Eur. J. Biochem.

84, 377-383 (1978)

A Role of Mitochondrial Glutathione Peroxidase

in Modulating Mitochondrial Oxidations in Liver
Helmut SIES and Karen M. MOSS
Institut fur Physiologische Chemie, Physikalische Biochemie und Zellbiologie der UniversitPt Munchen
(Received November 4, 1977)

1. The inhibitory effect of t-butyl hydroperoxide on 0 2 uptake by perfused rat liver and
by isolated hepatocytes was investigated with isolated mitochondria.
2 . 0 2 uptake by mitochondria oxidizing the ketoacids, 2-oxoglutarate and pyruvate, was substantially decreased upon addition of t-butyl hydroperoxide, that of isocitrate was only slightly decreased,
and that of succinate and of 3-hydroxybutyrate was practically unchanged. The reduction product
of the hydroperoxide, t-butyl alcohol, showed no effect. The inhibitory effect of the hydroperoxide was reversed upon addition of dithioerythritol, a thiol reductant. The inhibitory effect of
the hydroperoxide was mimicked by the penetrant disulfide, cystamine, and by the thiol-oxidizing
agent, diamide.
3. Mitochondrial extracts from rats fed a selenium-deficient diet were shown to have virtually
no measurable GSH peroxidase activity. The hydroperoxide had almost no inhibitory effect on
2-oxoglutarate-dependent 0 2 uptake in mitochondria from selenium-deficient rats.
4. These observations demonstrate effects of GSH peroxidase activity in the mitochondrial matrix
on the pattern of substrate oxidations, possibly with the ketoacid oxidases, dependent on coenzyme A
and lipoamide, as main target sites. In view of the known steady-state formation of mitochondrial
0 2 and H202, a connection between the resulting oxidation of mitochondrial GSH and NADPH
and the regulation of mitochondrial substrate oxidations is proposed.

The concept of the physiological occurrence of

hydrogen peroxide and organic hydroperoxides in
mammalian tissues has received experimental support
in recent years (cf. review in [l,1 a]). The mitochondrial generation of H 2 0 2 has been demonstrated by
Chance and his colleagues [2 - 41, and subcellular
distribution studies by Flohe and Schlegel [5] revealed
the presence of GSH peroxidase and GSSG reductase
in the mitochondrial matrix space, with catalase
activity being quite low [5,6]. A continuous flow of
reducing equivalents through the mitochondrial
2 GSH/GSSG redox system, ultimately expressed as
a utilization of NADPH, must be considered to take
place under aerobic conditions [7,8]. It is therefore
of interest to investigate the effects of hydroperoxides
on mitochondrial metabolism.
When introducing externally added hydroperoxides, e.g. t-butyl hydroperoxide or cumene hydroperoxide, for steady-state perturbation of the intracellular glutathione system, we observed a reversible
decrease of 0 2 uptake by the isolated perfused rat
liver [7,9]. The nature of this effect is analysed in the
Trivial Name. Diamide, diazenedicarboxylic acid bis(N,N-dimethy lamide) .

present paper using isolated hepatocytes and isolated

mitochondria.
Jocelyn [lo] has found that GSH is indeed oxidised
in the presence of t-butyl hydroperoxide in isolated
rat liver mitochondria. Thus, the present investigation
was directed to contribute to an understanding of the
possible relationships betwen the formation of hydroperoxides and the effects of disulfides on mitochondrial oxidations which were studied some years previously, e.g. by addition of cystamine, by Eldjarn
and his colleagues [11 - 131 and others (cf. review in
[14]). Parts of this investigation have been presented
~151.

MATERIALS AND METHODS

Animals

Male Wistar rats were either fed on Altromin chow

(normal rats) or on a Torula yeast-based seleniumdeficient diet (Se-deficient rats) or on the same diet
supplemented with 0.5 mg selenium, as sodium selenite, per kg (Se-supplemented rats) according to
Burk and Masters [16] for four weeks prior to the

Mitochondria1 GSH Peroxidase and Substrate Oxidations

378

1- Butyl hydroperoxide

experiment. Animals were used at a body weight of

150- 200 g.

