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The role of methionine in glutathione biosynthesis by isolated hepatocytes
Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE
ROLE OF M E T H I O N I N E
BIOSYNTHESIS
Donald
IN G L U T A T H I O N E
BY I S O L A T E D
J. R e e d I
HEPATOCYTES
and Sten O r r e n i u s
D e p a r t m e n t of F o r e n s i c Medicine, K a r o l i n s k a Institutet, Stockholm, S w e d e n and i D e p a r t m e n t of B i o c h e m i s t r y and Biophysics, O r e g o n State University, Corvallis, O r e g o n 97331 Received
June
22,1977
Summary: H e p a t o c y t e s freshly i s o l a t e d from d i e t h y l m a l e a t e - t r e a t e d rats have b e e n shown to p e r f o r m net b i o s y n t h e s i s of i n t r a c e l l u l a r g l u t a t h i o n e at a p p r o x i m a t e l y an in vivo rate. 3SS from I 3SSIcys teine or 1 3 5 S l m e t h i o n i n e each c o ~ r i b u t e d to the f o r m a t i o n of g l u t a t h i o n e to about the same extent, up to 75 and 61%, r e s p e c t i vely, d e p e n d i n g u p o n the amino acid c o n c e n t r a t i o n in the medium. The sulfur a t o m of m e t h i o n i n e m a k e s a s i g n i f i c a n t c o n t r i b u t i o n to the s y n t h e s i s of the thiol of g l u t a t h i o n e p r o b a b l y via the formation of c y s t a t h i o n i n e . Glutathione, portant sent
role
L-y-glutamyl-L-cysteinyl-glycine,
in hepatic
in r e l a t i v e l y
depletion
the
plenishment in regards
of the c y s t e i n e
to and m e t a b o l i s m cursors
include
This
pool
extracellular protein
about
turnover
such as
2-
about
3 hrs
interest
(4) during
or c y s t i n e
which
is pre-
of liver being
of chemicals.
cysteine
an im-
(1). A f t e r
the p r e c u r s o r s
is of keen
variety
within
content
conjugation
of a w i d e
and i n t r a c e l l u l a r ly in liver
cysteine
knowledge
to g l u t a t h i o n e
agents
can be c o m p l e t e d
intracellular (1,3),
and as such
(4 to 6 mM)
in rat liver w i t h
resynthesis
ly 0.2 to 0.3 m M
detoxification
concentrations
of g l u t a t h i o n e
chloroethanol, (2). W i t h
high
drug
plays
is known
on-
for re-
especially
human
exposures
Possible
pre-
and m e t h i o n i n e to occur
rapid-
(5,6).
report
describes
thione
in h e p a t o c y t e s
thione
with
after
diethylmaleate.
Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
the rate of net b i o s y n t h e s i s in v i v o
depletion
The c o n t r i b u t i o n
of liver
of glutagluta-
of e x t r a c e l l u l a r
1257 1SSN 0006-291X
Vol. 77, No. 4, 1977
cysteine
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and m e t h i o n i n e
sulfur
to the thiol of g l u t a t h i o n e
was
evaluated. METHODS
AND MATERIALS
Hepatocytes. One hr prior to h e p a t o c y t e isolation, m a l e SpragueD i w i e y rats, 210 to 230 g m body wt., w e r e a d m i n i s t e r e d i.p. 0.6 m m o l e / K g body wt. of d i e t h y l m a l e a t e (Aldrich) as a 20% s o l u t i o n in corn oil (7). The i s o l a t i o n p r o c e d u r e and cell v i a b i l i t y mea s u r e m e n t s w e r e those p r e v i o u s l y d e s c r i b e d (8). I n c u b a t i o n procedures w e r e as p r e v i o u s l y d e s c r i b e d except that the m e d i u m was a K r e b s - H e n s e l e i t b u f f e r s u p p l e m e n t e d w i t h 2 m M Hepes, p e n i c i l lin (500 I.U./ml), h e p a r i n (i0 I.U./ml) and an amino acid mixture (i X concentration) (9). In all experiments the serine c o n c e n t r a t i o n was 0.35 mM. W h e n 135Slmethionine was the substrate, c y s t e i n e in the m e d i u m was 0.2 m M and w h e n 13SSlcysteine was the substrata, m e t h i o n i n e in the m e d i u m was 0.i mM. All other amino acids w e r e p r e s e n t at the c o n c e n t r a t i o n s s p e c i f i e d (9). Total 13SSleysteine or 13SSlmethionine uptake by h e p a t o c y te s u s p e n s i o n s was d e t e r m i n e d a c c o r d i n g to a f i l t r a t i o n procedure p r e v i o u s l y d e s c r i b e d (10) except that 47 m m glass fiber filters (Whatman GF/C) w e r e c o u n t e d in a toluene fluor. Incorp o r a t i o n of 35S into p r o t e i n was m e a s u r e d by the same p r o c e d u r e except that 3 x 6 ml w a s h e s of the cells w i t h 10% t r i c h l o r o a c e