Alteration of enzymes in ageing human fibroblasts in culture. V. Mechanisms of glutathione peroxidase modification

Mechanisms of Ageing and Development, 58 ( 1991) 93--109

93

Elsevier Scientific Publishers Ireland Ltd.

A L T E R A T I O N OF E N Z Y M E S IN A G E I N G H U M A N F I B R O B L A S T S IN C U L T U R E . V. M E C H A N I S M S OF G L U T A T H I O N E P E R O X I D A S E MODIFICATION

E T I E N N E P I G E O L E T * and JOSI~ R E M A C L E Facultds Universitaires N.-D. de la Paix, 61 rue de Bruxelles, B-5000 Namur (Belgium)

(Received July 2nd, 1990) (Revision received October I lth, 1990) SUMMARY Ageing of WI-38 fibroblasts in culture was used as a model in order to investigate the evolution and the alteration of the key antioxidant enzyme glutathione peroxidase. The activity of glutathione peroxidase is influenced by the presence of selenium in the culture medium and we have also shown that the specific activity of this enzyme does not decrease during ageing, but rather slightly increases. No alteration could be detected by immunotitration. Also the kinetic parameter K for tertbutyl hydroperoxide has not changed. However, the heat resistance of the enzyme dramatically decreases with ageing. Dilutions of the enzyme preparations had the same influence on the thermosensitivity of the enzyme. This dilution effect is most probably linked to the dissociation of the enzyme subunits into dimers and monomers. Moreover, the kinetic of thermoinactivation curves are best explained by consecutive reactions of inactivation with an intermediary enzyme form. These observations strongly support the hypothesis that ageing is associated with an increased dissociation constant of the tetrameric glutathione peroxidase leading to an easier dissociation of the enzyme in old cells. K e y words: Glutathione peroxidase; Cellular ageing; Enzyme alteration; Thermoinactivation; Tetramer/dimer equilibrium

l NTRODUCTION Cellular ageing is certainly a complex process involving stochastic and programmed aspects [1,2]. One hypothesis is that it could result from a failure of the *Fellow of the I.R.S.I.A. Correspondence to: Laboratoire de BiochimieCellulaire, Facultds Universitaires N.-D. de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium. 004%6374/91/$03.50 Printed and Published in Ireland

© 1991ElsevierScientific Publishers Ireland Ltd.

94 mechanisms of cell maintenance and defense; but the primary molecular modifications leading to these failures are not yet clearly identified [1]. In the cell, the most important maintenance mechanisms that are candidates for primary alteration are biochemical pathways for energy production, detection of errors, repair mechanisms during replication and translation of genetic information, and defense against free radicals and oxidation. Numerous results suggest a decrease of cellular reductive potential during ageing: decrease of GSH and increase of GSSG in aged red ceils [3], decrease of GSH during the latest passage of WI-38 human fibroblasts in culture [4] increase of NAD+/NADH, GSSG/GSH, NADP+/NADPH ratios in the muscle of mice during ageing [5] and premature ageing of red cells in animals receiving deficient diets in vitamin E [6]. For example, alteration of glyceraldehyde-3-phosphate dehydrogenase during ageing seems to be due to oxidative conditions since chemical oxidation of the 'young' enzyme gives an enzyme similar to the one purified from old rats, both in its specific activity and in NAD + affinity [7]. Cellular ageing is thus related to the capability of maintaining cellular constituents in a reduced state and the capability of destroying oxidative molecules. Cells are well protected against these molecules by enzymatic and non-enzymatic defense systems. Glutathione peroxidase is one of the most important antioxidant enzymes. It was shown that glutathione peroxidase is the most effective enzyme to protect cells submitted to oxidative stress [8]. We have also shown [9] that the three antioxidant enzymes are themselves sensitive to the reactive oxygen species and we have suggested that, above a critical level of production, these reactive species will inactivate antioxidant enzymes leading to an autocatalytical, irreversible, destructive process. This process would also occur in the case of a decrease in the activity of glutathione peroxidase. It was thus interesting to investigate the quantitative and qualitative modifications of this enzyme and to estimate their importance in a possible decreased protection of old cells. The evolution of the activity of the enzyme was observed with and without addition of selenium in the medium since it is known to be a limiting factor in cell culture. MATERIALS AND METHODS Glutathione reductase (type IV from yeast), glutathione adenine dinucleotide phosphate (NADPH) are from sigma MO). tert-Butyl hydroperoxide, ethylenediaminetetraacetic phate and other chemicals are from Merck AG (Darmstadt,