(0.20rnM )

(0.33 rnM)

Liver Perjhsion and Preparation

of Hepatocytes and Mitochondria

Perfusion of livers was performed with bicarbonate-buffered saline as described [17] except that
the concentrations of L-lactate and pyruvate (sodium
salts) were 2.1 mM and 0.3 mM, respectively. The
temperature was 37 "C.
Hepatocytes were prepared by a modification [I81
of the method of Berry and Friend [19]. Mitochondria
were prepared according to Estabrook and Holowinsky [20].
O J

Assays
concentration and 0 2 uptake (d02jdt) were
recorded simultaneously using a thermostated stirred
chamber at 37 "C equipped with a Clark-type electrode. Hepatocytes were suspended in incubation
medium (incubation condition I of [18]) and mitochondria according to [20].Additions were made from
stock solutions using microsyringes. Protein was
measured by the biuret method. NADPH oxidation
in mitochondria was measured at 340- 380 nm, as
described [17]. Glutathione peroxidase activity was
determined at 30 "C according to [21], using extracts
of mitochondria1 suspensions in 50 mM potassium
phosphate pH 7.0,l mM EDTA and 1 %Triton X-100
after centrifugation at 105000 x g for 30 min. Assay
conditions were: 50 mM potassium phosphate pH 7.0,
1 mM EDTA; 1 mM NaN3. 1 mM GSH, 1 U glutathione reductase/ml, 0.25 mM NADPH, and hydroperoxide as indicated (Fig. 4). Extract volume, 5 20 yl, total volume 0.7 ml. t-butyl hydroperoxide [17]
and ATP were assayed as described.
0 2

5rnin

c---------ti

5rnin

Fig. 1. Reversible decrease of 0 2 uptake in isolated rat hepatocytes

by added hydroperoxide. Hepatocytes were incubated as described
in Materials and Methods. 100 % OZuptake corresponds to approx.
2 pmolO2 x min-' x g-'

200 1

5 rnin

Chemicals and Biochemica165

t-Butyl hydroperoxide was a gift from Peroxid

Chemie Miinchen GmbH (Hollriegelskreuth). Diamide was from Sigma (Munchen). Other chemicals,
biochemicals and enzymes were from Merck (Darmstadt), Boehringer (Mannheim) and Serva (Heidelberg). The selenium-deficient diet was kindly provided
by Dr R. Burk, University of Texas Southwestern
Medical School (Dallas).
RESULTS
Reversible Decrease of 0 2 Uptake
by isolated Hepatocytes Using Added Hydvoyeroxide

Fig. 1 shows the reversible decrease of 0 2 uptake

by isolated hepatocytes by added t-butyl hydroper-

Fig.2. Lack qf inhibitory effect qf t-but-vl hydroperoxide on state 3

and state 4 succinate oxidation by isolated rat liver mitochondria.
Mitochondria were incubated in the incubation buffer [20] with
10 nmol rotenone/ml. Additions of succinate (5 mM), t-butyl hydroperoxide (0.3 mM) and ADP (0.2 mM) as indicated. At 4 min,
0 2 uptake approached zero because of complete Oz reduction

oxide, similar to the earlier observations with the

perfused liver [7,9]. The duration of the effect increases with the concentration of the hydroperoxide
and lasts until the hydroperoxide is reduced to tbutyl alcohol, as was ascertained by assays of the remaining hydroperoxide and by following the decrease
of .dihydroband absorbance of NADPH [17]. The
extent of the decrease of 0 2 uptake was about 3040 of the control rate.

H. Sies and K. M. Moss

379
A

2-Oxoqlutarate

3 - Hvdroxv bi it u r i to

2-Oxoglutarate
I ADP

\
0

100

- 5rnin

5 rnin

5min

Fig. 3. Effects of t-hutyl hydroperoxide on oxidation of2-oxogluturute and hydroxybutyratr by isolated rut liver mitochondria. Mitochondria
were incubated in the incubation buffer [20] with 5 mM malonate and 2-oxoglutarate (5 mM) in A and B or DL-3-hydroxybutyrate(10 mM)
in C. Dithioerythritol was 2 mM, other additions as in Fig. 2