tic acid w e r e used to remove a c i d - s o l u b l e 3SS. P r e p a r a t i o n of 2 ~ 4 - d i n i t r o p h e n y l derivatives. Hepatocytes, l0 G C elis/ml, w e r e r e m o v e d f r o m i n c u b a t i o n m i x t u r e s by c e n t r i f u g a tion (50 x g for 2 min) and w a s h e d once by r e s u s p e n s i o n in K r e b s - H e n s e l e i t b u f f e r c o n t a i n i n g 2% bovine serum a l b u m i n followed by a second c e n t r i f u g a t i o n . The cell p e l l e t was t r e a t e d w i t h 1.0 ml of 3.3% p e r c h l o r i c acid and the p r o t e i n r e m o v e d by c e n t r i f u g a t i o n . A 0.5 ml a l i q u o t of the s u p e r n a t a n t was reacted w i t h i0 Z1 of p e r f o r m i c acid (20:1 V / V of 98% formic acid, 30% HzO 2 at room t e m p e r a t u r e for 1 hr prior to use) for 15 m i n and the excess p e r f o r m i c acid d e s t r o y e d w i t h 5 ~i of 48% hydr o b r o m i c acid. The r e a c t i o n m i x t u r e was s a t u r a t e d w i t h s o d i u m b i c a r b o n a t e and r e a c t e d w i t h an a l c o h o l i c s o l u t i o n of l-fluoro2,4-dinitro-benzene (FDNB] for 4 hrs in the dark (ii). H i g h - p e r f o [ m a n c e !iquid c h r o m a t o g r a p h y . A n a l i q u o t of the F D N B r e a c t i o n m i x t u r e was i n j e c t e d onto a 4 x 250 m m Micro B o n d a p a k amine c o l u m n (Waters) and the DNP d e r i v a t i v e s i s o l a t e d by gradient elution (0.05 to 0.4 M sodium acetate, pH 4.6, in 80% methanol) using a Spectra Physics model 3500 liquid c h r o m a t o g r a p h e q u f p p e d w i t h a UV d e t e c t o r (350 nm) and a Spectra Physics S y s t e m I integrator. A p p r o p r i a t e fractions were c o l l e c t e d and a s s a y e d for 35S w i t h A q u a s o l (New E n g l a n d N u c l e a r Co.) in a liquid s c i n t i l l a t i o n counter. G l u t a t h i o n e sulfonic a c i d DNP was q u a n t i t a t e d by internal s t a n d a r d i z a t i o n and c o m p a r i s o n w i t h a u t h e n t i c g l u t a t h i o n e s u l f o n a t e (12) after c o n v e r s i o n to the DNP derivative. Cysteic a c i d DNP (Sigma) was u s e d as a reference to q u a n t i t a t e cysteic acid formed by p e r f o r m i c acid o x i d a t i o n of i n c u b a t i o n m e d i a c o n t a i n i n g cysteine. F u r t h e r details of these p r o c e d u r e s including the s e p a r a t i o n of DNP d e r i v a t i v e s of g l u t a t h i o n e disulfide, c y s t e i n e g l u t a t h i o n e m i x e d disulfide, and y - g l u t a m y l cysteic a c i d will be d e s c r i b e d e l s e w h e r e (13).
1258
Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60- 5S] CYSTEINE UPTAKE / 10B CELLS 50
40-
'°'~'o~O~o
30, c 20-
/o/m~o~m
TCA INSOLUBLE/ 106CELLS
10-
T..E I.RSl
T,ME IHRSI
Figure 1. Uptake of 3SS during the i n c u h a t i o n of k e p a t o c y t e s w i t h [3Ss]cysteine. The nmoles of 35S w e r e c a l c u l a t e d from the specific a c t i v i t y of the [35S]cysteine in the mediLzm. [35S]cysteine c o n c e n t r a t i o n s in the m e d i u m were A-A, 0.2 ~ ; ~-~, 1.0 mM; o-o, 3.0 ng~; e-o, 5.0 ~M.
RESULTS The c r i t e r i a ne or m e t h i o n i n e
used for a s s e s s i n g
the c o n t r i b u t i o n
sulfur to net g l u t a t h i o n e
tocytes was the rate of amino acid uptake, soluble
~SS i n t r a c e l l u l a r
sis and the
ass specific
pool,
biosynthesis
rate of net g l u t a t h i o n e
activity
of g l u t a t h i o n e
amino acid in the medium.
take of
at 0.2 m M by h e p a t o c y t e s
acid-soluble
3SS pool that did not exceed
by hepa-
the size of the acid-
that of the substrata 135Slcysteine
of c y s t e i -
synthe-
relative
to
The limited upresulted
5 nmoles/106
in an cells
w h i l e more that 20 nmoles of g l u t a t h i o n e
were being s y n t h e s i z e d
(Fig. i, T a b l e
ratio of i n t r a c e l l u l a r
i). The specific
J 3SSJglutathione
relative
to
activity
J35Slcysteine
in the m e d i u m did not
exceed 0.19 w h i c h agreed very well with the cysteine lues
(Fig. i). A similar r e l a t i o n s h i p
J3 ~ S J g l u t a t h i o n e cysteine
specific
incubations
between
3SS uptake and
a c t i v i t y was o b s e r v e d
(Fig. i, Table
1259
i).
uptake va-
for 1.0 ~
j3SSJcysteine
L3SsI
at 3.0 and
Vol.
77,
No.
