phgpx and phospholipase a2/gpx: comparative importance on the reduction of hydroperoxides in rat...

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Free Radical Biology & Medicine, Vol. 19, No. 5, pp. 669-677, 1995 Copyright (~3 1995 Elsevier Science Inc. Printed in the USA. All rights reserved

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Brief Communication

PHGPx AND PHOSPHOLIPASE AJGPx: COMPARATIVE IMPORTANCE ON THE REDUCTION OF HYDROPEROXIDES

IN RAT LIVER MITOCHONDRIA

FERNANDO ANTUNES,* ARMINDO SALVADOR,* and RUY E. PINTO *t

*Grupo de Bioquimica e Biologia Te6ricas, lnstituto de lnvestiga@,o Cientiflca Bento da Rocha Cabral, Lisboa, Portugal; and *Departamento de Quimica e Bioquimica da Faculdade de Ci~ncias da Universidade de Lisboa,

R. Ernesto de Vasconcelos, Lisboa, Portugal

(Received 12 January 1995; Accepted 27 February 1995)

Abst ract The comparative importance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) and of "classic" glutathione peroxidase (GPx) in the reduction of phospholipid hydroperoxides is unclear. Although GPx activity is 500-fold higher than that of PHGPx in rat liver, ~ the reduction of phospholipid hydroperoxides by glutathione (GSH) through GPx may be strongly limited by a low PLA2 activity. We address this issue using a moderately detailed kinetic model of mitochondrial lipid peroxidation in rat liver. The model was based on published data and was subjected to validation as reported in the references.-' It is analysed by computer simulation and sensitivity analysis. Results suggest that in rat liver mitochondria PHGPx is responsible for almost all phospholipid hydroperoxide reduction, Under physiological conditions, the estimated flux of phospholipid hydroperoxides reduction through PHGPx is about four orders of magnitude higher than the estimated hydrolysis flux through PLA2. On the other hand, virtually all hydrogen peroxide is reduced through GPx. Therefore, a functional complementarity between PHGPx and GPx is suggested. Because the results are qualitatively robust to changes of several orders of magnitude in PLA2 and PHGPx levels, the conclusions may not be limited to mitochondria.

Keywords---Free radicals, Lipid peroxidation, Kinetic model, Mathematical model, Phospholipid hydroperoxide, Hydrogen peroxide, Glutathione peroxidase, Computer simulation, Antioxidant

INTRODUCTION

The role and compara t ive impor tance o f GPx (E.C. : I .11.1 .9) and P H G P x ( E . C . : I . l l . I . 9 ) on the reduction of phosphol ip id hydroperox ides is mat ter of debate. Tappel 3 tentat ively p roposed that the reduct ion o f phosphol ip id hydroperox ides would require the se- quential action o f P L A 2 (E.C.:3.1.1.4) and GPx. In- deed, several works 4-7 showed that the hydro lys i s is a

requirement for the reduct ion o f phospho l ip id hydroperoxides by G S H via GPx. Based on these and other findings, van Kui jk et al. s p roposed that the consecu- t ive action o f P L A 2 and GPx const i tutes a pa thway for the reduct ion of phosphol ip id hydroperoxides .

On the other hand, PHGPx, a 20 k D a monomer ic se leno-enzyme first identif ied by Ursini et al., 9 is able

Address correspondence to: Fernando Antunes, Grupo de Bioquim- ica e Biologia Te6ricas, lnstituto de InvestigaqAo Cientffica Bento da Rocha Cabral, Cq. Bento da Rocha Cabral, 14, P-1200 Lisboa, Portugal.

to cata lyse the reduct ion of d iacy lphosphol ip id hydroperoxides ~° direct ly. Al though, in rat l iver GPx act ivi ty is about 500-fold h igher than PHGPx activity, ~ the reduct ion rate of phosphol ip id hydroperox ides through GPx may be s t rongly l imited by PLA2 activity. On the other hand, PLA2 is act ivated by peroxidat ion, 4'~'~J

and ox id ised phosphol ip ids are preferent ia l ly hydro lysed by this enzyme. ~2 J6

In this work, we use a modera te ly deta i led kinetic modeP 7 o f l ipid peroxidat ion in inner mi tochondr ia l membranes to compare the impor tance of these two pathways. The model is based on publ ished kinetic data and a part ial val idat ion is repor ted in ref. 2.