E f f c t s of t-Butyl Hydroperoxide
on 0 2 Uptake by Isolated Mitochondria
0 2 uptake by isolated mitochondria incubated at
37 "C was studied using various substrates. As shown
in Fig.2, in the presence of succinate ( 5 mM) and
rotenone (10 nmol/ml), 0 2 uptake in state 4 (cf. [23])
is slightly increased, and state 3 respiration is obtained
after addition of 0.15 mM ADP. In contrast, 2-0x0glutarate-dependent 0 2 uptake (Fig. 3 A) in the presence of malonate ( 5 mM) is almost completely halted
by the hydroperoxide. This inhibition is not observed
when the thiol reductant, dithioerythritol is added
prior to the hydroperoxide (Fig. 3 B) : similar observations were made with pyruvate as substrate (not
shown). Hydroxybutyrate-dependent 0 2 uptake
(Fig. 3 C) is unaffected by the hydroperoxide. t-Butyl
alcohol in concentrations up to 1 mM did not exhibit
effects on 0 2 uptake (not shown).

Comparison between the Effects on Mitochondria

from Livers of Selenium-Deficient
and Selenium-SupplementedRats
In order to substantiate the assignment of the
observed decrease of O2 uptake to the action of
glutathione peroxidase, isolated mitochondria from
selenium-deficient rats were used as a control. The
activity of the selenoenzyme GSH peroxidase, as
assayed with HzOz as substrate, is drastically decreased in livers of rats fed a diet deficient in selenium

[21,24]. However, Lawrence and Burk [21] discovered

a non-selenium dependent GSH peroxidase activity
in liver cytosol which reduces organic hydroperoxides,
and which has recently been identified with GSHS-transferase B [25,26]. Therefore, we determined
GSH peroxidase activity in Triton extracts of the
isolated mitochondria (Fig. 4) : with H202 as substrate,
there was no detectable activity, while with t-butyl
hydroperoxide (2.1 mM) and cumene hydroperoxide
(1 mM) there was approx. 5 % of the control activity
of 0.4 U/mg mitochondria1 protein. The corresponding residual activities in the liver cytosol of the
selenium-deficient rats were 8 % and 40% of the
controls, with H202 and cumene hydroperoxide respectively, confirming the results of [21]. As shown
in Fig. 5, 2-oxoglutarate-dependent 0 2 uptake is only
slightly affected by 0.5 mM t-butyl hydroperoxide in
the mitochondria from selenium-deficient rats as
compared to the drastic inhibition in mitochondria
from selenium-supplemented rats (5 ppm selenium
as sodium selenite in diet), the latter being similar
to that described above for chow-fed rats (Fig. 3A).

Oxidation of Nicotinamide Nucleotides

The oxidation of nicotinamide nucleotides by
added hydroperoxides in liver cells is due to NADPH
rather than NADH [27], in agreement with the nucleotide specifity of GSSG reductase. Incubation of
isolated mitochondria also leads to a significant

Mitochondria1 GSH Peroxidase and Substrate Oxidations

380
H202

t - Butyl hydroperoxide (2.1 mM)

(0.35mM)

I
t

Blank

Blank

Se - deficient

Se- def___
i cient
Se - supplemented

0.36 Ulmg protein

1-

1 min

Fie.4. GSH . oeroxidase uctivitv in mitochondria1 rxtracts from livers of selenium-deficient and selmium-supplemented rats. Conditions as deschbed in Materials and Methods

f -Butyl
hydroperoxide
2-Oxoglutarate

A
Se-supplemented

Se-deficient

2-Oxoglutarate

2- Oxoglutarate
P

M a l o r t e l ;A

Dithioerythritol

ADP

erythritol
t-Butyl
hydroperoxide

hydroperoxide

OJ

c---------l
5 min

10

Time (min)

Fig. 5. Oxygen uptake in liver mitochondria ,from selenium-deficient

and selenium-supplemented rats oxidi,sing 2-oxogluturatr. Conditions
and additions as for mitochondria from normal rats described
in Fig. 3

Fig. 6. N A D P H oxidation upon addition of t-butyl hydroperoxide in

mitoclzondriu oxidising 2-oxoglutarate and the reversal of the oxidation by dithioerythritol. Conditions as described for Fig. 3

oxidation of nicotinamide nucleotides [8], but enzymatic analyses for NADPH and NADH have not yet
been performed. Succinate and octanoate were found
to be more effective in supporting the re-reduction
of nicotinamide nucleotides than glutamate and
malate 181. The oxidation of nicotinamides by t-butyl
hydroperoxide in the presence of 2-oxoglutarate and
pyruvate was prolonged (no increase in dihydroband
absorbance up to 30 min) unless a thiol reductant,
e.g. dithioerythritol, was added (Fig. 6).