4,
1977
BIOCHEMICAL
AND
BIOPHYSICAL
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Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Z0 ,5 .[30% "I,M O ]/EoTH O I/N N IO EUP 'T ~AKE1/0%ELLS 30 "6
20
TCA N ISOLUBLE1/0BCELLS
/ a / " ~ a
TIME (HRS)
TIME (HRS)
F i g u r e 2. Uptake of 3SS during the i n c u b a t i o n of h e p a t o c y t e s w i t h [3SS]methionine. The nmoles of 3SS w e r e c a l c u l a t e d from the specific a c t i v i t y of the [3SS]meth~on~ne in the medin/m. [3SS]methionine c o n c e n t r a t i o n s in the m e d i u m w e r e A-A, 0.i raM; A-A, 0.3 mM; ~-~, 1.0 mM; o-o, 3.0 m M and e-e, 5.~ mM.
5.0 m M gave first hr specific respectively,
again r e f l e c t i n g
these higher c o n c e n t r a t i o n s .
not to a d e t e c t a b l e 13SSlmethionine
2, T a b l e
at 0.3,
i). Less of the
This
termediates,
about
activity
into
of
135Slcys -
25% at 0.2 mM c y s t e i n e
and formed an a c i d - s o l u b l e became
pool of
acid-soluble
13SSlglutathione
i n d i c a t e d that a s i g n i f i c a n t
trans-
13SSlglutathione
I35Slmethionine
pool of
~sS (Fig.
pool of
than that of
part of w h i c h could be thiols,
but
concentrations.
1.0 and 3.0 m M was readily
but not completely,
3SS was t r a n s f o r m e d teine.
a g r e a t e r uptake of amino acid at
amount at higher c y s t e i n e
into h e p a t o c y t e s
that rapidly,
ratios of 0.75 and 0.73
The specific
teine in the m e d i u m d e c r e a s e d
ported
activity
13SSlcys -
3Ss labeled in-
accumulated
in the
cells.
This was c o n f i r m e d by thiol analyses
using the Saville
method
(14). By the third hr of incubation,
maximum
tivity ratios were a c h i e v e d
(0.37,
and 3.0 m M
respectively).
activity
I 3SSlmethionine,
0.52 and 0.61 for 0.3,
ratios r e f l e c t the r a p i d i t y
is t r a n s f o r m e d
ac-
1.0
The one hr specific
in w h i c h m e t h i o n i n e
into the thiol of g l u t a t h i o n e
1261
specific
sulfur
by the hepatocytes.
Vol. 77, No. 4, 1977
Efflux
of g l u t a t h i o n e
was m e a s u r e d /hr/106
and found
cells w h e n
medium were values
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to be nearly
cysteine
0.2 and
respectively.
of the e x t r a c e l l u l a r
The total
lular)
was
tration
cells.
of g l u t a t h i o n e
Efforts
glutathione
exists
cells.
plus
Increasing
activity the
glutathiextracel-
the concen-
in the m e d i u m
increased
of 7 to 8 n m o l e s / h r / 1 0 6
in p r o g r e s s
to d e t e r m i n e
and to e s t a b l i s h
as a m i x e d
in the
essentially
(cellular
to a m a x i m u m
state of this g l u t a t h i o n e
concentrations The specific
or m e t h i o n i n e
are c u r r e n t l y
at 3.8 ± 0.8 nmoles
were
synthesized
cysteine
into the m e d i u m
for the i n t r a c e l l u l a r
ii to 22 n m o l e s / h r / 1 0 6
of either
the e f f l u x
glutathione
values
glutathione
constant
and m e t h i o n i n e
0.1 mM,
same as the c o r r e s p o n d i n g one.
from the h e p a t o c y t e s
disulfide
whether
the redox
any of the
of cysteine.
DISCUSSION Freshly thione,
isolated
rapidly
22 n m o l e s / h r / 1 0 6 (8) this
rate
rate of a b o u t activity
provide
Assuming
for p e r f u s e d
liver
data
support
may
provide
the c o n c l u s i o n from p r o t e i n
nearly
source
are known (16,17)
in normal
50% of the i n t r a c e l l u l a r
derived
and each amino
rates
tely
onine
liver
one half
free amino that w i t h
turnover of the
thesis.
1262
of liver
synthesis
(2). The
of sulfur
high
(5,6,
approxima-
(5). These
hepatocytes
and the i n c u b a t i o n sulfur
In vi-
hepatocytes
contributes acid pool
may
formed.
to be very
isolated
specific
acid at low con-
and i s o l a t e d
fed rats
of ii to
w e t wt.
w i t h an in vivo of
of gluta-
at a rate
one half of the g l u t a t h i o n e
turnover
turnover
depleted
108 c e l l s / g m
that an endogenous
approximately
Protein
tripeptide
w e t wt.
of g l u t a t h i o n e
indicated
as also
(18).
cells.