The react ions and the enzymes considered in the model are prevalent within cells and the results were qual i ta t ively robust upon changes of several orders of magni tude in the most re levant parameters . Therefore, the conclus ions of this work may also be val id for cel lular membranes other than inner mi tochondr ia l membranes .

669

670 F. ANTUNES et al.

METHODS

Description of the model

In addition to the orthodox reactional scheme of lipid peroxidation, the model (97 reactions) takes into account the following sets of reactions and species (for a detailed description see ref. 17).

1. The various unsaturated fatty esters are explicitly considered. The corresponding radicals were considered to have the same reactivity.

2. Reactive oxygen species: superoxide, perhydroxyl, hydroxyl radicals, and hydrogen peroxide.

3. a-Tocopfierol, ascorbate, and ubiquinone. 4. Superoxide dismutase (E.C.:I.15.1.1), dehydroas-

corbate reductase (E.C.: 1.8.5.1), semidehidroascor- bate reductase (E.C.:1.6.5.4), PLA2, GPx, PHGPx, and glutathione reductase (E.C.:1.6.4.2).

5. "F r e e " iron involved in Fenton-type reactions with hydroperoxides, in the ascorbate oxidation, in the reduction of dioxygen, and so forth.

6. Initiation of lipid peroxidation by perbydroxyl, hydroxyl, and a-tocopheroxyl radicals.

7. Reaction of superoxide, perhydroxyl, and hydroxyl radicals with proteins and DNA.

Whenever several experimental measurements for a kinetic parameter were available in the literature, the determinations that most closely approached the physiological conditions were chosen.

A lipid and an aqueous compartment, each with a characteristic composition and reactivity, were considered. All lipid species, a-tocopherol, ubiquinone, and PLA2 are present only in the lipid phase. Perhydroxyl radical and dioxygen were considered to distribute in- stantaneously between the two compartments, with partition coefficients (lipid to aqueous phase) of 1 and 3 (ref. 18), respectively. All other species were assumed to be present only in the aqueous phase. Some interfacial reactions were allowed. Unless otherwise stated, local concentrations and rates are referred to the compartment where the species is present.

Estimation of the main parameters

For the problem under study, the reactions in Table 1 are the most important. Concentrations and kinetic parameters for the enzymes appearing in Table 1 were estimated as follows.

Modelling the action of PLA2 is difficult. De Winter et al. 2° isolated PLA2 from rat liver mitochondria but data about kinetics or physiological enzyme concentration are lacking. Therefore, we assumed a rate law as k × [phospholipid]. The pseudo-first-order rate con-

stant, k, aggregates the information about the turnover number and the enzyme concentration, and was estimated from PLA2 specific activity, which can be measured either with endogenous or with exogenous substrate. For the current purpose, the first alternative is preferable because it represents the physiological flux better. The specific activity of the enzyme, measured with endogenous phosphatidylethanolamine, is 0.4 nmol fatty acid released/min/mg mitochondrial protein. zt A similar value--0 .53 nmol fatty acid released/ min/mg mitochondrial prote in--was obtained in inner mitochondrial membranes by measuring the release of polyunsaturated fatty acids. 2z

We estimated k from the value determined in ref 21. To do that, this activity had to be first converted to a flux with units referred to the lipid phase. The value 47 /zMs -~ was obtained considering that: (a) PLA2 activity distribution between inner and outer mitochondrial membranes is similar to total protein distribution (i.e., 84% of membrane protein is in the inner membrane)2~; (b) 21% of mitochondrial protein is present in the inner membrane23; (c) the concentration of P-containing lipid is 0.34 mol/mg inner membrane pro- tein24; and (d) phospholipid concentration in the inner membrane is 0.6 M (ref. 17).