(GSH), nicotinamide Chemicals (St Louis, acid (EDTA), phosGermany).

Cell cultures WI-38 fibroblasts were from the American Type Culture Collection, culture medium from Eurobio (Paris, France) fetal bovine serum from Sera-lab (Sussex, U.K.) aureomycin from Lederle S.A. (Brussels, Belgium) and trypsin 0.25% in Puck's solution from Gibco (Paris, France). Plastic culture flasks were obtained from Cell Cult

95

(Feltham, U.K.). WI-38 cells were serially cultivated in Eagle's minimum essential medium with 10% fetal bovine serum in 75-cm2 plastic flasks as described by Hayflick [10]. When each culture flask was confluent, the cells were detached and transfered into 4 other flasks. This was defined as 2 passage numbers. Aureomycin (50 mg/ml) was added to this medium. For thermolability experiments and immunotitration, cells were harvested in PBS buffer (KH:POffK2HPO4 10 raM, pH 7.4, NaC1 0.15 M) with EDTA 1 mM after a rinsing with PBS. The cells were homogenized with a Dounce tight homogenizer. The homogenates were then centrifuged at 39 000 rev./min during 30 min in a Beckman L5-65 centrifuge (Beckman Instruments Inc, Spinco Division, Palo Alto, CA, rotor type 40) and the supernatants were considered as the soluble fraction. Cells were subcultivated according to Hayflick [1 l]. In this work, cells from passage number 29 to 39 were considered as young or phase II cells since they were in the exponential growth phase.

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Time (days) Fig. 1. Evolution of the specific activity of glutathion¢ peroxidase in WI-38 fibroblasts after 1/4 subcultivation. A passage number 38; B passage number 45. Confluency is reached after 6 days for the passage number 38 and 8 days for the passage number 45. Each point is the mean of 2 measurements.

96

Glutathione peroxidase assay The glutathione peroxidase activity was assayed using the consecutive glutathione reductase reaction and following the oxidation of NADPH as described in detail earlier [9]. We assayed the glutathione peroxidase specific activity along with the ageing of the cultures but since the cell cycle influences the activity of most enzymes, we first measured the specific activity of the glutathione peroxidase during the growth of young (phase II) and old (phase III) cells. The specific activity increased from the first day after subcultivation until confluency was reached and it then remained stable at least for 4 days, in young cells as well as in old cells (Fig. 1). Because of this stable level of activity, the activity of the enzyme was assayed at confluency in the long range experiment (see Results section). Immunotitration Rabbit antiserum was prepared by the method of Vaitukaitis [12]. The antibodies were purified by affinity chromatography. Glutathione peroxidase was fixed on sepharose 6B (Pharmacia, Uppsala, Sweden) after activation by BrCN (1 mg glutathione peroxidase/3 ml gel). The column (1.2 × 8 cm) was equilibrated with PBS.

Antiserum was passed through the column which was then rinsed with PBS. The antibodies were eluted by formic acid 0.5%, neutralized with a small volume of Tris--HC1 1 M (pH 8.0). The antibodies were dialyzed against PBS and centrifuged for 10 min at 12 000 rev./min. The final solution contained 2.58 mg antibodies/ml. Increasing volumes of this solution were added to 0.4 ml of soluble fractions prepared from the cells and incubated at 4°C overnight. The samples containing 0.5 ml total volume were then centrifuged for 10 min at 12 000 rev./min and glutathione peroxidase activity of the supernatant was measured. The control was obtained by addition of rabbit antibodies anti-mouse IgG to 0.4 ml of the soluble fraction.