Ejfect of Hydroperoxide on A T P Formation

The formation of ATP from added ADP by isolated mitochondria was measured (Table 1). Whereas
the ATP concentration found after 10 min incubation
with the reduction product, t-but$ alcohol. was
approx. 0.2 mM, regardless of the substrate provided
or of the selenium state, large differences were observed in incubations with t-butyl hydroperoxide.
Very low levels of ATP were found in the presence of

H. Sies and K. M. Moss

381

Table 1. ATP formation by isolated mitochondria in the presence

of t-butj-1 hydroperoxide and t-butyi alcohol and the reduction

of t-hutyl hydroperoxide using various substrates

Liver mitochondria (approx. 5 mg/ml) from rats kept on seleniumdeficient or selenium-adequate diets were incubated at 37 "C, as
described in Materials and Methods, in the presence of 5 mM
substrate, 5 mM malonate (except when succinate was the substrate),
0.25 mM ADP and the i-butyl derivative (0.3 mM) for 10 min.
ATP and t-butyl hydroperoxide were assayed in neutralized perchloric acid extracts
Substrate
(5 mM)

Diet
pretreatment

ATP concentration
at 10 min
- __ -t-butyl
t-butyl
alcohol
hydroperoxide

i-Butyl
hydroperoxide
at
10 min
Fig. 7. Decrease oJ staie 3 0 2 uptake in zsoiated mitochondriu by
cystalnine (0.Sm M ) . Conditions as in Fig.3A except that the
ADP concentration was 0.8 mM. The cysteamine concentration
was 4 m M

mM
Pyruvate
Pyruvate
DL-3-Hydroxybutyrate
DL-~-H~droxybutyrate
2-0Y.oglutarate
Isocitrate
Succinate

Se-supplemented
or chow
Se-deficient
Se-supplemented
or chow

0.20
0.18

0.01
0.18

0.21
0.27

0.20

0.16

<0.02

0.19

0.19

0.26

0.19
0.23
0.22

0.08
0.12
0.20

0.25
0.11
< 0.02

Se-deficient

chow
chow
chow

reduction occurred, approx. 60 % of the added hydroperoxide being present a t the end of the incubation.
Intermediate values were obtained with isocitrate as
substrate. With mitochondria from selenium-deficient
rats, there was virtually no change in the hydroperoxide concentration at the end of the incubation
with all substrates tested.
Obervations with Cystamine and Diamide

the hydroperoxide when pyruvate or 2-oxoglutarate

were substrate in mitochondria from control rats as
well as from rats fed the selenium-supplemented diet,
but ATP was formed in mitochondria from seleniumdeficient rats under these conditions, as is expected
from the 0 2 uptake measurements. Dithioerythritol
(2 mM) was effective in counteracting the inhibitory
effect of the hydroperoxide on pyruvate-dependent
ATP-formation.
Also in agreement with the 0 2 uptake measurements, 0.3 mM hydroperoxide was without effect on
ATP formation when succinate was the substrate,
while there was somewhat less ATP formed with
3-hydroxybutyrate and isocitrate. These observations
extend the finding of Summer [28] of a transient
decrease of the ATPIADP ratio upon hydroperoxide
infusion into perfused liver.
t-Butyl Hydroperoxide Reduction
by Isolated Mitochondria
In mitochondria from normal or selenium-supplemented rats, the hydroperoxide was almost completely
reduced to t-butyl alcohol when 3-hydroxybutyrate
or succinate were used as substrate (Table 1). With
pyruvate as substrate, considerably less hydroperoxide