From the relative activities of PLA2 toward different phospholipids, zs an average flux of 31 #Ms-L was calculated. This value is achieved upon full activation by Ca 2~ . For phospfiatidylethanolanline, the kinetics of activation is of Henri-Micfiaelis-Menten type with half-activation obtained at 20 /zM Ca2+. 2j Therefore, at a physiological mitochondrial concentration of 16 #M free Ca 2+,26 a flux around 14 #Ms-~ was estimated, by assuming that the hydrolysis of the other phospholipids follows the same kinetics of activation by Ca 2+ as that of phosphatidylethanolamine.

Considering a phospholipid concentration of 0.6 M in the inner membrane, t7 we roughly estimated the pseudo-first-order rate constant as 2.3 x 10 5 s-~. With this value, and by assuming that the fatty acids turnover is mainly determined by PLA2, a half-life of 8.4 h is estimated for fatty acids in inner mitochondrial membranes. This value is in the range of experimental determinations, 2v and so our estimate for k seems accurate at the order of magnitude level.

Owing to lack of quantitative-adequate data, PLA2 activation by lipid peroxidation was not taken into con- sideration. Therefore, a rate constant of 2.3 X 10 -5 s--' was assumed for the hydrolysis of oxidised phospholipids (reactions 1 and 2 in Table 1). The error associated with this assumption is probably less than one order of magnitude (see discussion section).

Because mitochondria do not contain acyl-CoA: 1-

Reduct ion of hydroperoxides

Table 1. React ions Invo lv ing PLA2, GPx, and P H G P x Considered in the Model

671

N u m b e r React ion Kinetic Paramete r Reference

PI-A2

1 P - L O O H ---' L O O H k~ = 2.3 × 1 0 5 s ~ see text

2 P -LOH ~ L O H k_, = 2.3 × 10 ~ s t see text 3 L O O H + GPx,~ + H + ~ GPx,, + L O H k3 = 2.1 × 107 M ~ s ~ ref. 19 4 GPxo + G S H ~ G S G P x + HaO k4 = 4 × l04 M L s ~ Est imated f rom ref. 19 5 G S G P x + G S H - - , G P x ~ a + G S S G + H + k5 = 1 × 10 v M ~ s ~ Est imated f r o m r e f . 19 6 H202 + GPx~j + H+ ~ GPxo + H20 I% = 2.1 × 10 v M ~ s t Est imated f rom ref. 19 7 L O O H + PHGPx~a + H + ~ PHGPx,, + L O H ks = 3 × 10 7 M t s * ref. 10 8 P - L O O H + P H G P x ~ + H ÷ --' PHGPx,, + P -LOH k8 = 1.0 × l 0 7 M * s * ref. 10 9 PHGPx,, + G S H ~ G S P H G P x + H~O k,~ = 1 × 105 M- * s * Est imated f rom ref. 10

10 G S P H G P x + G S H ~ P H G P x ~ + G S S G + H + k,~ = 1 × 107 M '~ s ~ Est imated f rom ref. 10 11 H20~_ + PHGPx~d + H +--* PHGPx,, + H2 0 k** = 3.2 × 106 M -f s -* ref. 10

For the comple te list of reactions see ref. 17.

acylphospholipid acyltransferase activity nor an acylation-deacylation cycleY the model does not take into account the deacylation-reacylation cycle of fatty acids.

More data is available for peroxidases. The kinetics of GPx can be described 19'29"3° as

- - - + - - ( 1 )

v [LOOH] [GSH]'

where E is the enzyme, v is the reaction rate, and LOOH is the hydroperoxide. These and other experimental data led Floh63~ to propose reactions 3 - 5 in Table I as a minimal model for catalysis. ~bj is related to the reaction rate of the reduced form of the enzyme with the hydroperoxide (~b~ = l/k3), while ~b2 is related to the rate of reactions 4 and 5 (qbz = l/k4 + 1/ks). 3~ In the model this mechanism was considered explicitly. k4 and k5 were estimated based on kinetic studies 3z suggesting k5 "> k4 and, therefore, k4 ~ 1/4~2. The values in Table 1 were calculated from the experimental values for ~b~ and ~b2 for rat liver enzyme, using linoleic acid hydroperoxide as substrate] 9 Reaction 3 was assumed to occur in membrane-aqueous interface.

