ARCHIVES
Vol.
OF BIOCHEMISTRY
277, No. 1, February
Inhibition Elizabeth
AND
BIOPHYSICS
15, pp. 149-154,199O
of Glutathione
J. Palmer,
J. P. MacManus,”
Reductase and Bulent
by Oncomodulin Mutus?’
*Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada KIA OR6, and TDepartment of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4
Received
July
5,1989,
and in revised
form
September
30,1989
Evidence for a specific interaction between oncomodulin and glutathione reductase is presented. Glutathione reductase (EC 1.6.4.2) isolated from either the bovine intestinal mucosa or the rat liver was bound in a Ca2+-dependent manner to oncomodulin which was covalently attached to Sepharose. In addition, glutathione reductase was able to catalyze the reduction of the disulfide-linked dimer of oncomodulin. The interaction of these proteins could also be indirectly demonstrated by monitoring glutathione reductase activity since oncomodulin was shown to inhibit the enzyme in a dosedependent manner with an apparent I& of -5 PM. The kinetic analysis of the oncomodulin-dependent effects on glutathione reductase activity indicates that oncomodulin interacts at a site other than the active site as the oncomodulin-induced inhibition was of the noncompetitive type. The in uiuo inhibition of glutathione reductase appears to be an oncomodulin-specific effect as closely related members of the troponin C superfamily such as rabbit (~15.5) or carp (~14.25) parvalbumins, as well as calmodulin, failed to affect the activity of this enzyme. The present in vitro study indicating that oncomodulin can regulate the activity of glutathione reductase could be very significant with respect to the elucidation of a physiological role for oncomodulin. o isso Academic
Press,
Inc.
Oncomodulin is an oncofetal Ca’+-binding protein of the troponin C superfamily (1). Unlike the parvalbumins, oncomodulin can undergo a Ca2+-specific conformational change (2-4). Oncomodulin (ONC)2 is ex’ To whom correspondence should be addressed. * Abbreviations used: ONC, oncomodulin; ONC-d, oxidized dimer of ONC; GSSGredase, glutathione reductase; CaM, calmodulin; SAMONC, S-iodoacetamide-labeled oncomodulin; cPV, carp P-parvalbumin; EGTA, ethylene glycol bis(@aminoethyl ether)-N,N,N’,N’tetraacetate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; It&,, the inhibitor concentration at which the activity is inhibited by 50%; KBetlVltY, apparent dissociation constant = the con0003-9861/90
Copyright All
rights
$3.00 1990 by of reproduction
0
Academic in any
Press, Inc. form
reserved.
pressed early in development and upon neoplastic transformation. It has been detected in tumors from rats, mice, guinea pigs, and humans and also in the rat placenta (1, 2,5, 6). ONC shows extensive homology to the /3-parvalbumin subclass of the troponin C superfamily, and is known to activate several calmodulin-dependent enzymes, for example, bovine heart phosphodiesterase (7) and bovine brain calcineurin (8). It is also able to stimulate DNA synthesis in Ca2+-deprived nonneoplastic cells with higher potency than calmodulin (9). Recently, our laboratory has demonstrated that in vitro ONC can undergo intermolecular disulfide bond formation. The oxidized dimer of ONC (ONC-d) appears to be the molecular species responsible for CaM-like behavior as it was shown to interact with high affinity with the amphiphilic peptide melittin, and activated two CaMdependent enzymes with Kactivity 5 to 20-fold higher than those observed for CaM (8). Despite this line of evidence, it is unlikely that in viuo this protein shares the regulation of the same processes as CaM. This is in part due to the lower affinity displayed, relative to CaM, by ONC-d for the CaM-dependent enzymes (8), but also to the fact that the disulfide bridge of ONC-d is not expected to survive in the highly reducing intracellular environment (10,ll). Therefore, elucidation of a biological role for oncomodulin must await the discovery of processesthat demonstrate absolute specificity for this protein. In this paper, we present evidence for the in vitro ONC-specific modulation of glutathione reductase, which catalyzes the NADPH-dependent reduction of glutathione disulfide (GSSG), thereby maintaining a high (300/l) intracellular [GSH]/[GSSG] ratio (12). Glutathione reductase has been extensively studied (13, 14). It is a homodimer with a subunit M, of 52.4 kDa. Each subunit contains 478 amino acids and one FAD molecule. Its catalytic mechanism and amino acid se-
centration of an activator rPV; rabbit parvalbumin.