Thermolability Aliquots (0.35 ml) of the soluble cell fraction were placed in calibrated tubes and incubated in a thermostatized bath during indicated periods of time. They were then placed in ice and assayed for glutathione peroxidase activity. Results are presented on a logarithmic scale expressing the residual activity with regard to a sample unexposed to high temperature, and versus time of incubation. RESULTS One estimation of the role of glutathione peroxidase in ageing can be obtained by testing the evolution of the total enzyme activity in ageing cells. Since it was already shown that the glutathione peroxidase activity is dependent on the selenium concentration of the medium [13--17] another experiment was performed in the normal

97

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Fig. 2, Evolution of the specific activity of glutathione peroxidase in WI-38 fibroblasts with continuous subcultivation. A: culture without addition of selenomethionine. B/C: culture with selenomethionine 2 × 10-7 M added to the medium. Each point is the mean of 2 measurements. Cells were subcultivated 1/4 every week.

98 TABLE I APPARENT K m OF PURE GLUTATHIONE PEROXIDASE OR GLUTATHIONE PEROXIDASE OF SOLUBLE FRACTION OF PHASE II OR PHASE 111 FIBROBLASTS FOR tcrt-BLrfYL HYDROPEROXIDE AT 0.87 mM GSH. Sample

App K m (raM)

Phase II Fibroblasts Phase Ill Fibroblasts Pure GPX

0.118 + 0.030 0.101 :t: 0.062 0.112

culture medium and two experiments with addition of 2 x

l0 -7 M s e l e n o m e t h i o -

n i n e to the m e d i u m . T h e results a r e p r e s e n t e d in Fig. 2. A d d i t i o n o f s e l e n o m e t h i o n i n e i n c r e a s e d t h e specific a c t i v i t y o f the e n z y m e by a r o u n d 50% in all the p a s s a g e s b u t did n o t c h a n g e the e v o l u t i o n o f the a c t i v i t y w i t h age. A slight i n c r e a s e in the specific a c t i v i t y o f t h e g l u t a t h i o n e p e r o x i d a s e w a s o b t a i n e d in a g e i n g cells in all three e x p e r i m e n t s . T h e c u l t u r e m e d i u m c o n t a i n s its s e l e n i u m m a i n l y t h r o u g h the p r e s e n c e o f 10% fetal b o v i n e s e r u m . H o w e v e r , the c o n c e n t r a t i o n o f s e l e n i u m in s e r u m is a l r e a d y so l o w t h a t it c a n b e c o m e the l i m i t i n g f a c t o r for the g l u t a t h i o n e p e r o x i d a s e e x p l a i n i n g p r o b a b l y s o m e c o n f l i c t i n g results o b t a i n e d o n m o d e l s u s i n g c e l l u l a r in v i t r o a g e i n g [41.

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Antibody votume (gl) Fig. 3. lmmunotitration of soluble fractions of fibroblasts at passage number 38 ( a ) and passage number 50 ( • ) with purified antibodies by affinity chromatography. Aliquots of 0.4 ml of soluble fraction werc incubated with 0.1 ml of a solution containing the indicated volume of purified antibodies. After overnight incubation and centrifugation, glutathione peroxidase was assayed in the supernatant. The control ( • ) contains 0.4 ml of soluble fraction with 0.1 ml of purified rabbit antibodies anti-mouse IgG Each point is the mean of 3 assays.