this
2 ~moles/hr/gm
vo liver p r o t e i n 15)
replenished
is in good a g r e e m e n t
ratios
centration
hepatocytes, that w e r e
meth~
medium
for g l u t a t h i o n e
syn-
Vol. 77, No. 4, 1977
Studies
on intact
F o r example, methionine found
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
into rat
m a y be r e l a t e d
vatives
and m a j o r
effect
of c y s t i n e
rapid
Even
(20,21),
in rat liver
synthetase,
via
of g l u t a t h i o n e
as
caused
glutathione
of the serious
of a m a j o r
the c y s t a t h i o n i n e during
(22),
methionine-sparing
Thus,
to the c o n s e q u e n c e s
trans-sulfuration
prevented
in rats
and k i d n e y
35S
equally
for y - c y s t a t h i o n a s e
established
biosynthesis.
of
a c i d deri-
methionine
by e t h i o n i n e
I~5SI
results
was
of m e r c a p t u r i c
(24) may be an i n d i c a t i o n
be g i v e n
these
that m e t h i o n i n e
a substrate
the firmly
fraction
(19). Also,
of y - g l u t a m y l c y s t e i n e
in g l u t a t h i o n e
biosynthesis
in dogs
decreases
(23).
should
shown
this conclusion. introduced
the m a j o r
in the f o r m a t i o n
sulfoximine,
levels
tion
with
of liver g l u t a t h i o n e
as an i n h i b i t o r
thionine
tissue
has b e e n
with bromobenzene
and m e t h i o n i n e
rapid
liver
with
of p a r e n t e r a l l y
to the o b s e r v a t i o n s
as c y s t i n e
the d e p l e t i o n
well
are c o n s i s t e n t
the t r a n s f o r m a t i o n
in g l u t a t h i o n e
effective
rats
extensive
role
of me-
considerarole
pathway
of
for the
glutathione
con-
jugation. ACKNOWLEDGEMENTS The w o r k r e p o r t e d in this paper was u n d e r t a k e n during the tenure of one of us (D.J.R.) on an A m e r i c a n C a n c e r S o c i e t y Eleanor Roosevelt-International C a n c e r F e l l o w s h i p a w a r d e d by the I n t e r n a t i o n a l U n i o n A g a i n s t Cancer. The a u t h o r s w i s h to thank the S w e d i s h M e d i c a l R e s e a r c h C o u n c i l for f i n a n c i a l support (grant no. 03X-2471) and Spectra Physics Co. for the loan of a liquid c h r o m a t o g r a p h and a S y s t e m I i n t e g r a t o r for use in this study. We thank Dr. J o h a n H ~ g b e r g for very f r u i t f u l discussions, Mrs. G u n - B r i t t Sundby and Mrs. A n n i k a K r i s t o f e r s o n for e x c e l l e n t t e c h n i c a l a s s i s t a n c e and Dr. D e a n Jones and D o c e n t Sten Jakobsson for s u g g e s t i o n s d u r i n g m a n u s c r i p t p r e p a r a t i o n . REFERENCES i. 2. 3. 4.
Meister, A. and Tate, S.S. (1976) Ann.Rev. Biochem. 45, 559604. White, I.N.H. (1976) C h e m . - B i o l . Interactions, 13, 333-342. T a t e i s h i , N., Higashi, T., Shinya, S., Naruse, A. and Sakamoto, Y. (1974) J.Biochem. 75, 93-103. Boyland, E. and Chasseaud, L.F. (1969) A d v . E n z y m o l . 32, 173-219.
1263
Vol. 77, No. 4, 1977
5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Gan, J.C. and Jeffay, H. (1971) Biochim.Biophys.Acta, 252, 125-135. Glass, R.D. and Doyle, D. (1972) J.Biol.Chem. 247, 5234-5242. Boyland, E. and Chasseaud. L.F. (1967) Biochem.J. 104, 95-102. H~gberg, J. and Kristoferson, A. (1977) Eur.J.Biochem. 74, 77-82. Seglen, P.O. (1976) Biochim. Biophys.Acta, 442, 391-404. Czech, M.P. (1976) Molecular and Cellular Biochem. ii, 51-63. Kesner, L., Muntwyler, E., Griffin, G.E. and Quaranta, P. (1967) Methods of Enzymol. ii, 94-108. Calam, D.H. and Waley, S.G. (1962) Biochem.J. 85, 417-419. Reed, D.J. (1977) Manuscript in preparation. Saville, B. (1958) Analyst, 83, 670-672. Scornik, O.A. and Botbol, V. (1976) J.Biol.Chem. 251, 28912897. Mortimore, G.E. and Mondon, C.E. (1970) J.Biol.Chem. 245, 2375-2383. Woodside, K.H. and Mortimore, G.E. (1972) J.Biol.Chem. 247, 6474-6481. Seglen, P.O. (1975) Biochem. Biophys.Res.Commun. 66, 44-52. Shtutman, Ts.M. and Biber, A. (1973) Ukr. Biokhim. Zh. 45, 439-443. White, A. and Lewis, H.B. (1932) J.Biol.Chem. 98, 607-624. Stekol, J.A. (1937) J.Biol.Chem. 117, 147-159. Glaser, G. and Mager, J. (1974) Bioehim.Biophys.Acta, 372, 237-244. Palekar, A.G., Tate, S.S. and Meister, A. (1975) Biochem. Biophys.Res.Commun. 62, 651-657. Finkelstein, J.D. and Mudd, S.H. (1967) J.Biol.Chem. 242, 873-880.