which
gives rise to half-maximal
activation;
149
150
PALMER,
quence, as well as its three-dimensional been determined (13,15,16). MATERIALS
AND
structure,
MACMANUS,
AND
have
MUTUS
12
123456
123
METHODS
Oncomodulin was isolated from rat Morris hepatoma 5123tc (17). Bovine brain calmodulin was purified according to the method of Sharma and Wang (18). Glutathione reductase was purified from rat liver by the method of Carlberg and Mannervik (19). Carp fl-parvalbumin (~14.25) from skeletal muscle was a gift from Dr. L. Lee, University of Windsor. Glutathione reductase (EC 1.6.4.2) from bovine intestinal mucosa and rabbit skeletal muscle parvalbumin were purchased from Sigma as were all other reagents and biochemicals. Oncomodulin dimer was prepared by incubating oncomodulin in buffer A (Tris HCl, 20 mM; KCl, 150 mM, pH 7.5) containing 1 mM Ca*+ in the absence of reducing agents. The dimer was then separated from reduced oncomodulin by organomercurial-Sepharose chromatography. After application of the protein, the column was washed with buffer A to remove the disulfide-linked dimer. The monomer was then eluted with buffer A containing 10 mM 2-mercaptoethanol. The purity of all proteins was assessed by 10% SDS-PAGE on a Bethesda Research Laboratories V12 vertical slab apparatus according to the method of Laemmli (20). Protein determination was performed according to Bradford (21). Oncomodulin-Sepharose was prepared as follows. Sepharose 4B (5 ml) was activated by CNBr treatment and incubated with oncomodulin (-1 mg) overnight at 4°C. Glycine was then added to react with any remaining active groups on the Sepharose. Before use, the column was washed with 100 mM Tris-HCl, pH 7.0, containing 10 mM 2-mercaptoethanol to ensure that all the bound oncomodulin was in the monomeric form. The column was then equilibrated in 100 mM Tris-HCl, 1 mM calcium chloride, pH 7.0. The glutathione reductase (containing 1 mM calcium chloride) was applied to the column and the column was washed with equilibrating buffer. The bound glutathione reductase was then eluted with 100 mM Tris-HCl containing 2 mM EGTA. Calmodulin-Sepharose chromatography of GSSGredase was performed in the same manner. Glutathione reductase was assayed according to previously published procedures (19). The reaction mixture (1 ml) contained 40 mM Tris-HCl, pH 7, 0.1 mM NADPH, GSSG, and modulator proteins as indicated. The reaction was initiated by the addition of GSSGredase (-2 nM) and allowed to proceed for 60 s. The reaction was then terminated by the addition of 50 ~1 10% SDS. The assays were performed at 30°C in either 1 mM EDTA or 1 mM Ca2+ as indicated. The change in absorbance was monitored at 340 nm on a Shimadzu UV-160 spectrophotometer. To ensure that the reduced oncomodulin used for the titrations and kinetics was indeed reduced, it was separated from excess reducing agent by Sephadex G-25 chromatography immediately prior to use. In addition, a sample was taken at the end of the titration for SDSPAGE analysis to determine whether oxidation had occurred during the course of the experiment. Samples for SDS-PAGE to study the interaction of oncomodulin dimer with glutathione reductase were prepared as follows. Oxidized oncomodulin (15 pg) was incubated at 30°C in the presence of GSH (10 mM) f glutathione reductase (10 nM) and NADPH (0.1 mM). Control samples of oxidized oncomodulin in the presence and absence of NADPH were also incubated without enzyme. Aliquots were removed at various time intervals and the reaction was terminated by decreasing the pH to -4.0. The samples were then run on 10% SDS-PAGE under nonreducing conditions (20).
