Comp. Biochem. Physiol. Vol. 88B, No. 3, pp. 851-854, 1987 0305-0491/87 $3.00 + 0.00 Printed in Great Britain © 1987 Pergamon Journals Ltd

NADPH/NADP RATIO COULD REGULATE THE GLYOXYLATE CYCLE IN TETRAHYMENA P YRIFORMIS

ELISA REVILLA, ISABEL FABRF.GAT and ALnEgTO MACHAIX)* Departamento de Bioquimica, Facultad de Farmacia, Universidad de Sevilla, Spain

(Received 20 January 1987)

Abstract--1. The glyoxylic acid cycle pathway could be regulated through the modulation of the isocitmte dehydrogenase-NADP activity. This enzyme is inhibited by NADPH.

2. The effect on the glyoxylate cycle flux of variations in the rate of the NADPH-consuming pathways has been studied.

3. Increase in the rate of NADPH-consuming activity by addition of H202 produces inhibition of the glyoxylate cycle and decrease in the NADPH]NADP ratio.

4. These results suggest that the glyoxylate flux in Tetrahymena could be modulated by regulation of NADP-dependent isocitrate dehydrogenase by the NADPH/NADP ratio.

INTRODUCTION

The glyoxylic acid cycle is a pathway that leads to the production of dicarboxylic acids required for ana- plerotic functions when two carbon sources such as acetate or ethanol are used as carbon substrates (Kornberg, 1966). The cycle includes several Krebs cycle reactions and two specific enzymes: isocitrate lyase (E.C. 4.1.3.1.5) and malate synthase (E.C. 4.1.1.3.2). This cycle is regulated by the level of the specific enzymes, which are induced by the pres- ence of their nutritional inductor in several micro- organisms such as Escherichia coli (Kornberg, 1966), Tetrahymena pyriformis (I-Iogg and Kornberg, 1963), Candida tropicalis (Nabeshima et al., 1977), Sacharo- myces cerevisiae (Betz and Weiser, 1976) and Co- prinus lagopus (Casselton et al., 1969).

However, an increase in enzyme levels alone can- not account for the operation of the cycle, because the kinetic characteristics of these enzymes clearly improve the flux of isocitrate through the tricar- boxylic acid cycle (Bautista et al., 1979; Lttscher et al., 1979). NADP-isocitrate dehydrogenase has a higher activity and a lower Km for isocitrate than isocitrate lyase (Cooper and Beevers, 1969). In this case, there is a need for NADP-isocitrate dehy- drogenase inhibition for the functioning of this cycle with the replenishment of oxalacetate. This inhibition could be produced in three different ways:

(a) by concerted inhibition by oxalacetate and glyoxylate (Dhillon and Silver, 1972; Machado and Satrfistegui, 1981; Montoya-Villarroya and Ltpez- Ptrez, 1975)

(b) phosphorylation of the enzyme (Borthwick et al., 1984; Garnak and Reeves, 1979)

(c) inhibition by NADPH (Bautista et al., 1979;

*To whom correspondence should be sent: Dpto. Bioqui- mica. Fac. Farmacia, c/Tramontana s/n, 41012 Sevilla, Spain.

Machado and Satrfistegui, 1981; Rose, 1960; Satrdstegui et al., 1983).

We have found a relationship between the flux through the glyoxylate cycle and the NADPH/NADP ratio (Bautista et al., 1979; Machado and Satrdstegui, 1981; Satrdstegui et al., 1983). For this reason and in order to look for new data that support this possi- bility, we have studied the variation in the glyoxylate cycle flux of Tetrahymena pyriformis when the NADPH consuming rate is modified. We have found that the inhibition of the glyoxylate bypass takes place when the rate of NADPH consuming pathway is increased with the concomitant reduction of the NADPH/NADP ratio.

MATERIALS AND METHODS

Chemicals NADPH, NADP were purchased from Btehringer.

Glucose 6-phosphate, glucose 6-phosphate dehydrogenase, glucose oxidase and phenazine methosulfate were obtained from Amersham (U.K.). Proteose peptone and bacto- tryptone were obtained from Difco. All other reagents were of the highest available from commercial sources.

