting results leading to suggest that the same situation can be applied Tetrahymena pyriJormis. In the present work, the activation of the glyoxylate bypass resulted from differences in growth conditions (shaken versus static [11-I2] cultures) with no change in the carbon source.

M a t e r i a l s a n d Methods .

Materials.

NADH, NADPH and glucose-6-phosphate were obtai- ned from Boehringer ; DL-isocitrate, oxoglutarate (mono- sodium salt), NAD, NADP and lactate dehydrogenase were obtained from Sigma. Proteose peptone and bacto tryptone were obtained from Difco. Other reagents were of the highest purity available from commercial sources.

Culture conditions.

Tetrahymena was grown at 28°C under static and shaken conditions, essentially as described Raugi et aI. [12]. Growth medium contained 0,5 per cent proteose peptone, 0,026 per cent K~P HO,-3H~O, 0,1 per cent sodium acetate and 0,25 per cent glucose.

248 A. Machado and J. Satrustegui.

When grown under static conditions Tetrahymena cells were cultivated in 11 erlenmeyer flasks containing 200 ml media, in an immobilized temperature-controlled chamber. Growth under shaken conditions was carried out in 1 1 erlenmeyer flasks containing 100 ml media. Shaking was provided by a rotatory incubation shaker set at 75 r.p.m. Cultures were harvested after 48 h.

carried out following Saez and Lagunas [15], except that lactate dehydrogenase was used instead of alcohol dehy- drogenase, with 0.1 M Tris-HC1 pH 7.4 as buffer.

Glycogen and D N A were also measured on the lyo- philized cultures by the methods of Keppler and Decker [16], and Giles and Myers [17], respectively.

Enzyme assays and metabolite determinations.

For assay of enzymatic activities the cells were collec- ted by centrifugation at 15,000 × g, 4°C, and washed in cold Ringer phosphate solution [13]. Crude extracts were obtained by cell disruption in a MSR (B. Braun) cell homogenizer at 2 000 r.p.m, for 4 minutes and 4 000 r.p.m. for 1 minute and centrifuged at 3 000 × g for 10 minutes. The supernatants were used for enzyme assays.

The activity of NADP isocitrate dehydrogenase was determined by the method of Bautista et al. [10]. The iso- citrate lyase was assayed as reported by Dixon and Korn- berg [14].

R e s u l t s a n d D i s e u s s i o n .

Characteristics oJ growth conditions.

A s s h o w n in tab le I, an inc rease in i soc i t ra te lyase ac t iv i ty and in g lycogen c o n t e n t is p r o d u c e d w h e n Tetrahymena is g r o w n u n d e r s tat ic c o n d i - t ions. T h e s e resul ts agree wi th those r e p o r t e d by

TABLE I.

Effect of culture conditions on the activities of NADP-isocitrate dehydrogenase, isocitrate lyase, and glycogen content in Tetrahymena pyriformis.

Culture N A D P - I D H IL Glycogen conditions nmoles x min -1 x mg of protein -t ,amol glycosyl units

X ~g D N A -~

Shaken 229 (2) 2.1 (2) 15 ~ 0.9 (4) Static 284 (2) 4 .0 (2) 29 __+ 1.8 (4)

p < 0.005

Number in parenthesis are the number of times each experiment was performed.

TABLE II.

Effect of culture conditions on the concentration of Pyridine Nucleotides in Tetrahymena pyrilormis.

N A D P H NADP N A D P H N A D H N A D N A D H /NADP / N A D

Shaken 0.0053 ! 0.0017 0.017 + 0.007 0 3 0 0030 ± 0.0014 0.032 + 0.00079 0.1 Static 0.034 -~- 0.0032 0.020 __+ 0 0 9 1.7 0 027 + 0.0032 0.0062 ~+ 0.0007 4.3

p ~ 0.0025 NS p ~ 0.0005 p ~ 0.005

Concentrations are expressed in nmoles × ~xg D N A -1. The values are the means _ SD of 4 expt.

For metabolite determinations, 200 ml samples of shaken or static Tetrahymena cultures were rapidly collected in precooled flasks and immediately immersed in liquid nitrogen. The frozen cultures were lyophylized, weighed, resuspended in acid or alkaline soultions and used for the determination of pyridine nucleotides [15]. The assay of nicotinanfide adenine nucleotides was

BIOCH1MIE, 1981, 63, n ° 3.