RESULTS
Intimate contact between ONC and bovine intestinal mucosa GSSGredase was first suspected during at-
C FIG. 1.
Electrophoretic analysis of ONC-d under a variety of conditions. Polyacrylamide (10%) electrophoresis was performed under denaturating conditions (0.1% SDS) in the absence of thiol reducing agents. The gels were stained with Coomasie brillant blue (R-250; 0.5%). (A) ONC-d (15 rg), lane 1; molecular weight standards, lane 2. (B) Molecular weight standards, lane 1; ONC-d, lane 5; ONC-d (15 fig) plus NADPH (0.1 mM), lane 3; bovine intestinal mucosa GSSGredase (15 fig), lane 4; ONC-d (15 rg) plus GSSGredase incubated at 30°C for 2 min, with (lane 6) or without (lane 2) NADPH. (C) Molecular weight standards, lane 1; ONC-d (15 rg) incubatedwith GSH (10 mM) at 30°C for 1 min, lane 2; 5 min, lane 3. Molecular weight standards: bovine serum albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,100), lactalbumin (14,200).
tempts at determining the stability of ONC-disulfide linked dimer (ONC-d) to reduction by physiologically relevant concentrations of GSH. In these experiments GSH, GSSGredase, and NADPH had been incubated with ONC-d for various time intervals. GSSGredase had been included in the incubation mixture to prevent the oxidation of GSH, in the event that the ONC-d disulfide was not readily accessible, and therefore required long incubation times for the reduction. The relative amounts of ONC and ONC-d in the aliquots removed at various time intervals were determined by nonreducing SDS-PAGE electrophoresis (Figure 1). From these experiments, it is apparent that the ONC-d (Fig. 1, gel A) would not survive in the intracellular environment as all of the ONC-d was converted to the monomer within 5 min in the presence of GSH (10 mM) (Fig. 1, gel Clanes 2 and 3). Surprisingly, ONC-d was also reduced when incubated only with GSSGredase (plus or minus NADPH) (Fig. 1, gel B-lanes 2 and 6). The reduction in the absence of GSH was dependent on GSSGredase as NADPH alone could not affect the conversion of ONC-d to ONC (Fig. 1, gel C-lane 3). These results suggest that GSSGredase is catalyzing the reduction of ONC-d. However, the fact that NADPH is not required for the reduction of ONC-d (Fig. 1, gel B-lane 2) would suggest that this reduction is not occuring at the active site of the enzyme. An alternate explanation would be that the ONC-d disulfide is being reduced via disulfide exchange with one of the noncatalytic thiols of GSSGredase (22). In addition, small-molecular-weight thiol contamination of the enzyme samples is not likely to be the source
GLUTATHIONE
0.000
REDUCTASE
INHIBITION
BY
0.000 0
5
10
15
20
25
5
0
10
VOLUME (ml)
0.000
151
ONCOMODULIN
15
20
25
VOLUME (ml)
0
5
V&ME
;L)
20
FIG. 2. Elution profiles of bovine intestinal mucosa GSSGredase from oncomodulinand 280 nm. (A) GSSGredase in buffer A containing 1 mM Cazf was applied to ONC-Sepharose enzyme was eluted by the application of buffer A containing 2 mM EGTA. (B) GSSGredase to ONC-Sepharose (in the same buffer). Buffer A containing 2 mM Ca2+ and 2 M magnesium any bound protein. (C) GSSGredase was applied to calmodulin-Sepharose in buffer A, 1 mM of buffer A containing 2 mM EGTA.