Preparation of Tetrahymena cells Tetrahymena were grown at 28°C under shaken con-

ditions, as previously described (Raugl et al., 1975). Growth medium contained 0.5% proteose peptone, 0.5% bacto- tryptone and 0.026% K2PO4H. 3H20. The cells were culti- vated in 250 ml Erlenmeyer flasks containing 100 ml media. Shaking was provided by a rotatory incubation shaker set at 75 rpm. Cultures were harvested (after 3 days) by centri- fugation at 4000g for 20 min and resuspended in culture medium.

Cell viability Under all the experimental conditions we studied, the

viability of the cells was measured according to different parameters: cells number, change in their morphology and mobility. Moreover, in each experiment the acetate con- sumed by the cells was measured and the results calculated with respect to the acetate utilization.

851

852 ELISA REVILLA et al.

Counting of Tetrahymena cells was performed as follows: a thin layer of agar was added to the counting camera in order to diminish the mobility of the cells and cell number was calculated as the mean of at least 5-6 different mea- surements.

Analytical methods The rate of fatty acid synthesis has been calculated

from the (U-~4C)-acetate incorporation into lipids. The incubations were stopped by adding 2.4 ml of incubation medium to a mixture containing: 10ml DOLE solution (200 mi hexane, 800 ml isopropylic alcohol and 20 ml 1 N H2SO4), 6 ml hexane and 3 ml H20. Triglycerides and fatty acids were extracted as described by Rodbell (1964).

The rate of the glyoxylate cycle has been estimated by measurement of the incorporation of (U-14C)-acetate into glycogen. For this reason, the incubation was stopped by adding 8 ml ethanol (at 0-4°C) to 3 ml culture. This suspen- sion was kept overnight at 0-4°C. Thereafter, the radiactive glycogen was purified by two precipitations in ethanol. To remove proteins, the last pellet was dissolved in 2M perchloric acid followed by centrifugation at 5000&. Super- natants were concentrated by precipitation with ethanol, dissolved in I ml 2 M H2 SO4 and counted by liquid scintil- lation.

Utilization of acetate by the cells was calculated from the radiactivity remaining in the medium after the incubation time. In each case, incubations stopped at zero time were included to enable correction for background radioactivity and to calculate the dpm of (U-14C)-acetate in the medium.

The concentrations of NADP and NADPH in Tetra- hymena cells were estimated as described in detail elsewhere by Greenbaum et al. (1965), according to a modification of the procedure by Fabregat et al. (1985).

Cell free extracts for enzymatic assays were prepared as follows: cells were collected by centrifugation at 15,000g, 4°C for 10rain, washed twice in cold Ringer phosphate buffer (Ryley, 1952), resuspended in this same solution and disrupted by sonication. Catalase activity was determined by measurement of H202 consumption, directly by the decrease in the absorption at 240 nm. Glutathione per- oxidase was assayed as described by Lawrence and Burk (1976). Malate synthase activity was determined as de- scribed by Durchslag et al. (1981). Isocitrate lyase was assayed by the method of Dixon and Kornberg (1959). H:O 2 was measured according to Krebs et al. (1964).

RESULTS AND DISCUSSION

System employed to produce a variation in the N A D P H / N A D P ratio

NADPH consumption was increased in Tetra- hymena by incubating the cells in the presence of H20 2. It is well known that hydroperoxides and H:O 2 are reduced by glutathione peroxidase and catalase. Glutathione reductase regenerates reduced glutathione from oxidized glutathione at the expense of NADPH (L6tscher et al., 1979). NADPH can also be rapidly oxidized by H202 by means of a heme- undecapeptide from cytochrome-c (Bodanes, 1983). Tetrahymena is able to metabolize H202: we have measured their catalase specific activity (67/z mol/min per mg) and their glutathione peroxidase specific activity (25 nmol/min per rag).

Hydrogen peroxide was produced by the reaction of glucose (3.3 mM) with glucose oxidase. This sys- tern has the advantage that the hydrogen peroxide production is continuous during the 60 min of incu- bation. By varying the glucose oxidase added, the H2 02 production can be modulated. We have proved that the utilization of (U-]4C)-acetate and its incorpo-

3"5O e. o

u t- o u

-I-

.J /

/ 4

1 2 GOD un i ts

Fig. 1. H~O2 concentration in the incubation medium, with (/X) and without cells (O), produced by different amounts of glucose oxidase (in the presence of 3.3 mM glucose)

during 60 rain of incubation.

ration into triglycerides, fatty acids and glycogen did not change in the presence of 3.3 mM glucose. The H202 produced under the different conditions and its consumption by Tetrahymena are summarized in Fig. 1. Moreover, these H202 concentrations do not produce any change in the isocitrate lyase and malate synthase activities measured in cell free extracts at the end of the incubations.