L e v y and W a s m u t h [11], and R a u g i et al. [12], and p r o v e tha t i nc rea sed g luconeogenes i s and gly- oxy la t e cycle ac t iva t ion are b r o u g h t a b o u t by this cu l tu re cond i t ion . T h e r ise in g luconeogenes i s does n o t d e p e n d on the inc rease of i soc i t ra te lyase s ince R a u g i et al. h a v e s h o w n tha t it is no t affec- t ed b y inh ib i to r s of p ro t e in synthesis [12].

Tet rahymena glyoxy la te cyc le and N A D P H / N A D P ratio. 249

EJJect of g r o w t h condi t ions on the concen tra t ion of pyr idine nucleot ides .

Table I I shows the concentrations of N A D , N A D H , N A D P and N A D P H in shaken and static T e t r a h y m e n a cultures (*). A marked increase in N A D H is produced in static cultures in agreement with the virtual anaerobiosis of that condition. N A D H oxidation is probably greatly reduced in spite of the observed induction of N A D H utilizing systems [18]. Krebs and Hems [19] have shown that an increase in pigeon liver gluconeogenesis f rom N A D H - p r o d u c i n g sources is due to a rise in the N A D H / N A D ratio that drives glyceraldehyde- 3-phosphate dehydrogenase towards glucose for- mation instead of oxidation. In T e t r a h y m e n a however, the limiting steps in acetate incorpora- tion into glycogen are those included in the gly- oxylate bypass ; and the N A D H / N A D ratio would possibly have no regulatory significance since its effect on the bypass reactions are not apparent.

Table I I also shows a pronounced increase in the N A D P H concentration. NADP-isoci t ra te dehy- drogenase is inhibited by the produc t of the decar- boxylating reaction, N A D P H [20] with a K~ similar to its Km for N A D P . This indicates that the rise in the N A D P H / N A D P ratio would cause the activation of the glyoxylate bypass through the inhibition of NADP-isoci t ra te dehydrogenase.

These results support our previous reports [10] and suggest that the glyoxylate cycle could be regu- lated by the N A D P H / N A D P ratio through the inhibition of NADP-isoci t ra te dehydrogenase ;

(*) The concentrations of free nucleotides, or rather, the free N A D ( P ) H / # e e NAD(P) ratios give a better esti- mate of the true redox state of pyridine nucteotides than the ratio o[ the bound forms. However, in the present work, the conditions ]or a correct measure o[ the <~ free ~> N A D P H / N A D P ratio were lacking. This free ratio is usually determined through the mass action ratios and equilibrium constants of 6-phosphogluconate dehydro- genase [21-22], malic enzyme, or NADP-isocitrate dehy- drogenase that catalyze equilibrium reactions in rat liver [23]. The first two enzymes are virtually absent h~ Tetra- hymena pyriformis ([24] and unpublished results). The aim of this work was to study the possible inhibition o/ the third by N A D P H or by oxaloacetate + glyoxylate. Inhib#ion by the latter might create conditions under which NADP-isocitrate dehydrogenase no longer nzain- tains equilibrium between reactants and products, and therefore the estimate oJ the ~< Jree >~ N A D P H / N A D P ratio through the isocitrate/oxoglutarate ratio could be inaccurate. Whatever changes took place in the iso- citrate/oxoglutarate ratio, conclusions about the origin o[ such changes could never be attained without an independent estimate of the c~ #ee >> ratio.

the net flux of isocitrate beng directed through the dehydrogenase under low N A D P H / N A D P ratios, and through the lyase under high N A D P H / N A D P ratios. An increase in the redox ratio of pyridine nucleotides ( N A D H / N A D and N A D P H / N A D P ) is possibly a primary result of the decrease in oxy- gen tension of the static cultures.

Acknowledgments,

This work was supported by grants from the Comisidn Administradora de[ Descuento Complementario del hzsti- tuto Nacional de Previsirn and from the Comisi6n Ase- sora de b~vestigaeidn Cienti[ica 3' Tdcnica. We thank Miss Maria Victoria Mora-Gil [or her technical assistance and Dr. Gerald Frank for his help with the English version of the manuscript.

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BIOCH1MIE, 1981, 63, n c' 3.