Microbiology Group, Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, Wales, UK1
Department of Measurement and Control, Technische Universiteit, Eindhoven, The Netherlands2
Biochemical Engineering Laboratory, National Institute of Bioscience and Human Technology, Higashi 1-1, Tsukuba, Ibaraki, 305-8566 Japan3
Author for correspondence: Hiroshi Kuriyama. Tel: +81 298 546087. Fax: +81 298 546080. e-mail: kuriyama{at}nibh.go.jp
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ABSTRACT |
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Keywords: Oscillation, redox regulation, glutathione, NADH, yeast
Abbreviations: BSO, DL-buthionine-(S,R)-sulphoximine; NF, 5-nitro-2-furaldehyde
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INTRODUCTION |
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During oscillation, calculated metabolic fluxes through the tricarboxylic acid cycle and oxygen uptake are closely coupled (Satroutdinov et al., 1992 ). However, studies using the effectors of our system, i.e. ethanol (Keulers et al., 1996a
), acetaldehyde (Keulers & Kuriyama, 1998
), O2 and CO2 (Keulers et al., 1996b
) did not reveal either a mechanism for synchronisation or regulation. Individually, the effectors did not meet the requirements of a synchronisation agent (Richard et al., 1996
), i.e. the substance must be emitted, sensed and dissipated. It should also oscillate and produce phase shifts when introduced at above or below its normal oscillating concentration (Winfree, 1990
). It was suggested that the intracellular redox state plays an important role in the regulation of oscillation and it was shown that NAD(P)H fluorescence oscillates in phase with dissolved oxygen (Murray et al., 1998
).
Respiratory oscillation can be characterized on the basis of many metabolic variables. When cultures were grown on ethanol media, GSH oscillated about 20° in advance of dissolved oxygen and in phase with ethanol (Murray et al., 1998 ). Acetaldehyde and acetate oscillated 180° out of phase with dissolved oxygen. As ethanol and GSH accumulate, acetaldehyde and acetate are quickly utilized, and as ethanol and glutathione start to be consumed, acetaldehyde and acetate increase. These observations of phase relationships provided evidence of a close link between glutathione metabolism and ethanol metabolism during oscillation.
Recently, it was found that pulse addition of nanomolar concentrations of sodium nitroprusside, a source of NO+, perturbed oscillation by inhibiting respiration, whereas the pulse addition of reagents that generate NO· did not (Murray et al., 1998 ). A similar perturbation of oscillation occurred when GSH was injected, leading to the suggestion that redox sensing (Rouault & Klausner, 1996
) might be involved. GSH is synthesized from glutamate, cysteine and glycine. In yeast, the regulation of cytosolic and intramitochondrial redox balances are achieved by alteration of the ratios of GSH to its oxidized dimer GSSG (Gilbert, 1995
; Penninckx & Elskens, 1993
). This redox balance is vital for correct protein folding (Raina & Missiakas, 1997
), transcriptional regulation (Demple, 1998
) and protection against reactive oxygen species (Meister, 1995a
). These mechanisms of thiol:dithiol switching are highly conserved in nature.
In this paper we report an investigation of the role of redox cycling in the regulation of the respiratory oscillation. It is shown that glutathione redox balance rather than the redox state of the NAD+/NADH couple is involved in oscillation regulation.
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METHODS |
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Sample preparation, instrumentation and analytical techniques.
Acid-stable metabolites, e.g. NAD+, GSH and GSSG, were extracted from cell samples (1 ml) using perchloric acid (0·25 ml; 3 M) followed by three freeze (-70 °C)/thaw (4 °C) cycles. Samples were centrifuged (15000 g; 5 min) and the supernatant was neutralized to pH 7·8 with tri-potassium phosphate (2 M). The samples were centrifuged (15000 g; 5 min) and analysed.
Alkaline-stable metabolites, e.g. NADH, were extracted from cell samples (1 ml) using potassium hydroxide (2 M)/methanol (70%, v/v) mix (4 °C; 0·5 ml) followed by three freeze (-70 °C)/thaw (4 °C) cycles. Samples were centrifuged (15000 g; 5 min) and before analysis, the supernatant was brought to pH 6·6 with triethanolamine (2 M).
The same instrumentation and analytical techniques were used as previously described (Keulers et al., 1994 , 1996a
; Satroutdinov et al., 1992
). All enzyme assays were modified for a microtitre-plate reader and carried out in triplicate (Ashour et al., 1987
).
NAD+, NADH, NADPH and NAD+ were determined enzymically (Bergmeyer & Braßl, 1984 ). GSSG and GSH were determined enzymically (Baker et al., 1990
). The intracellular glutathione redox potential was calculated from levels of GSH and GSSG using the Nernst equation (Hwang et al., 1995
):
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where E0 is the standard potential of glutathione (-0·24 V), k is Boltzmanns constant (8·31 J mol-1 K-1), T is the absolute temperature (303 K), n is the number of electrons transferred (2) and F is the Faraday constant (96406 J V-1). Intracellular concentrations of metabolites from extracted cells, were calculated assuming that cytosolic volume was 1·6 cm3 (g dry cell wt)-1 (Pampulha & Loureiro-Dias, 1989 ).
NAD(P)H fluorescence.
NAD(P)H fluorescence was measured using a fibre-optic probe supplied by the Biomedical Instrumentation Group of the Department of Biochemistry and Biophysics, University of Pennsylvania (Chance et al., 1975 ). Fluorescence and reflectance data were normalized then smoothed exponentially (factor 0·90) using Microsoft Excel 97. NAD(P)H fluorescence was calculated by subtraction of normalized reflectance from fluorescence data.
