Download Hydrolysisof Glutathioneby Human Liver `y

CLIN. CHEM. 25/1, 75-79 (1979)
Hydrolysisof Glutathioneby Human Liver ‘y-Glutamyltransferase
Leslie M. Shaw and David A. Newman
We studied the catalytic hydrolysis of glutathione by human
tathione. Reasoning from these data, we propose a catabolic
pathway for glutathione and glutathione conjugates in human
liver. This may have important metabolic implications
for the
production from glutathione was maximal at pH 7.4 (37 #{176}C). diseased liver, because the activity of this enzyme in liver is
significantly
increased in various hepatic diseases (8).
Kinetically, the liver enzyme is similar to human kidney
liver
-y-glutamyltransferase
acid -y-glutamyltransferase,
[(y-glutamyl)-peptide:amino
EC 2.3.2.21. Glutamate
‘y-glutamyltransferase:
their respective Km values with
glutathione as substrate are similar (0.096 X iO
mol/L
and 0.097 X iO
mol/L, respectively).
S-Methylglutathione was hydrolyzed
at a slightly higher rate than glutathione by liver y-glutamyltransferase.
From these findings
and other established properties of liver and kidney yglutamyltransferase
we propose that human liver is an
important site of glutathione catabolism and that ‘y-glutamyltransferase
in liver catalyzes the first step of the
catabolism of glutathione and glutathione conjugates in this
organ.
AdditionalKeyphrases:
liver disease
-
catabolic
pathways
Recent studies in several laboratories have demonstrated
that the physiological
role of y-glutamyltransferase
(EC
2.3.2.2) is to catalyze the first step of the breakdown of glutathione or its S-substituted
conjugates to produce L-glutamate and L-cysteinylglycine
or S-substituted
L-cysteinylglycine (1-4). The original proposal (5) that the enzyme
functions in vivo as a transpeptidase,
and as such plays a key
role in amino acid transport across cell membranes, has never
been proven. In fact, recent evidence strongly argues against
this as its physiological
role in man (6) as well as in experimental animals (1, 2). Most biochemical and clinical studies
of it make use of the synthetic substrates -y-glutamyl-4-nitroanilide
or ‘y-glutamyl-3-carboxy-4-nitroanilide
as y-glutamyl donor substrates and glycylglycine
as the acceptor
substrate. Assays with these substrates are convenient and
straightforward,
especially when compared to the much more
tedious procedures
in which the physiological
substrate,
glutathione,
is used.
In studies of y-glutamyltransferase
in which glutathione
or glutathione
conjugates were the substrate, the enzyme
source was kidney. The kidney is now considered
in the
rat-and,
by implication,
in man-to
be the major site of catabolism of glutathione
and glutathione
conjugates (4, 7). In
this work we demonstrate
that human liver y-glutamyltransferase has kinetic properties very similar to that in kidney with glutathione
as substrate and that the human liver
enzyme catalyzes efficiently
the hydrolysis of S-methylgluToxicology and Special Enzymology Laboratory, Division of Laboratory Medicine, Wm. Pepper Laboratory, Department of Pathology,
School of Medicine, University of Pennsylvania, Philadelphia, PA
19104.
Received Oct. 3, 1978; accepted Oct. 24, 1978.
Materials
Glutathione,
S-methylglutathione,
the sodium salt of
adenosine-5’-diphosphate
(Grade I), tris(hydroxymethyl)
aminomethane
hydrochloride,
3-nicotinamide
adenine dinucleotide (Grade III), imidazole (Grade III), and maleic acid
were obtained from Sigma Chemical Co., St. Louis, MO 63178;
glycine and dithiothreitol
from Bio-Rad Laboratories,
Richmond, CA 94804; hydrazine hydrate and perchioric acid from
Fisher Scientific Co., Philadelphia,
PA 19406; and glutamate
dehydrogenase
(EC 1.4.1.3, from bovine liver, in glycerol!
water, 1:1) from P-L Biochemicals Inc., Milwaukee, WI 53205.
All other chemicals used were of the highest analytical quality
available. -y-Glutamyltransferase
was prepared from human
liver through the batch diethylaminoethylcellulose
step and
from human kidney through the DE-52 chromatography
step,
both as described by Shaw et al. (9).