of the reducing potentialas the GSSGredase samples were subjected to Sephadex G-25 chromatography prior to these experiments. Although this observation is interesting, it is thought not to have any physiological relevance, in light of the demonstrated unstability of ONCd to reduction by GSH (Fig. 1, gel C). In an extension of these studies, the interaction between ONC (monomer) and GSSGredase was explored. This was attempted by subjecting the enzyme to chromatography on a column of Sepharose to which ONC was covalently linked. The ONC-Sepharose elution profiles are presented in Fig. 2. When the column was equilibrated with buffer which contained Ca2+ (1.0 mmol/liter) -$ of the GSSGredase was recovered in the breakthrough. The remainder of the enzyme was bound to the column in a Ca2+-dependent manner as it could be eluted in the presence of the Ca2+ chelator, EGTA (Fig. 2A). There are several possible explanations for the partial binding of GSSGredase to ONC-Sepharose. Of these, the most likely is that the affinity of the enzyme for immobilized ONC is probably low. Alternatively, partial binding could indicate that the enzyme population is not homogeneous. Such heterogeneity could result from either different allosteric forms of GSSGredase or, more likely, a mixture of native and denatured forms of the enzyme. The requirement of Ca2+ in the
25
calmodulin-Sepharose. Protein was monitored at (preequilibrated in buffer A with 1 mM Ca*+). The (in buffer A containing 1 mM EGTA) was applied chloride were then applied in an attempt to elute Ca’+. Bound protein was eluted by the application
binding of GSSGredase to ONC-Sepharose was further suggested by the observation that ONC-Sepharose, which was preequilibrated with buffer containing EGTA, failed to bind any GSSGredase (Fig. 2B). Furthermore, CaM does not appear to interact with GSSGredase as this enzyme failed to bind to a column of CaMSepharose (Fig. 2C). The functional consequences of ONC/GSSGredase interactions were probed by monitoring the catalytic activity of the enzyme as a function of [ONC] (Fig. 3). GSSGredase from the bovine intestinal mucosa as well as that from the rat liver was inhibited by ONC in a dosedependent manner with an estimated I&, of -5 pmol/ liter (Fig. 3B). This relatively low affinity interaction could account for the partial binding of the enzyme to ONC-Sepharose. In addition, ONC did not completely inhibit the activity of the enzymes from both tissue sources. At the highest [ONC] employed (-0.6 mM) the bovine intestinal mucosa and the rat liver enzymes retained 37 and 30% of their activity, respectively. This observation as well as the partial binding of GSSGredase to ONC-Sepharose (Fig. 2), could arise as a consequence of the heterogeneity of the enzyme samples. Further evidence for the requirement of Ca2+ in the ONC/GSSGredase interaction could be demonstrated by the fact that the ONC-induced inhibition was not ob-
152
PALMER,
c? 0 $
60.. 70.. 60.. 60..
c 0 c-l
40.. 30.. 20..
MACMANUS,
Log~%%n]
(mol/L)-4.w0
110 x
loo
.-> 2
.
BI
EGTA
90 60.. 70..
MUTUS
the absence of ONC (Fig. 4A). However, in the presence of Ca2+, increasing amounts of ONC increased both the slope and y intercept of the double-reciprocal plots of the data while not affecting the x intercept, a behavior typical of a noncompetitive inhibitor (Fig. 4B). Under identical conditions (buffer containing Ca2+) CaM or PV had no effect as the kinetic data were identical to those obtained in their absence (Fig. 4C). The inability of closely related proteins to effect the same changes on the catalytic properties of the enzyme strongly suggest that this effect is specific to the oncodevelopmental protein oncomodulin.
-6.500 X
AND
\
Reduced glutathione is present in millimolar levels in virtually all cells and is responsible for maintaining intracellular reducing conditions. Glutathione has many functions, one of which is the control of its own biosynthesis through feedback inhibition of y-glutamylcysteinyl synthase, the first committed step of the glutathi-
-x
60.. 50.. 40..
, f
30.. 20 . . to-3.500
DISCUSSION
-3.000
TABLE
FIG. 3.