Kinetic studies "~ support that PHGPx has the same mechanism as GPx. The kinetic parameters were calculated from experimental values for ~b~ and ~b2. t° Reac- tions 7 and 8 were assumed to occur in membrane- aqueous interface.

Total concentrations for both GPx and PHGPx (reduced plus oxidised enzyme plus enzyme adduct with GSH) were considered constants. GPx concentration was estimated from the following data: specific activity in mitochondria, 0.15 U/mg protein33; specific activity for the purified enzyme, 278 U/mg protein33; molecular weight, 76 kDa. 33 Assuming a concentration of 1 mg mitochondrial protein//A osmotically active matrix (es-

timated from data in ref. 34), we estimated the total concentration for GPx in mitochondria as 6.7/~M.

Only an indirect estimate for total PHGPx concentration could be obtained. In adult rat liver, GPx activity for hydrogen peroxide is about 500 times higher than PHGPx activity for phosphatidylcholine hydro- peroxideJ This activity was measured for a cellular fraction that excludes mitochondria and damaged cells, and a GSH concentration about 1000 times higher than hydroperoxide concentration was used. Because 13% of PHGPx (as in pig liver ~5) and 18% of GPx 33 activities are located in mitochondria, the above ratio of activities is representative for these organelles. Using the values obtained for k6, k8, and [GPx]tot, we roughly estimated the total concentration for PHGPx in mitochondria as 0.03/~M.

The activities of both peroxidases are proportional to the above estimated concentrations, because these concentrations reflect not the total quantity of protein but only the amount that is active, that is, able to catalyse reactions.

Setting up and analysis of the model

Setting up of the model, implementation of the system of ordinary differential equations, simulations, and analysis were carried out using PARSYS, 37 a set of Mathematica 38 and C programs developed in our labo- ratory. The system of differential equations was nu- merically integrated by using algorithm LSODA, which is able to switch between a nonstiff (Adams's) and a stiff (Gear's) method according to the behaviour of the integration process. 39 (For methodological aspects about kinetic modelling of biochemical processes see ref. 40.)

For any tested set of plausible initial concentrations, the simulations always converge to the same physio-

672 F. ANTUNES e t al.

logically meaningful steady state. Except for glutathione, which was assumed to have a half-life of approximately 30 h , 41 all other variables of the system relax to this steady state within a timescale of seconds or faster. For mathematical convenience we assumed that the physiological system stays in steady state.

Sensitivity analysis in the framework of Biochemi- cal Systems Theory 42-44 and simulations indicate PLA2, GPx, and PHGPx levels as the most influential parameters for the problem under study. Therefore, we studied how these parameters affect the steady state.

R E S UL T S

Simple calculations

The comparative importance of PHGPx and PLA2/ GPx in the reduction of phospholipid hydroperoxides can be estimated by simple calculations, without doing any simulation. The reduction flux of these hydroperoxides through PHGPx is approximately given by the steady- state rate expression [1]. The flux through GPx is not higher than the flux of PLA2-catalysed hydrolysis. In Figure 1 these two fluxes are plotted for a wide range of phospholipid hydroperoxide concentrations. For plausible concentrations (i.e., less than 1% of total phospholipids), the reduction flux through PHGPx is about 2-4 orders of magnitude higher than that of through GPx. Only for nonphysiological very high concentrations do the two fluxes approach similar values.

The reduction flux of phospholipid hydroperoxides through PHGPx and the hydrolisis flux of these species through PLA2 depend differently on the concentration

1E-04

1E-05

-t- O 1E-06 o -t, 1E-07 a.

"~ 1E-08 c- O

1 E-09

"o 1E-10 r,,

1E-11 . . . . . . . . I . . . . . . . . I = ~ . . . . . . . . I . . . . . . . . I . . . . . . .