Methods
For the study of glutamate production from glutathione
or
its S-methyl conjugate, -y-glutamyltransferase
prepared from
human liver as described above was incubated in 2 mL of a
reaction mixture, at 37 #{176}C,
containing,
per liter, 5 mmol of
glutathione
(or S-methylglutathione),
10 mmol of dithiothreitol,
and 100 mmol of tris(hydroxymethyl)aminomethane (pH 7.4 unless otherwise noted). The reaction was
terminated
at 30 mm (unless otherwise noted) by adding of
2 mL of perchloric acid (1.0 mol!L). After centrifugation,
a
2-mL aliquot of the supernatant fluid was adjusted to pH 9.0
with 0.5 mL of a K;1P04 solution (1.93 mol/L). Duplicate reaction blanks were included in each experiment.
The same
incubation,
reaction termination,
and final pH adjustment
procedures as above were followed except that distilled water
was used in place of the enzyme.
The i-glutamate
produced by the enzymic hydrolysis of
glutathione
was quantitatively
determined
by the enzymic
method of Bernt and Bergmeyer (10). In this procedure a
0.4-mL aliquotof terminated reaction
mixture is added to an
incubation medium (0.92mL) in each of two testtubes containinga pH 9.0glycine-hydrazinebuffer(perliter,
300 mmol
of glycine and 250 mmol of hydrazine), adenosine 5’-phosphate (1.0 mmol/L)
and nicotinamide
adenine dinucleotide
(1.6 mmol!L). The A340 nm (A1) of thismixture in one of the
two test tubes is determined. Glutamate dehydrogenase (0.02
mL; from bovine liver; in glycerol/water,
1:1; catalytic activity
4.5 kU/L) is added to the second test tube. The reaction
CLINICAL CHEMISTRY, Vol. 25. No. 1, 1979
75
140
120
//‘N
a,
(I)
0
E
80
a,
a
E
0
60
/[Glutathione]
(m mol/li’
40
20
D
68
7.6
84
92
>
pH
Fig. 1. Glutamate
production
from glutathione
as a function
of
pH
Glutamate was measured by the enzymic procedure described under Methods,
at pH values (37 #{176}C)
of 6.0, 7.0, 7.4, 8.0,8.6, and9.0. Incubationwasfor 30 mm
as described under Methods, with a glutathione concentration of 5 mmol/L and
buffer limidazole at pH 6.0; trls(hydroxymethyl)aminomethane
at the other pH
values] concentrationsof 100 mrnol/L Eachpointis the average glutamatevalue
for
duplicate incubationflasks.
I / [Glutathione]
Fig. 2. Double-reciprocal
plots: The reciprocal of initial velocity
(1 / v) is plotted vs. the reciprocal of glutathione concentration
(L/mmol) for liver and kidney -y-glutamyltransferase
In these experiments
mixture is
after which
A1 and A2
calculated.
then incubated for 45 mm at room temperature,
A:340 (A2) is determined.
The difference between
(A2 - A1) for each sample and reaction blank was
Then Asampie
Ablank
= LAgiutamate
was used
for the calculation of glutamate concentration
mula
[glutamate,
.Agiut.amate
mmol!L]
=
X TV
from the forX 2.5
6.22 X SV
where TV (1.34 mL) is the total volume of the glutamate assay,
SV (0.4 mL) is the sample volume, 2.5 is the dilution factor,
and 6.22 is the millimolar
absorptivity
of NADH at 340 nm.
All absorbance measurements
were made with a Stasar III
spectrophotometer
(Gilford Instrument
Labs., Oberlin, OH
44074).
In two experiments glutamate formation from glutathione
was measured with a Model D-500 amino acid analyzer
(Durrum Chemical Corp., Palo Alto, CA 94303). The same
incubation
conditions as above were used to study the production of glutamate by the action of ‘y-glutamyltransferase
on glutathione.
We terminated
the reactions by placing the
mixtures in a boiling water bath for 3 mm, then rapidly cooling
them in ice. At this point 0.1 mL of norleucine internal standard (6.5 mmol/L) was added to each reaction mixture, followed by 0.05 mL of a 0.01 mol!L solution of dithiothreitol.
lodoacetamide,
5 mg, was then added to each reaction mixture, with thorough mixing. After 15 mm at room temperature,
enzyme protein was removed from the reaction mixtures by
filtration
through collodion membranes (ii). These proteinfree samples were analyzed with the amino acid analyzer,
according to the regular protocol for amino acid analysis with
this system (12). The average elution times for standard so-
lutions of the 5-acetamido derivatives of glutathione and
L-cysteinylglycine, and of L-glutamate were 12 mm, 40 mm,
and 17 mm, 10 s, respectively.
Results
Precision
of the
enzymic
solution of L-glutamate
centration
76
of 100 smol/L.
(m mol/l1’
was
assay.