O2 uptake by mitochondria engaged in state 3

respiration supported by 2-oxoglutarate was restricted
upon addition of the permeable disulfide, cystamine
(Fig.7), but not by the non-penetrant GSSG (not
shown). The effect was reversed by the addition
of dithioerythritol. In agreement with these observations, 2 mM cystamine was found to inhibit ATP
synthesis using pyruvate or 2-oxoglutarate as substrate, whereas inhibition was low with P-hydroxybutyrate as substrate. GSSG was without effect on
ATP synthesis by mitochondria.
Diamide (0.15 mM), the thiol oxidising agent
described by Kosower et al. [29] was also found to
inhibit 0 2 uptake by mitochondria oxidising 2-0x0glutarate. Siliprandi et al. [30] found a substantial
decrease of the respiratory control index to less than
half in the presence of 0.1 mM diamide, using succinate or glutamate + malate as substrate. We did not
observe such striking effects of diamide (0.15 mM)
on the respiratory control index under the incubation
conditions at 37 OC; it decreased to SS%, 81 % and
61 % of the controls with succinate, hydroxybutyrate
and 2-oxoglutarate respectively.
Diamide was also found to inhibit ATP formation
by mitochondria oxidising certain substrates. In the
presence of pyruvate and 0.5 mM diamide, the amount
of ATP presents after 10 min incubation was 10%

Mitochondrial GSH Peroxidase and Substrate Oxidations

382
Table 2. ATP formation by isolated mitochondria in the presence
of diarnide using various substrates
Liver mitochondria (approx. 5 mg/ml) from normal rats were incubated at 37 "C, as described in Materials and Methods, in the
presence of 5 mM substrate, 5 mM malonate (except when succinate was the substrate), 0.25 mM ADP and varying concentrations of diamide. ATP was assayed in neutralized perchloric acid
extracts. The ATP concentration obtained after 5 min incubation
in the absence of diamide was 0.26 mM with all substrates tested
Additions

Pyruvate
Pyruvate
Pyruvate
Pyruvate
Pyruvate
+ dithioerythritol(2mM)
2-Oxoglutarate
DL-3-H ydroxybutyrate
DL-3-Hydroxybutyrate

Incubation Diamide
time
concentration

ATP
concentration

min

mM

5
10
10
10

0.2
0.5

0.26
0.27
0.26
0.02

10
5
5
10

0.5
0.5
0.5
0.5

0.23
0.12
0.25
0.16

of the control value in the absence of diamide

(Table 2), whereas when dithioerythritol (2 mM) was
also present, it was about 90% of the control value.
As was observed with t-butyl hydroperoxide, ATP
formation using hydroxybutyrate as substrate was
unaffected by the presence of 0.5 mM diamide after
5 min incubation, but after 10 min, the ATP concentration was only 60 of the control value.

DISCUSSION
Reversible Inhibition of Respiration
in Isolated Liver Cells and Isolated Mitochondria
upon Addition of Hydroperoxide :
The Role of Glutathione Peroxidase
The rate of 0 2 uptake was decreased upon addition
of t-butyl hydroperoxide in the perfused liver [7,9],
in isolated liver cells [8] (Fig. 1) and in isolated mitochondria oxidising 2-oxoglutarate (Fig. 3), pyruvate
(not shown) and other substrates. This effect is ascribed
to the action of mitochondrial GSH peroxidase,
and consequently to a perturbation of the mitochondrial matrix glutathione redox state, on the following
grounds.
Firstly the inhibition lasts until the hydroperoxide
has been reduced to the alcohol, and added t-butyl
alcohol has no effect. Secondly, the effect of the added
hydroperoxide on the glutathione system has been
documented [7 - 91 and has further been extended to
the mitochondrial matrix glutathione system [lo].
Thirdly, the inhibition was mimicked by the penetrant
disulfide, cystamine, and by the thiol-oxidizing agent,

diamide, and it was reversed upon addition of a

thiol-reductant, dithioerythritol (Fig. 6 and 3, and
Table 2, respectively). Fourthly, since mitochondrial
selenium-dependent GSH peroxidase activity has been
shown to be virtually absent in livers from seleniumdeficient rats (Fig. 4), the absence of the hydroperoxide
effect in such mitochondria (Fig. 5) as compared to
selenium-supplemented or control mitochondria
(Fig. 3A) strongly supports the hypothesis. This observation excludes a number of possible unspecific
explanations.
Nature of the Suljhydryl Groups Responsible
for the Inhibition of Respiration