Effect o f the H2 02 concentration on ( U-14 C)-acetate incorporation into: glycogen (glyoxylate cycle rate), fat ty acids (fatty acid biosynthesis rate) and tri- glycerides

The incorporation of (U-]4C)-acetate into the com- pounds mentioned above in Tetrahymena cells are

i i

0 u 50 ~

1; 2'7 . s 47 H,o 2~.. , 0.25 0,S 1 2 GOD (un | ts )

Fig. 2. Effect of the increase in the H202, produced by different units of glucose oxidase, in the incubation medium on (U-14C)-acetate incorporated into: glycogen (glyoxylate cycle rate: O), fatty acid (fatty acid biosynthesis rate: O) and triglycerides: ZX. Tetrahymena cells were incubated for 60rain in the presence of2mM acetate, 0.5/zCi (U-14C)- acetate, 3.3 mM glucoseand different amounts of glucose oxidase. The results have been calculated with respect to acetate utilization and are expressed as a percentage of control (without glucose oxidase). They are the mean +

SEM of at least five different experiments.

Regulation of the glyoxylate cycle in Tetrahymena 853

Table i. NADP N A D P H NADPH/NADP

Without glucose oxidase 1.14+0.28 2.04=1=0.65 1.74+0.30 With 1 unit of glucose oxida~ 1.90+0.44 0.84=t=0.19 0.54+0.15" Measure of NADP and NADPH concentrations (in nmol/10 s cells) in Tetrahymena

cells incubated for 60 rain in the presence of 2 mM acetate, 3.3 mM glucose and with or without glucose nxidase. The results are the mean of five different experiments + SE of the mean (*P < 0.05).

represented in Fig. 2. As can be seen, the incorpo- ration of (U-14C)-acetate into glycogen was linearly and strongly inhibited when the production of H2 O2 was increased. Total inhibition was reached at 47/z M H202 concentration, that was produced by two units of glucose oxidase. However, the effect on (U-14C) - acetate incorporation into fatty acids and trigly- cerides was really less pronounced. There is only a little decrease at higher concentration of glucose oxidase, but it is probably due to the decrease in the NADPH concentration. Similar results have been described for lipogenesis in adipose tissue when cells are incubated with phenazine methosulfate, a NADPH-consuming compound (Katz and Wals, 1971; Saggerson, 1972). When two units of glucose oxidase were used, there was a total inhibition of the glyoxylate cycle and the rate of fatty acids bio- synthesis resulted only in 40% inhibition with respect to controls without H202.

Variations in the N A D P H and N A D P concentrations /n Tetrahymena cells incubated with H2 02

Hydrogen peroxide (38.5 #M) produced no change in the total nicotinamide adenine dinucleotides phos- phate. However, there was a large decrease in the NADPH/NADP ratio, that became more than three times less than the control (Table l). In this con- dition, there was a 70% inhibition of the glyoxylate cycle rate and only 20% inhibition of the fatty acid biosynthesis rate (Fig. 2).

These results strongly suggest that the decrease in the NADPH/NADP ratio provokes the inhibition of the glyoxylate cycle flux activity, probably due to a de-inhibition of isocitrate dehydrogenase by means of the decrease in the NADPH concentration. In hepa- tocytes, we have shown that the pentose phosphate cycle, one of the main NADPH-producing path- ways in liver, is modulated by the NADPH/NADP ratio (Fabregat et al., 1985). This kind of regulation could also play an important role in Tetrahymena, where it has not been reported that the isocitrate dehydrogenase-NADP could be regulated by phos- phorilation. Besides, this reaction is the unique NADPH-producing pathway in this organism (Vidal and Machado, 1977). Therefore, taking into account the implication of the isocitrate dehydrogenase in the glyoxylate cycle regulation, the modulation of this enzyme by the NADPH/NADP ratio could indirectly regulate the flux of isocitrate through the glyoxylate cycle.

Acknowledgements--Tlds work was supported by grant from the CAICYT no. 1902.

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