An aqueous
having a nominal conof this solution were frozen.
glutamate
prepared
Aliquots
CLINICAL CHEMISTRY, Vol. 25, No. 1, 1979
glutamate was determined
with an amino acid analyzer
as described under Methods
The glutamate content of one of these aliquots was determined each day an experiment was conducted. The mean,
standard deviation, and coefficient of variation for seven determinations were 105.6 tmol!L, 6.3 mol/L, and 5.96%, respectively, indicating acceptable precision for this method.
Variation
of glutathione
hydrolysis
with time, enzyme
amount and pH. We incubated 0.49 U of liver in the glutathione reaction mixture for various intervals up to 45 mm. The
increase in glutamate production
with time was linear over
this interval. To study the variation of glutamate production
with enzyme amount, we incubated zero to 0.74 U of liver ‘yglutamyltransferase
with glutathione reaction mixture for 30
mm, and found the quantity
of glutamate produced to be
proportional
to the amount of enzyme over the entire range.
Glutathione
hydrolysis by liver -y-glutamyltransferase
was
measured as a function of pH over the range of pH 6.0 to pH
9.0. As shown in Figure 1, L-glutamate production is maximal
at pH 7.4.
Kinetic studies. Figure 2 illustrates double-reciprocal plots
of the rate of glutathione
hydrolysis vs. glutathione
concentration. Glutamate production by the catalytic action of liver
and kidney -y-glutamyltransferase
on glutathione
was measured over the range of glutathione
concentrations
of 0.025
to 5.0 mmol/L. We determined
Km values for the liver and
kidney enzymes, using the mathematical
model and computer
methods previously described (9, 13). Evidently
the kinetic
behavior of liver ‘y-glutamyltransferase
is very similar to that
of the kidney enzyme, because their respective Km values were
almost identical (0.096 X 10
mol!L for the enzyme from
liver, 0.097 X iO
mol!L for that from kidney).
We incubated the glutathione
conjugate S-methylglutathione in place of glutathione
in the standard incubation
system, to determine whether or not this compound is a substrate for liver y-glutamyltransferase.
As summarized in Table
1, S-methylglutathione
does serve as a substrate for the enzyme from liver. In fact, the rate of glutamate production was
slightly higher (14.2%) with this substrate than for glutathione. To test the effect of including maleate in the reaction
Glutathione and Its S-Methyl Conjugate by Liver
‘y-Glutamyltransf erase
Activity
Glutathione
Glutathione + maleate
S-Methylglutathione
S-Methylglutathione + maleate
Boiled enzyme control
0.097 X 10 molfL. This Km value is
far lower than the reported 5 mmol/L concentration of glutathione in mammalian liver. In a previous study (9) we
demonstrated that glutathione substantially
and rather
equally inhibits the formation of 4-nitroaniine by the enzyme
from human liver or kidney. A further indication of the similarity of the kinetic behavior of the two with glutathione as
substrate is the fact that y-glutamyltransferase in rat kidney
tubules catalyzes the transpeptidation
reaction under the
standard conditions of Szasz (14), with use of the synthetic
‘y-glutamyltransferase,
Table 1. Effect of Maleate on Hydrolysis of
a
118.9
114.8
135.8
127.0
4.2
Nanomolesof glutamateproducedper milliliter of reaction mixture per 30
mm at 37 #{176}C.
Each result is the average of duplicate determinations. The total
volume of each reaction mixture was 2.0 mL and contained 0.74 U of human
liver y-giutamyltransferase,and, per liter, 5 mmol of glutathione or S-methylglutathione, 10 mmol of dithiothreitol, and 100 mmol of tris(hydroxymethyl)aminomethanehydrochloride buffer, pH 7.4. Maleate,when present, hada final
reaction mixture concentration of 20 mmol/L. In the boiled enzyme control an
aliquot
ofliver ‘-gIutamyltransferase was first placed in a boiling water bath
for 5 mm before introduction into the glutathione-containingreaction mixture.
After 30 mm of incubation the reaction was terminated andthe glutamateconcentrations determined as described under Methods. The amount of enzyme
we included in each reaction mixture, 0.74 U. is based on activity determined
by the Szasz (14) method at 30 #{176}C.
mixture on the rate of glutathione hydrolysis we included this
compound in the standard reaction mixture at a final concentration
of 20 mmol!L. As shown in Table 1, maleate had
little effect on the hydrolysis of either glutathione
or its Smethyl conjugate.