As a multitude of key processes involves sulfhydryl groups (for reviews, see [14,31,32]), the analysis
of possible types of sulfhydryl groups is carried further
by the observation that the system of energy conservation is most probably not involved in the t-butyl
hydroperoxide effects, as is shown by the absence of
an effect on ATP synthesis with succinate or hydroxybutyrate as substrates, as compared with pyruvate or
2-oxoglutarate (Table 1).
The inhibitory effect of the hydroperoxide on
mitochondrial ATP synthesis was most marked with
the substrates pyruvate and 2-oxoglutarate. This observation may indicate a selective inhibition of 0 2
uptake by hydroperoxides on pathways dependent on
coenzyme A and lipoamide, due to mitochondrial
thiol oxidation arising from the perturbation of the
glutathione system. In preliminary experiments, the
coenzyme A level in mitochondria decreased substantially in the presence of t-butyl hydroperoxide with
pyruvate as substrate; no significant effects were found
with mitochondria from selenium-deficient rats.
The findings presented here, together with the
effects found for octanoate oxidation in liver cells
[8], clearly show that transitions in the mitochondrial
thiol redox state have differential effects on substrate
oxidations. Thus the pattern of mitochondrial metabolite oxidations and, therefore, also of metabolite
concentrations, can be influenced by the mitochondrial thiol redox state.
Possible Role of GSH Peroxidase
in Modulating Mitochondrial Oxidations
under Conditions of Endogenous Hydroperoxide
Production
Whether the above observations obtained with
added organic hydroperoxide may apply to the physiological condition remains an open question. Chance
and his colleagues [2-41 have demonstrated the
mitochondrial generation of H202. Under state 4
conditions, the rate was about 1 nmol Hz02 x min-'
x mg mitochondrial protein-'. The glutathione con-

H. Sies and K. M. Moss

383

tent of isolated mitochondria was found to be between

5 [32] and 14 [33] nmol GSH GSSG per mg protein. Thus, the turnover in the glutathione redox
system is about 10% per min if GSH peroxidase is
responsible for mitochondrial H 2 0 2 reduction. The
rate of H202 generation was shown to increase under
different conditions, e.g. with hyperbaric oxygen or
upon inhibition of the respiratory chain by antimycin
[3,41.
The continuous flow of reducing equivalents
through the glutathione system must be balanced by
the continuous formation of mitochondrial NADPH
in order to maintain a steady state. Thus the participation in the GSSG reductase reaction constitutes an
important metabolic function of mitochondrial
NADPH.
The present proposal of an involvement of mitochondrial glutathione peroxidase in modulating substrate oxidations, mainly at the site of lipoamidedependent ketoacid oxidases, relates to the proposal
by Olson and Allgyer [34] of a key role of dihydrolipoyl dehydrogenase in regulating NAD-linked substrate oxidation. In addition to the control by the
GTP level [34] the GSH/GSSG ratio may exert
effects on the same target site, in agreement with the
interpretation 1341of the effects observed with tellurite
S51.
Finally, the role for GSH peroxidase proposed
here extends its function from a protective one, similar to that ascribed to mitochondrial superoxide dismutase by Tyler [36], to a possible regulatory function
insofar as the detoxification of hydroperoxides is
coupled to changes in the type of substrate oxidised
by the mitochondria. Furthermore, such a function
may find application in phenomena related to Hz02
metabolism, e.g. in the processes of platelet aggregation and secretion [37].

The expert technical assistance by Annegret Marklstorfer and

Ingrid Linke is gratefully acknowledged. We thank Dr A. Wendel,
Tiibingen, for samples of GSH peroxidase, and Dr R. Burk, Dallas,
for the selenium-deficient diet. This study was supported by Deutsche
Forschung,rgrmeinschqft, Sonderjor.FchunssberPich 51, Medizinische
Molekularbiologie und Biochemie. K.M.M. is recipient of a Stipendium of the Alexander-v.-Humboldt-Stiftung.

REFERENCES
I. Sies, H. (1974) Angew. Chem. 86, 789-801.
l a . Sies, H. (1974) Angew. Chem. Int. Ed. Engl. 13, 706-718.
2. Chance, B. & Oshino, N. (1971) Biochem. J. 122,225-233.
3. Loschen, G., Flohe, L. & Chance, B. (1971) FEBS Lett. 18,
261 -264.
4. Boveris, A. &Chance, B. (1973) Biochem. J . 134, 707-716.