99 Other qualitative modifications have been found associated with ageing. Slight alterations of enzyme structure can be reflected in the changes of K m values toward the substrate. We compared in Table I the K m values for tert-butylhydroperoxide of glutathione peroxidase present in soluble fractions of young and old fibroblasts and of the purified enzyme. There is no significant difference between the three samples. More global qualitative changes of the enzyme structure can also be obtained by comparing the enzyme activity with the number of antigenic determinants in an immunotitration curve. Many altered enzymes in ageing cells or animals have been detected in this way [18--25]. A soluble fraction of young or old cells were diluted in order to contain the same glutathione peroxidase activity. Then increasing amounts of purified antibodies directed against the enzyme were added to these two preparations and after one night, the preparations were centrifuged and the activity of glutathione peroxidase recorded. Controls were performed using purified rabbit anti mouse antibodies. The immunotitration curves presented in Fig. 3 show that no difference exists between the inactivation of the enzyme of young or old cells by the antibodies. The experiment was repeated twice with the same results. Another sensitive technique used to detect alteration of enzymes is to follow its rate of inactivation at high temperatures. Somville et al, [23], Houben et al. [26,27] and Mbemba Fundu et al. [28] successfully used this technique to investigate the nature of the alteration of superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase during cellular ageing. We then performed a series of inactivation

100

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Time (rain)

Fig. 4. Evolution of the glutathione peroxidaseactivity with incubation time at 45 °C of a soluble fraction of young fibroblasts. The same soluble fraction was divided into 2 parts. One was freezed during 1 week at -70"C before the incubation at 45"C ( • ). The other was incubated immediatelywithout freezing ( [] ). Each point is the mean of 3 assays.

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Fig. 5. Residual activity of glutathione peroxidase after a 30-min incubation at 45°C with different protein concentrations. The same soluble fraction was divided in aliquots. Ovalbumin was then added at various concentrations. Protein content of the soluble fraction alone was 0.8 mg/ml. Aliquots were then divided in 2 parts. One was incubated at 45"C and the other maintained at 4°C. The results were calculated in regard the sample maintained at 4*C. Each point is the mean of 3 assays,

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Time (rain) Fig. 6. Evolution of the glutathione peroxidase activity with incubation time at 45 *C of a soluble fraction of fibroblasts divided in 5 parts. One part was diluted 1.7-fold (X,e), another was not diluted (d) and in the three others purified enzyme was added in order to obtain a glutathione peroxidase activity of: a: 21.4 U.E./ml; b: 17.5 U.E./ml; c: 11.9 U.E./ml. d: 7 U.E./ml; e: 4.1 U.E./ml.

101

curves by heating a young soluble preparation at various temperatures for 30 min and assayed for the residual activity. The enzyme inactivated between 40 and 50"C but with a large variability from one experiment to the other. We then decided to use 45°C as the heating temperature and to study the parameters influencing the inactivation rate. First, the freezing of the sample was found to increase the inactivation rate of the preparation as exemplified in Fig. 4. Since we also observed that dilution of the preparation changed the behaviour of the enzyme, we tested the in-

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Time (rain.) Fig. 7. Evolution of the glutathione peroxidase activity with incubation time at 45 °C of soluble fractions from fibroblasts at 58 (a), 74 (b), 87 (c) and 89 (d) % of their lifespan completed. These fractions were not freezed and their initial activity was identical at 7 U.E./ml.