Hydrolysis
of glutathione
was slightly
slower in the presence of maleate (3.4%), but the difference
was within the experimental
error of the glutamate assay. In
the case of S-methylglutathione
the hydrolysis
was also
slightly slower in the presence of maleate (6.5%) but this difference barely exceeded the experimental
error of the glutamate procedure. These results contrast with the data of Tate
and Meister (15), who obtained an almost eightfold increase
by maleate of the rate of hydrolysis of S-methylglutathione
by rat-kidney
‘y-glutamyltransferase.
Perhaps this reflects a
species difference.
Discussion
The synthetic -y-glutamyl donor substrates y-glutamyl4-nitroanilide
or y-glutamyl-3-carboxy-4-nitroanilide
are the
ones most commonly used in clinical and biochemical studies
of y-glutamyltransferase
because of the much simpler assay
techniques involved. Unfortunately,
use of such unphysiological substrates has not been very helpful in the study of the
physiological
role of ‘y-glutamyltransferase.
The pH optima
with these substrates is 8.25 (9, 14) and initial velocity rates
are maximal only with high concentrations
of the dipeptide
glycylglycine
(14). In a recent detailed kinetic study, we have
shown that ‘y-glutamyltransferase
does not catalyze the hydrolysis of these substrates, whereas this is now considered
to be the physiological action of this enzyme toward its natural
substrate,glutathione (13). Thus, to gain insight into the
physiologicalroleof y-glutamyltransferaseinhuman liverwe
used the physiological substrate glutathione in this study. This
work has further potential significance because the activity
of this enzyme is increased significantly
in diseased human
liver (8); such increases would be expected to increase proportionally
the rate of glutathione
catabolism in the liver.
We have shown that the pH optimum for glutathione
hydrolysis by human liver y-glutamyltransferase
is 7.4. This
contrasts with our earlier finding with human liver -y-glutamyltransferase
of the unphysiological
pH optimum of 8.25
with the synthetic substrates (9). The Km value for liver ‘yglutamyltransferase
with glutathione
as substrate is 0.096 X
iO mol!L and isclose to the value we obtained for kidney
substrate ‘y-glutamyl-4-nitroanilide
and the acceptor substrateglycyiglycine,at 94-fold the rate of glutathione hydrolysis by kidney tubules (4). We obtained a value of 93 for
the ratio with human liver y-glutamyltransferase,
based on
the glutathione
hydrolysis data in Table 1.
Recent studies make it appear that the catabolism of glutathione and its S-substituted
derivatives
occurs primarily
in the lumen of the proximal tubule of kidney (2-4). The first
step of this pathway is the cleavage of the y-glutamyl moiety
by ‘y-glutamyltransferase
(16) to produce L-glutamate and
L-cysteinylglycine (when glutathione is substrate) or S-substituted L-cysteinylglycine (when S-substituted glutathione
is substrate) (17). The L-cysteinyl dipeptides are then hydrolyzed by a dipeptidase to produce glycine and L-cysteine
or S-substituted L-cysteine (18). According to this model
glutathione
is hydrolyzed outside of the cells in which
synthesized; one prerequisite
for this is the synthesis of
tathione by organs such as the liver, followed by its efflux
plasma. That such glutathione synthesis and efflux from
it is
glu-
into
liver
actually occurs is strongly supported by the evidence for the
inter-organ transport of glutathione in dogs (19) and by the
demonstration of glutathione efflux from the perfused rat liver
(20) and from
isolated viable rat hepatocytes
(21).
Another important feature of this model of glutathione
catabolism is the assumption that the orientation of -y-glutamyltransferase is toward the lumen of the proximal tubule.
This assumption is favored by experiments that show the
rapid hydrolysis of glutathione by isolated rat kidney tubules
(4). The finding of marked glutathionuria and glutathionemia
in a patient who lacks ‘y-glutamyltransferase
in his tissues
provides support for the inter-organ transport of glutathione
and emphasizes the importance of y-glutamyltransferase
in
the hydrolysis and conservation
of glutathione
in man (7).
In establishing this scheme for the catabolism of glutathione, the kidney has been postulated as the primary site for
the degradation of glutathione or its related S-substituted
conjugates, for good experimental reasons. The first of these
is that rat kidney contains the highest y-glutamyltransferase
activity, as compared to any other tissue (22). The second is
that when glutathione
was introduced
into the perfusate of
the perfused rat kidney it was rapidly broken down into its
constituent
amino acids (23, 24). On the other hand, when
glutathione was introduced into the perfusate of perfused rat
liver or incubated with freshly prepared erythrocytes it was
not metabolized
(4, 25).
We believe that serious consideration should be given to the
role of ‘y-glutamyltransferase
in glutathione catabolism in
human liver.