1E-6 1E-5 1E-4 1E-3 1E-2 1E-1 1E0

Fraction of P-LOOH

Fig. 1. Reduction of membrane lipid phospholipids by GSH through PHGPx and GPx. Curve 1, reduction flux through PHGPx; calculated from the steady-state rate expression [ll, with 11 mM GSH, 3~ ~bl (1 x 10 7 Ms) and ~b2 (1 X 10 -5 Ms) for diacylphospholipid mono- hydroperoxides, ~° and a PHGPx concentration of 3 X 10 -8 M Curve 2, reduction flux through GPx; estimated from the hydrolysis flux catalysed by PLA2 (k x [P-LOOH] with k = 2.3 x 10 -5 Ms -I, see methods section); P-LOOH, phospholipid hydroperoxide.

of phospholipid hydroperoxides (Fig. I ). The curve for the reduction flux arises from the saturation of PHGPx for high hydroperoxide concentrations. The curve for the hydrolysis does not present this behaviour because the concentration of substrate (a phospholipid) for PLA2 is independent of the oxidised phospholipid fraction, if we discard the different molecular surface areas for nonoxidised and oxidised phospholipids, j4 There- fore, the relationship between the hydrolysis flux of oxidised phospholipid and the fraction of phospholipid oxidised is linear.

These simple calculations have intrinsic limitations. They were done only for the estimated PLA2 and PHGPx physiological levels, which are rough esti- mates. The effect of changing these parameters is not known. More importantly, these calculations do not consider the compartmentation or the nonlinear dy- namics of lipid peroxidation. For example, when the concentration of phospholipid hydroperoxides increases, they will catalyse its own production. More- over, the effect of competition of peroxidases for two substrates, hydrogen and lipid hydroperoxide, is not known. The results can thus be different, even at the qualitative level. Therefore, it is important to carry out simulation studies, which test the robustness of the results and bring more insight to the detoxification mechanism of hydroperoxides because they integrate all the main processes of lipid peroxidation.

Simulation results

At the reference steady state the following results were obtained from simulation. Rates of phospholipid hydroperoxide production, initiation of lipid peroxidation, and production of hydrogen peroxide were 1.3 x 10 -7 Ms -~ (referred to as lipid phase); 1.3 x 10 -8 Ms -~ (referred to as lipid phase); and 8 x 10 -6 Ms -I (referred to as aqueous phase), respectively. Steady- state concentrations of phospholipid hydroperoxides and hydrogen peroxide were 4.2 x 10 -7 M (lipid phase) and 5.7 x 10 8 M (aqueous phase), respectively. PHGPx catalysed the reduction of virtually all membrane lipid (phospholipid plus fatty acid) hydroperoxides produced. Only about 0.01% of these hydroperoxides were decomposed into radicals or underwent PLA2-catalysed hydrolysis followed by GPx-catalysed reduction, On the other hand, GPx catalysed the reduction of almost all hydrogen peroxide produced while only about 0.07% was reduced through PHGPx and decomposed to radicals.

Figure 2 shows the effect of PHGPx and GPx concentrations on the hydrogen peroxide and membrane lipid hydroperoxides steady-state concentrations.

Reduction of hydroperoxides 673

~" 1E-2

~. 1E-3

t~' 1E-.4

1E-5

'~' 1E-6

x 1E-7' ,,9. ~. 1E-8

1E_9I ~ IE 10 L

1E-11 1E-10

~" 1 E-04

. l _0s I

~"~ 1E-07 i

• I 1 E-09 Q, 0

J 1E-9 1E-8 10E-8 10E-7 10E-6 10E-5

[PHGPx]tot (M)

b

1

3

,~ 1E-10

~1E-11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1E-9 1E-8 1E-7 1E- .6 1E-5 1E-4 1E-3 1E-2

[GPx]tot (M)

Fig. 2. Effect of PHGPx (a) and GPx (b) concentrations on the steady-state concentration of hydroperoxides and on the rate of lipid peroxidation (Rp). All other parameters in the simulation are kept constant. The vertical line indicates the estimated physiological concentrations of PHGPx and GPx. The activities of these enzymes are proportional to these concentrations (see methods section), which include the reduced and oxidised form of the enzyme, and the enzyme adduct with GSH. Curve 1, membrane lipid hydroperoxides; curve 2, hydrogen peroxide; curve 3, Rp.