1264
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE
ROLE OF M E T H I O N I N E
BIOSYNTHESIS
Donald
IN G L U T A T H I O N E
BY I S O L A T E D
J. R e e d I
HEPATOCYTES
and Sten O r r e n i u s
D e p a r t m e n t of F o r e n s i c Medicine, K a r o l i n s k a Institutet, Stockholm, S w e d e n and i D e p a r t m e n t of B i o c h e m i s t r y and Biophysics, O r e g o n State University, Corvallis, O r e g o n 97331 Received
June
22,1977
Summary: H e p a t o c y t e s freshly i s o l a t e d from d i e t h y l m a l e a t e - t r e a t e d rats have b e e n shown to p e r f o r m net b i o s y n t h e s i s of i n t r a c e l l u l a r g l u t a t h i o n e at a p p r o x i m a t e l y an in vivo rate. 3SS from I 3SSIcys teine or 1 3 5 S l m e t h i o n i n e each c o ~ r i b u t e d to the f o r m a t i o n of g l u t a t h i o n e to about the same extent, up to 75 and 61%, r e s p e c t i vely, d e p e n d i n g u p o n the amino acid c o n c e n t r a t i o n in the medium. The sulfur a t o m of m e t h i o n i n e m a k e s a s i g n i f i c a n t c o n t r i b u t i o n to the s y n t h e s i s of the thiol of g l u t a t h i o n e p r o b a b l y via the formation of c y s t a t h i o n i n e . Glutathione, portant sent
role
L-y-glutamyl-L-cysteinyl-glycine,
in hepatic
in r e l a t i v e l y
depletion
the
plenishment in regards
of the c y s t e i n e
to and m e t a b o l i s m cursors
include
This
pool
extracellular protein
about
turnover
such as
2-
about
3 hrs
interest
(4) during
or c y s t i n e
which
is pre-
of liver being
of chemicals.
cysteine
an im-
(1). A f t e r
the p r e c u r s o r s
is of keen
variety
within
content
conjugation
of a w i d e
and i n t r a c e l l u l a r ly in liver
cysteine
knowledge
to g l u t a t h i o n e
agents
can be c o m p l e t e d
intracellular (1,3),
and as such
(4 to 6 mM)
in rat liver w i t h
resynthesis
ly 0.2 to 0.3 m M
detoxification
concentrations
of g l u t a t h i o n e
chloroethanol, (2). W i t h
high
drug
plays
is known
on-
for re-
especially
human
exposures
Possible
pre-
and m e t h i o n i n e to occur
rapid-
(5,6).
report
describes
thione
in h e p a t o c y t e s
thione
with
after
diethylmaleate.
Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
the rate of net b i o s y n t h e s i s in v i v o
depletion
The c o n t r i b u t i o n
of liver
of glutagluta-
of e x t r a c e l l u l a r
1257 1SSN 0006-291X
Vol. 77, No. 4, 1977
cysteine
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and m e t h i o n i n e
sulfur
to the thiol of g l u t a t h i o n e
was
evaluated. METHODS
AND MATERIALS
Hepatocytes. One hr prior to h e p a t o c y t e isolation, m a l e SpragueD i w i e y rats, 210 to 230 g m body wt., w e r e a d m i n i s t e r e d i.p. 0.6 m m o l e / K g body wt. of d i e t h y l m a l e a t e (Aldrich) as a 20% s o l u t i o n in corn oil (7). The i s o l a t i o n p r o c e d u r e and cell v i a b i l i t y mea s u r e m e n t s w e r e those p r e v i o u s l y d e s c r i b e d (8). I n c u b a t i o n procedures w e r e as p r e v i o u s l y d e s c r i b e d except that the m e d i u m was a K r e b s - H e n s e l e i t b u f f e r s u p p l e m e n t e d w i t h 2 m M Hepes, p e n i c i l lin (500 I.U./ml), h e p a r i n (i0 I.U./ml) and an amino acid mixture (i X concentration) (9). In all experiments the serine c o n c e n t r a t i o n was 0.35 mM. W h e n 135Slmethionine was the substrate, c y s t e i n e in the m e d i u m was 0.2 m M and w h e n 13SSlcysteine was the substrata, m e t h i o n i n e in the m e d i u m was 0.i mM. All other amino acids w e r e p r e s e n t at the c o n c e n t r a t i o n s s p e c i f i e d (9). Total 13SSleysteine or 13SSlmethionine uptake by h e p a t o c y te s u s p e n s i o n s was d e t e r m i n e d a c c o r d i n g to a f i l t r a t i o n procedure p r e v i o u s l y d e s c r i b e d (10) except that 47 m m glass fiber filters (Whatman GF/C) w e r e c o u n t e d in a toluene fluor. Incorp o r a t i o n of 35S into p r o t e i n was m e a s u r e d by the same p r o c e d u r e except that 3 x 6 ml w a s h e s of the cells w i t h 10% t r i c h l o r o a c e tic acid w e r e used to remove a c i d - s o l u b l e 3SS. P