102

fluence of the total protein content by adding increasing amounts of ovalbumin to a soluble preparation of young cells containing 0.8 mg protem/ml. The residual activity of glutathione peroxidase was then measured after 30 rain incubation at 45 °('. The results given in Fig. 5 clearly show that there is no intluence of ovalbumm on the enzyme stability. Yet another factor that could influence the behaviour of the enzyme is the concentration of the enzyme itself. This was verified by an experiment using a soluble preparation of young cells containing 0.8 mg/ml of protein and 7 units of glutathione peroxidase/ml. The preparation was divided in 5 parts: in three of them purilied glutathione peroxidase was added in order to obtain 11.9, 17.5 and 21.4 U.E./ml. The fourth part was not changed and the last one was diluted 1.7 times. The inactivation curves of these five samples are presented in Fig. 6. They clearly show that, the higher the initial glutathione peroxidase content, the higher the thermostability of the enzyme. It seems that the concentration of the enzyme plays a major role for the resistance of the enzyme to heat inactivation. Based on those observations, inactivation curves were performed for enzyme obtained for preparations of cells at various stages of their in vitro ageing but using the same initial glutathione peroxidase activity. Four of these inactivation curves are compared in Fig. 7. The rate of inactivation of the enzyme at 45°C is strongly dependent of the age of the cell preparation. It is very slow in young cells but dramatically increases in old cell preparations. This higher thermosensitivity of the enzyme seems also a continuous process extending from cells with 58"/,, of their lifespan completed until the latest subcultivations. DISCUSSION Glutathione peroxidase activity increased in cells cultivated with selenium added to the medium. Neve et al. [29] demonstrated that a correlation exists between glutathione peroxidase activity and selenium concentration when selenium is the limiting factor. Culture medium is probably deficient for selenium which is not unexpected since the selenium content in the 10% fetal bovine serum is very low. The evolution of glutathione peroxidase with ageing in the three experiments shows that the activity of the enzyme does not decrease but rather slightly increases when reported to the protein content (Fig. 2). If G S H content is also maintained, this would suggest a very good protection of the cells during all their passages in culture. Other studies have compared the enzyme content of young and old cells or animals [ 3 0 4 1 ] . The results are however very different with increased, decreased or unchanged activities and do not allow any correlation between ageing and the glutathione peroxidase content. These results have to be reinvestigated in conditions where selenium is not a limiting factor so that perhaps new conclusions could be obtained. In our experiments, the evolution of the enzyme activity in the basic medium was also parallel to the evolution with supplementation of selenium so that selenium

103

incorporation into the enzyme does not seem to be affected by ageing. The qualitative approaches of the enzyme modification using comparison of the enzyme K m or the inactivation by specific antibodies were negative so that neither subtle nor strong modifications of the tertiary structure of the enzyme seems to take place in the enzyme of old cells. Factors affecting heat inactivation curves

However, the rates of heat inactivation of the enzyme preparations were first affected by factors affecting the preparation. We first observed that freezing destabilized the enzyme. Also the concentration of the preparation seems to play a crucial role since diluted preparations always inactivated faster than concentrated one. Several experiments were performed in order to display the factors responsible for this behaviour. Two of them are shown in this paper (Figs. 5 and 6). In other experiments (not shown), the enzyme was diluted but the protein concentration was kept constant and the inactivation rate also increased with the dilution of the enzyme. The conclusions of all these experiments is that the initial enzyme concentration determines the rate of inactivation especially if the enzyme concentration falls below a value of about 10 U.E./ml. This behaviour is not characteristic for glutathione peroxidase alone. The same observation was made for AMP deaminase [42] and lipoprotein lipase [43]. The explanation of such a property is the equilibrium existing in multimeric enzyme between oligomeric and monomeric structures [44 49]. McHevily and Harrisson [50] clearly showed for high dilutions of citrate synthase, a dissociation of the dimeric enzyme into monomers. According to the laws of equilibrium, one can understand that dilution favours the dissociation of oligomeric structures in equilibrium, for example tetramers into dimers and finally monomers. As glutathione peroxidase is a tetrameric enzyme, we hypothesize that, like for the other multimeric enzymes, an equilibrium exists which is affected by high dilutions. The dissociation of the enzyme would then be responsible for the increased rate of inactivation. With ageing, the inactivation rate of glutathione peroxidase increases and parallels the evolution of the inactivation rate of diluted enzyme. Ageing seems to provoke a dissociation of the tetrameric enzyme. However, as all the inactivation curves of Fig. 7 were obtained from preparations with the same initial activity, this hypothesis means that in aged cells, the equilibrium constant between the tetramers and the dimers is modified in such a way that given the same enzyme concentration, the dissociation is higher for 'old' than for 'young' enzymes. Ageing of cells would cause an increase in the equilibrium constant between tetramers and dimers of glutathione peroxidase. Model for inactivation curve