5. Floht, L. & Schlegel, W. (1971) Hoppe-Seyler's 2. Physiol.

Chem. 352,1401 - 1410.
6 . Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey,
J . W., Fowler, S. & DeDuve, C. (1968) J . Cell B i d . 37,
482- 513.
7. Sies, H., Gerstenecker, C., Menzel, H., Flohe, L. (1972) FEBS
Lett. 27, 171 - 175.
8. Oshino, N. & Chance, B. (1977) Biochem. J . 162, 509-525.
9. Sies, H., Gerstenecker, C., Summer, K. H., Menzel, H. & Floht, L. (1974) in: Glutathione (Flohe, L., Benohr, H., Sies; H.,
Waller, H. & Wendel, A,, eds) pp. 261 -276, G . Thieme,
Stuttgart.
10. Jocelyn, P. C . (1975) Biochim. Biophys. A r m , 369,427-436.
11. Eldjarn, L. & Bremer, J. (1963) Actu Chem. Scand. 17, S59S 66.
12. Skrede, S., Bremer, J. & Eldjarn, L. (1965) Biochem. J . 95,
838 - 846.
13. Skrede, S. (1968) Biochem. J . 108, 693 - 699.
14. Jocelyn, P. C. (1972) Biochemistry of the SH Group, Academic
Press, London and New York.
15. Sies, H. (1976) 10th ZUB Canguess, Hamburg, Abstract 06-7129.
16. Burk, R. F. & Masters, B. S. S. (1975) Arch. Biochem. Biophys.
170, 124-131.
17. Sies, H. & Summer, K. H. (1975) Eur. J . Biochem. 57,503-512.
18. Sies. H. & Grosskopf, M. (1975) Eur. J . Biochem. 57, 513-520.
19. Berry, M. & Friend, D. S. (1969) J . Cell B i d . 43, 506 - 520.
20. Estabrook, R. W. & Holowinsky, A. (1961) J . Biochem. Biophys. C y t d . 9, 19 - 28.
21. Lawrence, R. A. & Burk, R. F. (1976) Biochem. Biophys. Res.
Commun. 71,952-958.
22. Haussinger, D., Weiss, L. & Sies, H. (1975) Eur. J . Biochem.
52,421 -431.
23. Chance, B. & Williams, G. R. (1956) Adv. Enzymol. 17, 65134.
24. Hafemdn, D. G., Sunde, R. A. & Hoekstra, W. G . (1974)
J . Nutr. 104, 580- 587.
25. Prohaska, J. R. & Ganther, H. E. (1977) Biochem. Biophys.
Res. Commun. 76,437 - 445.
26. Burk, R. F., Lawrence, R. A. & Parkhill, L. K . (1977) Gustroenterology, 73, 1215.
27. Sies, H., Summer, K. H. & Grosskopf, M. (1976) in Use of'
Isolated Liver Cells and Kidney Tubules in Metubolic Studies
(Tager, J. M., Soling, H. D. & Williamson, J. R., eds) pp.
317-322, North Holland Amsterdam.
28. Summer, K. H. (1974) Diplomarbeit, Fachbereich Chemie,
University of Tubingen.
29. Kosower, E. M., Correa, W., Kinon, B. J. & Kosower, N. S.
(1972) Biochim. Biophys. Acta, 264, 39 -44.
30. Siliprandi, D., Scutari, G., Zoccarato, F. & Siliprandi, N. (1974)
FEBS Lett. 42, 197-199.
31. Gautheron, D. (1973) Biochemie, 55, 727-745.
32. Vignais, P. M. & Vignais, P. V. (1973) Biochim. Biophys. Acta,
325, 357- 374.
33. Jocelyn, P. C. & Kamminga, A. (1974) Biochim. Biophys. Actu,
343, 356- 362.
34. Olson, M. S. & Allgyer, T. T. (1973) J . B i d . Chem. 248, 15901597.
35. Siliprandi, D., DeMeio, R. H., Toninello, A. & Zoccarato, F.
(1971) Biochem. Biophys. Res. Commun. 45, 1071 - 1075.
36. Tyler, D. D. (1975) Biochim. Biophys. Acta, 396, 335-346.
37. Holmsen, H. & Robkin, L. (1977) J . B i d . Chem. 252, 17521757.

H. Sies and K. M. Moss, lnstitut fur Physiologische Chemie,

Physikalische Biochemie und Zellbiologie der Ludwig-Maximilians-UniversitatMiinchen.
GoethestraBe 33, D-8000 Munchen 2, Federal Republic of Germany