hydroperoxides through PHGPx and GPx. At a suffi- ciently high PLA2 level, the reduction flux through GPx is about two orders of magnitude higher than that through PHGPx (Fig 3, curve 1), which is in agreement with a higher GPx activity ~ when compared with PHGPx activity. However, at the reference steady state, PLA2, in competition with PHGPx, was able to hydrolyse less than 0.01% of phospholipid hydroperoxides. Therefore, the action of GPx on these hydroperoxides was strongly limited. Moreover, to allow an equal contribution of GPx and PHGPx to the reduction of membrane lipid hydroperoxides it would be necessary to increase the PLA2 level by about four orders of magnitude (Fig. 3, curve 1). Even if it was assumed that the PHGPx concentration was overestimated by two orders of magnitude, it would be necessary to increase the PLA2 level by two orders of magnitude (Fig. 3, curve 2).

1 E6

#. o 1E4

'~ 1E2

"5 n, lEO

t, t~ IE-2

"~ 1E.-4. n,,,

1E-6 1E-S

a

1E-4 1E-3 1E-2 1E-1 lEO 1E1 1E2 1E3 k {S "1)

PHGPx effectively controlled the concentration of membrane lipid hydroperoxides (Fig. 2a, curve 1), but had almost no control over the hydrogen peroxide concentration (Fig. 2a, curve 2). The opposite happened with GPx (Fig. 2b, curves 1 and 2).

The sharp accumulation of membrane lipid hydroperoxides observed at low PHGPx concentrations (Fig. 2a, curve 1), further suggests a key role for this enzyme in the protection against lipid peroxidation. This accumulation is coincident both with the saturation of PHGPx (almost all enzyme becomes oxidised) and with an increase of the peroxidation rate. Moreover, when the initiation rate of lipid peroxidation or the iron concentration was increased, a higher PHGPx level was required for protection of the system (not shown). On the other hand, GPx had almost no effect on the peroxidation rate (Fig. 2b, curve 3).

Figure 3 shows the effect of increased PLA2 levels (characterised by the pseudo-first-order rate constant k; see methods section) on the reduction of phospholipid

x 1.0 G.

"~ 0.8 'o

i 0,6

Z o o (1.4 e¢

~ 0.2

u. 0.0 1E-5

b

? 1 _ _

1E-4 1E-3 1E-2 1E-1 lEO 1E1

k (s "1)

Fig. 3. Effect of the pseudo-first-order rate constant k, which character- ises the PLA2 level (see methods section), on the reduction of membrane lipid hydroperoxides (ROOH) through PLA2/GPx and PHGPx. (a) Ratio between the reduction of membrane lipid hydroperoxides through GPx and through PHGPx (log-log plot). (b) Fraction of membrane lipid hydroperoxides reduced through GPx - reduction through GPx over reduction through GPx plus PHGPx - (normal-log plot). Two different PHGPx concentrations are studied: curve 1, 3 x 10 -~ M (estimated physiological concentration); curve 2, 3 × 10 ~o M. The vertical lines indicate the estimated physiological level of PLA2. The horizontal lines indicate equal contributions of GPx and PHGPx for the reduction of membrane lipid hydroperoxides.

674 F. AN'rUNES et al.

10E-6~

10E-7~

10E-8~_

11::-5 1E-4 1E-3 1E-2 1E-1 lEO 1E1 k Is'b

Fig. 4. Combinations of PHGPx and PLA2 levels that allow equal reduction rates of membrane lipid hydroperoxides through PHGPx and GPx, for four initiation rates of lipid peroxidation (Ri). k as in Fig. 3. Curve 1, Ri = 1.3 x 10 8 Ms-~ (estimated physiological rate); curve 2, Ri = 1.3 x 10 7 Ms ~; curve 3, Ri = 1.3 x 10 _6 Ms ~; curve 4, Ri = 1.3 X 10 5 Ms ~. The vertical and horizontal lines show the physiological estimated PLA2 and PHGPx levels, respectively.