Another set of information can be obtained by the analysis of the kinetic of inactivation as exemplified on the semi-logarithmic curves of Figs. 6 and 7. In this presenation, a first order inactivation rate would give a straight line. This is observed

IO4 for the very low denaturation rate but not for the other experiments which give sigmo'l'd or convex curves. This complex behaviour can be included into the model proposed by Sadana and Henley [51] which proposes the inactivation of the enzyme as a consecutive inactivation E -- El -- E2 with the two following characteristics at the initial stage, the enzyme is only in the first form E and the final state E2 is not necessarily inactive. The mathematical equation which describes the evolution of the activity with time is the following:

E

kl

al

k2

a2

--

El

~

E2

Y(t) = [1 + al.kl/(k2 - kl) - a2.k2/(k2 - kl)]. exp(-kl.t) - [kl/(k2 - kl)]. (al - a2) . exp(-k2.t) + a2 with al and a2 being respectively the ratios of the specific activity of El and E2 to E. By computer simulation (Excel for Macintosh), we studied the impact of al, a2, kl and k2 on the configuration of the curves derived from the equation. We first limited the values o f a l around 1. The reasons for this simplification are the following: as the process represents an enzymatic inactivation, it is unlikely that al values would be above 1. al is the ratio El/E; a value superior than 1 would mean an activation of the enzyme. This has been observed for glucose-6-phosphate dehydrogenase for example [28] with activities higher than 100% for the first minute of incubation at high temperature. We never observed this phenomenon for glutathione peroxidase, On the other hand, by the ~imulation experiment, we found that it was possible to describe all the experimental results by keeping the a l value around 1. For values greater than 1.5, or smaller than 0.9, it was not any more possible to obtain all the configurations of the experimental curves by variations of the three other parameters. According to the principle which consists in searching for the simpliest explanation which accounts for all the experimental observations, we chose the value of a l equal to 1. Using this simplification, experimental curves were analysed by simulation. The passage from curve d to e of Fig. 6 and from the curves b to c and to d of Fig. 7 could be obtained only by decreasing the value of a2 while increasing k l and k2. The evolution of the other curves with dilution or with ageing could all be described by the same decrease of a2 and increase of the kinetic constant kl and k2 even if in this case other evolution of the parameters could also include the experimental values. Since dilution of the enzyme acts on its thermolability by a unique and gradual mechanism, we propose that the evolution of the values of parameters also evolves gradually along with the dilution. The dilution would then result in a decrease of a2 and an increase of kl and k2. Ageing of the cell would also lead to the same results.

105

General model taking into account the dissociation The first biological interpretation of these results is that glutathione peroxidase would inactivate following a consecutive process from a native form to a final form which is not necessarily inactive, and passing through an intermediate with a specific activity close or equal to that of the native form. Secondly, a decrease of a2 with dilution or with ageing means a decrease in the specific activity of the final form (E2). Biochemically, it is not possible to consider that a unique enzymatic form (E2) could possess different intrinsic specific activity. The continuous changes of a2 would mean a better continuous structural modification. Since we showed that dilution influences the equilibrium between the tetramers and the dimers we decided to introduce this information in the model and to consider E, E1 and E2 as representing structural evolution of both tetramers and dimers and not as unique molecular entity. The model of Sadana and Henley [51] does not take into account the effect of dilution on the inactivation. So, we completed this model to account for the results and we propose the following model: General model: kl E ~

al E1

k2 --

a2 E2

Detailed mechanism for glutathione peroxidase

Tetramer lI 2 Dimers

kiT alT -- interm, active prot. kiD aiD -- 2 interm, active prot.

k2T a2T -- active prot. k2D a2D -- 2 inactive prot.