r e p a r a t i o n of 2 ~ 4 - d i n i t r o p h e n y l derivatives. Hepatocytes, l0 G C elis/ml, w e r e r e m o v e d f r o m i n c u b a t i o n m i x t u r e s by c e n t r i f u g a tion (50 x g for 2 min) and w a s h e d once by r e s u s p e n s i o n in K r e b s - H e n s e l e i t b u f f e r c o n t a i n i n g 2% bovine serum a l b u m i n followed by a second c e n t r i f u g a t i o n . The cell p e l l e t was t r e a t e d w i t h 1.0 ml of 3.3% p e r c h l o r i c acid and the p r o t e i n r e m o v e d by c e n t r i f u g a t i o n . A 0.5 ml a l i q u o t of the s u p e r n a t a n t was reacted w i t h i0 Z1 of p e r f o r m i c acid (20:1 V / V of 98% formic acid, 30% HzO 2 at room t e m p e r a t u r e for 1 hr prior to use) for 15 m i n and the excess p e r f o r m i c acid d e s t r o y e d w i t h 5 ~i of 48% hydr o b r o m i c acid. The r e a c t i o n m i x t u r e was s a t u r a t e d w i t h s o d i u m b i c a r b o n a t e and r e a c t e d w i t h an a l c o h o l i c s o l u t i o n of l-fluoro2,4-dinitro-benzene (FDNB] for 4 hrs in the dark (ii). H i g h - p e r f o [ m a n c e !iquid c h r o m a t o g r a p h y . A n a l i q u o t of the F D N B r e a c t i o n m i x t u r e was i n j e c t e d onto a 4 x 250 m m Micro B o n d a p a k amine c o l u m n (Waters) and the DNP d e r i v a t i v e s i s o l a t e d by gradient elution (0.05 to 0.4 M sodium acetate, pH 4.6, in 80% methanol) using a Spectra Physics model 3500 liquid c h r o m a t o g r a p h e q u f p p e d w i t h a UV d e t e c t o r (350 nm) and a Spectra Physics S y s t e m I integrator. A p p r o p r i a t e fractions were c o l l e c t e d and a s s a y e d for 35S w i t h A q u a s o l (New E n g l a n d N u c l e a r Co.) in a liquid s c i n t i l l a t i o n counter. G l u t a t h i o n e sulfonic a c i d DNP was q u a n t i t a t e d by internal s t a n d a r d i z a t i o n and c o m p a r i s o n w i t h a u t h e n t i c g l u t a t h i o n e s u l f o n a t e (12) after c o n v e r s i o n to the DNP derivative. Cysteic a c i d DNP (Sigma) was u s e d as a reference to q u a n t i t a t e cysteic acid formed by p e r f o r m i c acid o x i d a t i o n of i n c u b a t i o n m e d i a c o n t a i n i n g cysteine. F u r t h e r details of these p r o c e d u r e s including the s e p a r a t i o n of DNP d e r i v a t i v e s of g l u t a t h i o n e disulfide, c y s t e i n e g l u t a t h i o n e m i x e d disulfide, and y - g l u t a m y l cysteic a c i d will be d e s c r i b e d e l s e w h e r e (13).
1258
Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60- 5S] CYSTEINE UPTAKE / 10B CELLS 50
40-
'°'~'o~O~o
30, c 20-
/o/m~o~m
TCA INSOLUBLE/ 106CELLS
10-
T..E I.RSl
T,ME IHRSI
Figure 1. Uptake of 3SS during the i n c u h a t i o n of k e p a t o c y t e s w i t h [3Ss]cysteine. The nmoles of 35S w e r e c a l c u l a t e d from the specific a c t i v i t y of the [35S]cysteine in the mediLzm. [35S]cysteine c o n c e n t r a t i o n s in the m e d i u m were A-A, 0.2 ~ ; ~-~, 1.0 mM; o-o, 3.0 ng~; e-o, 5.0 ~M.
RESULTS The c r i t e r i a ne or m e t h i o n i n e
used for a s s e s s i n g
the c o n t r i b u t i o n
sulfur to net g l u t a t h i o n e
tocytes was the rate of amino acid uptake, soluble
~SS i n t r a c e l l u l a r
sis and the
ass specific
pool,
biosynthesis
rate of net g l u t a t h i o n e
activity
of g l u t a t h i o n e
amino acid in the medium.
take of
at 0.2 m M by h e p a t o c y t e s
acid-soluble
3SS pool that did not exceed
by hepa-
the size of the acid-
that of the substrata 135Slcysteine
of c y s t e i -
synthe-
relative
to
The limited upresulted
5 nmoles/106
in an cells
w h i l e more that 20 nmoles of g l u t a t h i o n e
were being s y n t h e s i z e d
(Fig. i, T a b l e
ratio of i n t r a c e l l u l a r
i). The specific
J 3SSJglutathione
relative
to
activity
J35Slcysteine
in the m e d i u m did not
exceed 0.19 w h i c h agreed very well with the cysteine lues
(Fig. i). A similar r e l a t i o n s h i p
J3 ~ S J g l u t a t h i o n e cysteine
specific
incubations
between
3SS uptake and
a c t i v i t y was o b s e r v e d
(Fig. i, Table
1259
i).
uptake va-
for 1.0 ~
j3SSJcysteine
L3SsI
at 3.0 and
Vol.
77,
No.