To further study the robustness of the results, the combinations of PHGPx and PLA2 levels that, according to our model, allowed an equivalent reduction of membrane lipid hydroperoxides through GPx and PHGPx are plotted in Figure 4. This was done for four initiation rates of lipid peroxidation, to model more oxidative conditions. The concentration of membrane lipid hydroperoxides increases along each of the curves from high to low PHGPX and PLA2 levels, and only combinations for which oxidised phospholipids were below 0.3% of nonoxidised phospholipid are plotted. Therefore, for all the initiation rates modelled, an effi- cient protection against accumulation of membrane lipid hydroperoxides and an equal or superior partici- pation of GPx in their reduction, was simultaneously possible only when the PLA2 level was several orders of magnitude higher than our estimate.

DISCUSSION

Membrane lipid hydroperoxides were efficiently reduced by GSH because less than 0.01% were decomposed into radicals. PHGPx was the main enzyme involved in this detoxification (Figs. 1 and 2). When its level was below a certain value, membrane lipid hydroperoxides accumulated (Fig. 2a, curve 1), a higher proportion of these species was decomposed into radicals that feed the propagation cycle of lipid peroxidation, and consequently, the rate of lipid peroxidation increased (Fig. 2a, curve 3).

The key role of PHGPx in the reduction of membrane lipid hydroperoxides was a robust result. It was

not only observed for a particular set of parameters but for a wide range of PLA2 and PHGPx levels (Figs. 3 and 4), whose estimations were the least accurate from all the relevant parameters for the issue under study. Furthermore, it was also observed under situa- tions of strong oxidative stress (Fig. 4).

On the other hand, in liver mitochondria, which lack catalase, 45 GPx has an essential role in the reduction of hydrogen peroxide (as shown in Fig. 2b, curve 2) due to its higher activity when compared with that of PHGPx.J In addition, under conditions where the initiation of lipid peroxidation by the reactive species formed from hydrogen peroxide is significant, GPx is probably more important in the defence against pri- mary initiation of lipid peroxidation than in the reduction of membrane lipid hydroperoxides, because the latter action was strongly limited by PLA2 (Fig. 3).

The estimated physiological flux of hydrolysis of total phospholipids catalysed by PLA2 was about 14 /zM s-~ (see methods section), while the physiological flux through PHGPx, which was similar to the rate of lipid peroxidation, was about two orders of magnitude lower (0.13 #M s-~, estimated from simulation). Be- cause phospholipid hydroperoxides were 4.2 x 10 7 M/0.6 M = 7.0 x 10 5% of total phospholipids (estimated from simulation), its hydrolysis flux through PLA2 was only 9.8 pM s ~, and so the action of GPx on membrane lipid hydroperoxides was strongly limited.

Therefore, for GPx to have an equivalent role to PHGPx in the reduction of membrane lipid hydroperoxides, the hydrolysis flux of phospholipid hydroperoxides through PLA2 had to be increased by about four orders of magnitude (Figs. 3 and 4). PLA2 activation by lipid peroxidation and the higher PLA2 activity toward oxidised species, neither of which is considered in this work, are not sufficient to achieve this increase. Indeed, an activation by lipid peroxidation of only 60% for the mitochondrial PLA2 is observed with exogenous nonoxidised substrate.~r For venom PLA2, 2- to 5-fold activation is a typical result, 14 16.46 but an activation as high as 15 times is also observed, r6 A 2- to 4- fold preferential hydrolysis of oxidised over unoxi- dised phospholipids, in the same vesicle, s'~x~s was also reported. Furthermore, PLA2 activity in rat liver mitochondria was recently reported to be concentrated in Kupffer cells, 47 4,~ whereas almost no activity is found in hepatocytes, 49 as partially confirmed in ref. 48.

Due to the weak, 2- to 4-fold, PLA2 specificity toward oxidised phospholipids when compared to nonoxidised phospholipids, s' ~3' J s a four-order of magnitude increase in the hydrolysis flux of phospholipid hydroperoxides would imply a hydrolysis flux of total phos-


pholipids through PLA2 of 104/(2 to 4) × 14 #M s -~. A flux as high as this would represent a challenge for membrane integrity, particularly in mitochondria, which lack acyl-CoA: l-acylphospholipid acyltransferase activity. 28 So, the flux catalysed by PLA2 necessary for GPx to have an important role in the reduction of membrane lipid hydroperoxides, is not only much higher than our estimate, but also potencially danger- ous for cells.