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RESULTS |
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The combined addition of BSO (50 µM), GSH (50 µM) and NF (50 µM) at high dissolved oxygen (Fig. 3) produced an immediate cessation of oscillation and inhibited respiration. The duration of perturbation was twice as long as those produced by separate additions of individual components. When the BSO, GSH and NF cocktail was injected at low dissolved oxygen there was also an immediate inhibition of respiration.
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Glutathione perturbation caused a reduction in ethanol uptake and respiration
Perturbation of the oscillation by addition of GSH (100 µM) inhibited ethanol uptake and acetaldehyde production (Fig. 4a). Extracellular GSH was not detected 1 h after addition, indicating either GSH uptake or modification by the yeast (D. B. Murray, unpublished results). The duration of oscillation perturbation by GSH was concentration based, i.e. injections of 50 µM and 100 µM gave perturbation times of 2·8 h (Fig. 2
) and 6·5 h (Fig. 4
), respectively. Intracellular GSH increased from 10 to 25 mM and intracellular GSSG decreased from 1·5 to <0·1 mM (Fig. 4b
), indicating that the yeast takes up GSH. Intracellular GSH fluctuated markedly during the first 2 h of perturbation. Biomass concentration in the fermenter declined from 6·45 to 5·84 g l-1 during the period of respiratory inhibition due to wash out of yeast. Biomass then started to increase during the phase of increased respiration before levelling out once oscillation recovered (21·5 h). NAD(P)H fluorescence decreased after injection of GSH, reaching a minimum 20 min after injection, and ethanol uptake was immediately inhibited (Fig. 4c
). Then NAD(P)H fluorescence increased during the reduced-respiration period. When the oscillation was perturbed with GSH, almost all of the GSSG is metabolized, resulting in a decrease in intracellular glutathione redox from -0·3 to -0·35 mV. Injection of glutamate (Fig. 5a
) and cysteine (Fig. 5b
) at millimolar concentrations, inhibited respiration and perturbed oscillation: both caused immediate respiratory inhibition. Glycine had no influence probably because of the low uptake of glycine by yeast (Rose & Harrison, 1989
). However, to have a similar oscillation perturbation duration as GSH, 50- or 5-fold increases in the concentration of glutamate or cysteine, respectively, had to be used.
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DISCUSSION |
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Investigations on the mechanism of glutathione-induced perturbation of respiratory oscillation indicated that factors increasing GSH concentration, e.g. glutamate, cysteine, GSH or GSSG (Osuji, 1979 ; Udeh & Achremowicz, 1997
) inhibited respiration, and compounds reducing GSH concentration, e.g. BSO (Meister, 1995b
), either altered the oscillation period or elevated respiration during perturbation. Therefore glutathione or a product of glutathione metabolism directly perturbs oscillation.
Inhibition of glutathione reductase with NF produced a damped respiratory oscillation, indicating that the activity of glutathione reductase influences oscillation. These experiments indicate that the mechanism of oscillation regulation involves the redox cycling of GSH and GSSG. The perturbation phase delay observed when GSH or GSSG was injected at high and low dissolved oxygen did not occur when the BSO, GSH and NF, or GSH and NF cocktails were injected: therefore, glutathione reductase plays an important role in regulating the oscillation.
It is probable that the NAD+/NADH redox couple is not involved directly in either the regulation or the population synchronisation of respiratory oscillation, because during GSH perturbation glutathione redox dropped from -0·30 to -0·35 mV. This indicated that conditions were more reducing; however, during the same period NAD(P)H fluorescence fell. This also indicated that the respiratory chain was not blocked at the NADH-Q reductase. GSH perturbation caused an immediate cessation of ethanol uptake; however, NAD(P)H fluorescence and acetaldehyde concentration decreased. This indicated direct inhibition of alcohol dehydrogenase; inhibition may occur by disruption at the zinc finger (Crow et al., 1995 ).
Regulation of the respiratory oscillation occurs during ethanol metabolism and at a site in the respiratory chain beyond NADH-Q reductase. The synchronisation factor(s) must influence these regulation points. Period frequency may be caused by slow thiol-transfer rates and slow protein-folding rates observed in vivo (Raina & Missiakas, 1997 ). Pantothenate and pyridoxal-HCl both cause enhanced respiration, and glutathione and cysteine both caused reduced respiration during perturbation. These cofactors (Wainwright, 1969
) and metabolites are involved in sulphur metabolism in yeast. Temperature and aeration rate but not dissolved oxygen (within limits) (Keulers et al., 1996a
, b
), influence population synchrony, suggesting that the synchronisation mechanism may involve a volatile component. The volatile compound is a product of intracellular glutathione cycling, and therefore probably contains a thiol group. Further evidence for this was supplied by the perturbation of oscillation with GSH precursors cysteine and glutamate, i.e. cysteine injection produced a greater perturbation than glutamate.
In conclusion, the respiratory oscillation in ethanol-grown cultures of S. cerevisiae is regulated by intracellular redox potential. The regulation mechanism relies on the intracellular cycling of glutathione and glutathione reductase activity. It appears to have at least two sites of action, i.e. ethanol catabolism and the respiratory chain post NADH-Q reductase. The synchronisation mechanism probably involves a volatile compound with a thiol group. Respiratory oscillation provides an excellent opportunity to study the influence of intracellular redox on metabolism in vivo. Further work is being carried out to elucidate the molecular mechanisms involved in the regulation of respiratory activity in the oscillatory state and the mechanism of population synchronisation.
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ACKNOWLEDGEMENTS |
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Received 3 February 1999;
revised 3 May 1999;
accepted 1 June 1999.