4,
1977
BIOCHEMICAL
AND
BIOPHYSICAL
COMMUNICATIONS
R O
,rJ
4J
r~
~. O
p~. O
.q~ O
~ O
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O +1 ;.~
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O +1 00
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t--q
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c,q
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u)
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Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Z0 ,5 .[30% "I,M O ]/EoTH O I/N N IO EUP 'T ~AKE1/0%ELLS 30 "6
20
TCA N ISOLUBLE1/0BCELLS
/ a / " ~ a
TIME (HRS)
TIME (HRS)
F i g u r e 2. Uptake of 3SS during the i n c u b a t i o n of h e p a t o c y t e s w i t h [3SS]methionine. The nmoles of 3SS w e r e c a l c u l a t e d from the specific a c t i v i t y of the [3SS]meth~on~ne in the medin/m. [3SS]methionine c o n c e n t r a t i o n s in the m e d i u m w e r e A-A, 0.i raM; A-A, 0.3 mM; ~-~, 1.0 mM; o-o, 3.0 m M and e-e, 5.~ mM.
5.0 m M gave first hr specific respectively,
again r e f l e c t i n g
these higher c o n c e n t r a t i o n s .
not to a d e t e c t a b l e 13SSlmethionine
2, T a b l e
at 0.3,
i). Less of the
This
termediates,
about
activity
into
of
135Slcys -
25% at 0.2 mM c y s t e i n e
and formed an a c i d - s o l u b l e became
pool of
acid-soluble
13SSlglutathione
i n d i c a t e d that a s i g n i f i c a n t
trans-
13SSlglutathione
I35Slmethionine
pool of
~sS (Fig.
pool of
than that of
part of w h i c h could be thiols,
but
concentrations.
1.0 and 3.0 m M was readily
but not completely,
3SS was t r a n s f o r m e d teine.
a g r e a t e r uptake of amino acid at
amount at higher c y s t e i n e
into h e p a t o c y t e s
that rapidly,
ratios of 0.75 and 0.73
The specific
teine in the m e d i u m d e c r e a s e d
ported
activity
13SSlcys -
3Ss labeled in-
accumulated
in the
cells.
This was c o n f i r m e d by thiol analyses
using the Saville
method
(14). By the third hr of incubation,
maximum
tivity ratios were a c h i e v e d
(0.37,
and 3.0 m M
respectively).
activity
I 3SSlmethionine,
0.52 and 0.61 for 0.3,
ratios r e f l e c t the r a p i d i t y
is t r a n s f o r m e d
ac-
1.0
The one hr specific
in w h i c h m e t h i o n i n e
into the thiol of g l u t a t h i o n e
1261
specific
sulfur
by the hepatocytes.
Vol. 77, No. 4, 1977
Efflux
of g l u t a t h i o n e
was m e a s u r e d /hr/106
and found
cells w h e n
medium were values
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to be nearly
cysteine
0.2 and
respectively.
of the e x t r a c e l l u l a r
The total
lular)
was
tration
cells.
of g l u t a t h i o n e
Efforts
glutathione
exists
cells.
plus
Increasing
activity the
glutathiextracel-
the concen-
in the m e d i u m
increased
of 7 to 8 n m o l e s / h r / 1 0 6
in p r o g r e s s
to d e t e r m i n e
and to e s t a b l i s h
as a m i x e d
in the
essentially
(cellular
to a m a x i m u m
state of this g l u t a t h i o n e
concentrations The specific
or m e t h i o n i n e
are c u r r e n t l y
at 3.8 ± 0.8 nmoles
were
synthesized
cysteine
into the m e d i u m
for the i n t r a c e l l u l a r
ii to 22 n m o l e s / h r / 1 0 6
of either
the e f f l u x
glutathione
values
glutathione
constant
and m e t h i o n i n e
0.1 mM,
same as the c o r r e s p o n d i n g one.
from the h e p a t o c y t e s
disulfide
whether
the redox
any of the
of cysteine.
DISCUSSION Freshly thione,
isolated
rapidly
22 n m o l e s / h r / 1 0 6 (8) this
rate
rate of a b o u t activity
provide
Assuming
for p e r f u s e d
liver
data
support
may
provide
the c o n c l u s i o n from p r o t e i n
nearly
source
are known (16,17)
in normal
50% of the i n t r a c e l l u l a r
derived
and each amino
rates
tely
onine
liver
one half
free amino that w i t h
turnover of the
thesis.
1262
of liver
synthesis
(2). The
of sulfur
high
(5,6,
approxima-
(5). These
hepatocytes
and the i n c u b a t i o n sulfur
In vi-
hepatocytes
contributes acid pool
may
formed.
to be very
isolated
specific
acid at low con-
and i s o l a t e d
fed rats
of ii to
w e t wt.
w i t h an in vivo of
of gluta-
at a rate
one half of the g l u t a t h i o n e
turnover
turnover
depleted
108 c e l l s / g m
that an endogenous
approximately
Protein
tripeptide
w e t wt.
of g l u t a t h i o n e
indicated
as also
(18).
cells.