We propose that even though kidney isprobably
the major site of glutathionehydrolysisin the rat,otherorgans
such as the liver play a significant
role in glutathione
catabolism in man. This proposal is based on the following data:
(a) ‘y-Glutamyltransferase activityin human liverisabout
10-fold that in rat liver, and its activity in human kidney is
about 10-fold lower than in rat kidney (16).
(b) The kinetic properties of human liver ‘y-glutamyltransferase with glutathione
or 5-methylglutathione
as substrates are very similar to those of the kidney enzyme: (i)
Human liver y-glutamyltransferase catalyzes the hydrolysis
of glutathione and has a Km value similar to that of the kidney
CLINICAL CHEMISTRY,
Vol. 25, No. 1, 1979
77
)‘-GLu-CYS-GLY]
SH
I
Conjugation
y-GW-CYS-GLV
CH1I Glutothione-S-tronsferase
+
References
S-CH3
Glutoth,one
Hydrolysis
L-GLL)
GGT
GGT
Hydrolysis
#{149}
L -CYS-GLY
L-GLU.
L-CYS-GLY
SH
S-C H3
Dupeptidase
Dipeptudase
0
L-CYS.GLY
SH
C
erase
N-ocetyllronsf
L-CYS
‘
+
GLY
S-CH,
L-CYS
S-CH,
Mercopturuc
(.CH,
-
-s-coa)
Acid
Fig. 3. Proposed glutathione and conjugated glutathione catabolic
pathway in human liver
CH3I is used here as an exampleof an electrophilic compound that is conjugated
to glutathione with the ultimate production of the corresponding mercapturic
acid. I3LU, L-gfutamate;Ct’S, L-cysteine; GL V. glycine. GOT, -y-glutamyitrans-
ferase
SH
enzyme. (ii) In this work we have shown that human liver
y-glutamyltransferase
catalyzes hydrolysis of S -methylglutathione equally as efficiently
as hydrolysis of glutathione.
Similarly,
others have shown that -y-glutamyltransferase
in
isolated rat-kidney tubules efficiently catalyzes the hydrolysis
of S-methylglutathione
(3). (iii) As indicated above, the ratio
of the rate of p-nitroaniline
production in the standard Szasz
(14) assay to the rate of glutamate
production
from glutathione obtained with liver -y-glutamyltransferase
was comparable to that reported by others for rat kidney tubule y
glutamyltransferase.
(c) Glutathione added to the perfusate of perfused rat liver
is not metabolized. This should not he considered as evidence
that the liver is not involved in glutathione hydrolysis, because
the perfusate only comes into intimate contact with the sinusoidal side of hepatocytes, and y-glutamyltransferase
is not
present in the sinusoidal membrane of rat or human hepatocytes. Histochemically,
it has been shown to be located in the
canalicular plasma membranes and in the plasma membrane
of biliary duct epithelial cells of normal rat and human liver
(26, 27). A permeability
barrier exists between the canaliculi
and the space of Disse (28). Thus glutathione
added to a
perfusate of the perfused rat liver is unlikely to come into
contact with y -glutamyltransferase.
(d) The N-acetylcysteine, glutathione, L-cysteinylglycine,
and L-cysteine conjugates of a group of hydrocarbon epoxides
have been detected in the bileof ratstreated with the corresponding polycyclic hydrocarbons
(29-31).
This is but one
illustration
of the fact that glutathione
conjugates, once
formed in liver cells, are excreted in high concentrations in bile
(17). The presence in bile of the catabolites-namely,
the
N-acetylcysteine,
L-cysteinyl glycine, and L-cysteine conjugates-is
evidence in favor of the role of liver y-glutamyltransferase in glutathione
catabolism, because the first step
in the breakdown of conjugated glutathione is the production
of conjugated L-cysteinylglycine
by the catalytic action of
y-glutamyltransferase.
Numerous drugs and toxic agents are
metabolized
by this pathway.
A summary of the proposed steps in the catabolism of glutathione in human liver is displayed in Figure 3.
We are indebted to Lorette Petersen for the preparation of human
liver and kidney -y-glutamyltransferase used in this work; to Jack
London for the determination of the kinetic constants; to Donald
Fetterolf and Ada Bello for the amino acid analyses; and to Jeanne
Esposito for assistance with the manuscript.
78
CLINICAL CHEMISTRY. Vol. 25. No. 1, 1979
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Simultaneous assay of the hydrolysis and transfer reactions with
glu1amafeJ4Cjglutathione.
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6. Renal Metabolism
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to Renal Punctwn,
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unct ion: A Multifaceted
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