kl would be a combination of k i T and k i D , k2 would be a combination of k2T and k2D, al would be a combination of a l T and a i D , a2 would be a combination of a2T and a2D. Different proportions of tetramers and dimers would lead to different values of the global parameters al, a2, kl and k2. At high dilution, the final activity would tend to zero (Fig. 6e) suggesting a value of zero for a2D. Modelisation of experimental data does not prove that the model is correct but that it is possible. In order to test this model, we simulated the evolution of the rate of inactivation for a mixture of tetramer and dimer and compared the slope of the curve with experimental data. At high enzyme concentration, the tetrameric enzyme would be the main active form and the curve like Fig, 6a can be described for example with a l T = 1,a2T = 0.2, k i T = 0.05, k2T = 0.055 (Fig. 8A). At high dilution, however, the dimeric form is dominant and curve like Fig. 6e can be described for example with a i D = 1, a2D = 0, k i D = 0.6, k2D = 0.65 (Fig. 8B). If now, we consider a preparation containing both tetrameric and dimeric enzymes characterized by their own parameters described

106

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Fig. 8. Theoritical inactivation curves obtained by calculation from the equation of Sadana and Henley. Values of the parameters: curve A: al = I; a2 = 0.2: kl = 0.05; k2 = 0.055: curve B: al = 1; a2 = 0: kl = 0.6:k2 = 0.65" curve C was drawn with parameters being the average of curve A and B : al = 1:a2 = 0.1: kl = 0.325:k2 = 0.352.

here a b o v e a n d mixed in such a w a y that each form p a r t i c i p a t e s for 50% to the global p a r a m e t e r s (al = 1, a2 = 0.1, kl = 0.325, k2 = 0.352) then the curve shown in Fig. 8C is o b t a i n e d . T h e c o n f i g u r a t i o n o f this theoritical curve can be f a v o r a b l y comp a r e d to the e x p e r i m e n t a l curves o f Fig. 6d, 7b a n d c a n d this similarity is a g o o d a r g u m e n t for the validity o f the model. In this simulation, we did n o t t a k e into a c c o u n t the d y n a m i c e q u i l i b r i u m between t e t r a m e r s and dimers which can be also m o d i f i e d d u r i n g the 30 min o f heating especially because the i n a c t i v a t i o n o f the d i m e r is so drastic. This e q u i l i b r i u m does n o t q u e s t i o n the m o d e l b u t d o e s n o t allow the q u a n t i f i c a t i o n o f the t e t r a m e r s and d i m e r s in the initial p r e p a r a t i o n since this p r o p o r t i o n can v a r y with time, if the rate o f d i s s o c i a t i o n o f the t e t r a m e r s into d i m e r s is significant c o m p a r e d to the rate o f inactivation. A n y o t h e r simplification o f the m o d e l c o u l d not be m a d e w i t h o u t losing the c o r r e s p o n d e n c e with the e x p e r i m e n t a l data. T h e m e c h a n i s m b e h i n d the higher d i s s o c i a t i o n c o n s t a n t

o f the tetrameric

g l u t a t h i o n e p e r o x i d a s e in the old cells is n o t k n o w n . It c o u l d result from a modification o f one or m o r e a m i n o acids or the fixation o f a p a r t i c u l a r m e t a b o l i t e affecting the a s s o c i a t i o n o f the subunits. T h e b i o l o g i c a l i m p l i c a t i o n o f the m o d i f i c a t i o n in the q u a t e r n a r y structure o f such e n z y m e is also very intriguing since m a n y o t h e r enzymes have a l r e a d y been shown to be affected in the same respect, with m o d i f i c a t i o n o f the d i s s o c i a t i o n o f the t e t r a m e r i c g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e [26,27] or dimeric s u p e r o x i d e d i s m u t a s e

[521. Such subtle m o d i f i c a t i o n s affecting q u a t e r n a r y

107

enzyme structure have perhaps physiological implications for ageing and studies of more polymeric enzymes would be required before such implication could be assessed in the complex ageing process. These modifications could represent an evolution in the decreasing ability of the old cells to cope with their environment. REFERENCES 1 2 3 4

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