this
2 ~moles/hr/gm
vo liver p r o t e i n 15)
replenished
is in good a g r e e m e n t
ratios
centration
hepatocytes, that w e r e
meth~
medium
for g l u t a t h i o n e
syn-
Vol. 77, No. 4, 1977
Studies
on intact
F o r example, methionine found
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
into rat
m a y be r e l a t e d
vatives
and m a j o r
effect
of c y s t i n e
rapid
Even
(20,21),
in rat liver
synthetase,
via
of g l u t a t h i o n e
as
caused
glutathione
of the serious
of a m a j o r
the c y s t a t h i o n i n e during
(22),
methionine-sparing
Thus,
to the c o n s e q u e n c e s
trans-sulfuration
prevented
in rats
and k i d n e y
35S
equally
for y - c y s t a t h i o n a s e
established
biosynthesis.
of
a c i d deri-
methionine
by e t h i o n i n e
I~5SI
results
was
of m e r c a p t u r i c
(24) may be an i n d i c a t i o n
be g i v e n
these
that m e t h i o n i n e
a substrate
the firmly
fraction
(19). Also,
of y - g l u t a m y l c y s t e i n e
in g l u t a t h i o n e
biosynthesis
in dogs
decreases
(23).
should
shown
this conclusion. introduced
the m a j o r
in the f o r m a t i o n
sulfoximine,
levels
tion
with
of liver g l u t a t h i o n e
as an i n h i b i t o r
thionine
tissue
has b e e n
with bromobenzene
and m e t h i o n i n e
rapid
liver
with
of p a r e n t e r a l l y
to the o b s e r v a t i o n s
as c y s t i n e
the d e p l e t i o n
well
are c o n s i s t e n t
the t r a n s f o r m a t i o n
in g l u t a t h i o n e
effective
rats
extensive
role
of me-
considerarole
pathway
of
for the
glutathione
con-
jugation. ACKNOWLEDGEMENTS The w o r k r e p o r t e d in this paper was u n d e r t a k e n during the tenure of one of us (D.J.R.) on an A m e r i c a n C a n c e r S o c i e t y Eleanor Roosevelt-International C a n c e r F e l l o w s h i p a w a r d e d by the I n t e r n a t i o n a l U n i o n A g a i n s t Cancer. The a u t h o r s w i s h to thank the S w e d i s h M e d i c a l R e s e a r c h C o u n c i l for f i n a n c i a l support (grant no. 03X-2471) and Spectra Physics Co. for the loan of a liquid c h r o m a t o g r a p h and a S y s t e m I i n t e g r a t o r for use in this study. We thank Dr. J o h a n H ~ g b e r g for very f r u i t f u l discussions, Mrs. G u n - B r i t t Sundby and Mrs. A n n i k a K r i s t o f e r s o n for e x c e l l e n t t e c h n i c a l a s s i s t a n c e and Dr. D e a n Jones and D o c e n t Sten Jakobsson for s u g g e s t i o n s d u r i n g m a n u s c r i p t p r e p a r a t i o n . REFERENCES i. 2. 3. 4.
Meister, A. and Tate, S.S. (1976) Ann.Rev. Biochem. 45, 559604. White, I.N.H. (1976) C h e m . - B i o l . Interactions, 13, 333-342. T a t e i s h i , N., Higashi, T., Shinya, S., Naruse, A. and Sakamoto, Y. (1974) J.Biochem. 75, 93-103. Boyland, E. and Chasseaud, L.F. (1969) A d v . E n z y m o l . 32, 173-219.
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5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Gan, J.C. and Jeffay, H. (1971) Biochim.Biophys.Acta, 252, 125-135. Glass, R.D. and Doyle, D. (1972) J.Biol.Chem. 247, 5234-5242. Boyland, E. and Chasseaud. L.F. (1967) Biochem.J. 104, 95-102. H~gberg, J. and Kristoferson, A. (1977) Eur.J.Biochem. 74, 77-82. Seglen, P.O. (1976) Biochim. Biophys.Acta, 442, 391-404. Czech, M.P. (1976) Molecular and Cellular Biochem. ii, 51-63. Kesner, L., Muntwyler, E., Griffin, G.E. and Quaranta, P. (1967) Methods of Enzymol. ii, 94-108. Calam, D.H. and Waley, S.G. (1962) Biochem.J. 85, 417-419. Reed, D.J. (1977) Manuscript in preparation. Saville, B. (1958) Analyst, 83, 670-672. Scornik, O.A. and Botbol, V. (1976) J.Biol.Chem. 251, 28912897. Mortimore, G.E. and Mondon, C.E. (1970) J.Biol.Chem. 245, 2375-2383. Woodside, K.H. and Mortimore, G.E. (1972) J.Biol.Chem. 247, 6474-6481. Seglen, P.O. (1975) Biochem. Biophys.Res.Commun. 66, 44-52. Shtutman, Ts.M. and Biber, A. (1973) Ukr. Biokhim. Zh. 45, 439-443. White, A. and Lewis, H.B. (1932) J.Biol.Chem. 98, 607-624. Stekol, J.A. (1937) J.Biol.Chem. 117, 147-159. Glaser, G. and Mager, J. (1974) Bioehim.Biophys.Acta, 372, 237-244. Palekar, A.G., Tate, S.S. and Meister, A. (1975) Biochem. Biophys.Res.Commun. 62, 651-657. Finkelstein, J.D. and Mudd, S.H. (1967) J.Biol.Chem. 242, 873-880.
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