A low ratio between the fluxes of the reactions catalysed by PLA2 and by PHGPx leads to a high steady- state concentration of hydroxydiacylphospholipids. This agrees with experimental observations by Hughes et al. 5° in mice liver. These authors do not detect phospholipid hydroperoxides but do find hydroxyphospho- lipids at levels well above the limit of detection of their method. In simulations, at the reference steady state, the phospholipid hydroperoxide and hydroxy- phospholipid levels were 7 × 10-5% and 0.9% of total phospholipids, respectively. The experimental value observed in rat liver mitochondria (0.45% of phospholipid is oxidised 51) is in the same order of magnitude.

Although both hydroxydiacylphospholipids and diacylphospholipid hydroperoxides induce structural changes in membranes, ~4"52 the latter species are chemi- cally more unstable and can catalyse their own production via decomposition to radicals. Therefore, whereas cellular membranes can accumulate hydroxydiacylphospholipids to some extent, a diacylphospholipid hydroperoxide peroxidase activity becomes critically important in the protection of membranes against lipid peroxidation. Indeed, under strong selenium deficiency, PHGPx activity is less affected than GPx activity) 3 The same happens with the mRNA levels of each one, under moderate selenium deficiency. 54

The only enzyme with diacylphospholipid hydroperoxide peroxidase activity considered in this work was PHGPx. However, the membrane-bound GSH S- transferase 5-s-57 may also play a role, 5~'s9 namely in the microsomal fraction and in the outer mitochondrial membrane where it is concentrated. 6° This activity was not considered owing to lack of suitable data for modelling. Because in rat liver the activity of phospholipid hydroperoxide glutathione peroxidase is modulated by selenium, 53 the role of the membrane-bound GSH S- transferase (which is not a selenium-enzyme) does not seem important in this respect.

In summary, in liver mitochondria PHGPx and GPx probably have a complementary role in the reduction of hydroperoxides: PHGPx reduces mainly membrane lipid hydroperoxides produced in lipid peroxidation while GPx reduces mainly hydrogen peroxide. A similar scheme has also been proposed by Panfili et al. 6r

for brain mitochondria, based on the distribution of these enzymes. It is noteworthy that both enzymes are probably modulated by a common factor; that is, selenium s ta tu s . 153"54"62"63 Because the results are qualitatively robust upon changes of several orders of magnitude in the most relevant parameters, they may be valid for organelles other than rnitochondria.

Acknowledgements - - We are grateful to H. Susana Marinho for helpful discussions and critical review of the manuscript, and to Prof. Fulvio Ursini for providing unpublished data. A. S. and F. A. acknowledge support from Grants PRAXIS XXI-BD/3457/94 and FMRH-BD-399-92/JNICT, respectively, IICBRC and JNICT (FACC) contribute to the support of GBBT.

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ABBREVIATIONS

G P × - - " c l a s s i c " glutathione peroxidase

G S H - - r e d u c e d glutathione G S S G - - o x i d i s e d glutathione

G S X - - a d d u c t of glutathione with X

k- -Pseudo- f i r s t -o rder rate constant characterising the

action of PLA2 in the model

k i - - R a t e constant of reaction i L O H - - h y d r o x y fatty acid

L O O H - - f a t t y acid hydroperoxide

P - L O H - - hydroxylphosphol ip id ( lysophosphol ip ids are not inc luded)

P - L O O H - - p h o s p h o l i p i d hydroperoxide ( lysophosphol ipids are not inc luded)

P H G P × - - p h o s p h o l i p i d hydropero×ide g lu ta th ione peroxidase

P L A 2 - - p h o s p h o l i p a s e A2

R p - - r a t e of l ipid peroxidat ion

R O O H - - m e m b r a n e lipid hydroperoxides (P -LOOH + LOOH)

R i - - i n i t i a t i o n rate of l ipid peroxidat ion X , - - o x i d i s e d form of species X

X r d - - r e d u c e d form of species X

[ X ] t o t - - s u m of the concent ra t ions of all the forms cons idered for species X

phgpx and phospholipase a2/gpx: comparative importance on the reduction of hydroperoxides in rat...

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