Dose-dependent effects of oncomodulin, calmodulin, and parvalbumin on GSSGredase activity. The reaction mixture (30°C) contained GSSG (3 mM), NADPH (0.1 mM), and GSSGredase (2 nM). (A) Increasing amounts of calmodulin (a) or rabbit parvalbumin (0) were added in the presence of Ca*+ (1.0 mM). (B) increasing amounts of ONC were added in the presence of Ca2+ (1.0 mM) (A) or EGTA (0).
Kinetic
EDTA
(1
Ca2+ (1
mM)
(1
ONC
(1 PM)
ONC
(10
CaM
(1
rPV
(1
50.1 2.35
pM)
in
EDTA
(1
mM)
wM)
PM) pM)
B. Bovine EDTA
(1
Ca2+ (1
mM)
ONC
(1
ONC
(10
CaM
(1
rPV
(1
n Unless
GSSGredase
mM)
ONC
intestinal
mucosa
mM)
PM) FM)
FM) pM)
stated,
Reductase
K m,app(PM) V max,app (nmol/min/md
Condition A. Rat liver
served in the absence of Ca2+ (EGTA 0.1 mmol/liter) (Fig. 3B). The inhibition of GSSGredase appears to be a property specific to ONC as closely related Ca2+-binding proteins such as rabbit parvalbumin and calmodulin did not alter enzyme activity (Fig. 3A). In the next stage of these studies, the effect of ONC on the steady-state kinetic behavior of GSSGredase was examined with a view of gaining information on the ONC binding site (Table I; Fig. 4). The steady-state kinetics were performed in the presence of EDTA or Ca’+, as a function of [GSSG] with constant [NADPH] (0.1 mmol/liter). The Km,*,,* and Vmax,appvalues estimated in the absence of ONC with the liver enzymes and intestinal mucosa are summarized in Table I. The GSSGredase assay is routinely performed in the presence of EDTA (19). Since this study concerns the interaction of Ca2+binding proteins with this enzyme, its catalytic parameters were initially estimated in the presence of Ca’+. While the Km,app values were identical, within experimental error, in the presence of EDTA or Ca2+, the V max,app estimated for the enzymes from the rat liver and bovine intestinal mucosa was 2.3- and 1.67-fold lower, respectively, in Ca2+than in EDTA (Table I). When the kinetic study was repeated in the presence of ONC, with buffer containing EDTA, the double-reciprocal plots of the data were nearly superimposable to that obtained in
I
Parameters of Glutathione Utilization of GSSG”
all kinetics
were performed
f +
2.9 0.04
67.1 + 1.01 +
4.0 0.03
57.0 2.37 62.1 0.72 62.1 0.55 68.0 1.35 70.6 1.03
2.4 0.09 5.9 0.04 5.5 0.04 2.3 0.04 1.0 0.12
+ f f f f k f ic + *
GSSGredase 85.9 5.08+90.3 3.04 88.9 1.91+ 95.1 1.40
+14
94.1 3.11 98.9 3.16
f & +f
in 1
mM
0.05 f 7 k 0.02 2~ 15 0.16 +15 + 0.16
05 0.01 10 0.02 Cd%.
GLUTATHIONE
REDUCTASE
INHIBITION
BY
=iiN
1 /[GSSG]
(M-’
-02E4 -8
-iN
0
l/[GSSG]
)
-1E4
0
lE4
I/[GSSG]
153
ONCOMODULIN
2E4
lE4
2E4
I
SE4
(M-l)
?
(M-l)
FIG. 4. Kinetic analysis of GSSGredase activity in the presence of oncomodulin, calmodulin, and parvalbumin. All kinetics were - 2 nM). (A) Kinetics performed in EDTA (1.0 mM) in the presence (0) and absence (0) at 30°C (NADPH, 0.1 mM; GSSGredase FM). (B) Kinetics performed in buffer containing CaCl, (1.0 mM) and EDTA (0.01 mM) in the presence of 1 pM ONC (0) or 10 pM in the absence of ONC (0). (C) Kinetics performed in Ca” (1.0 mM) in the presence of calmodulin (1 pM) (V), rabbit parvalbumin or Ca*+ alone (0).
one biosynthetic pathway (23). In neoplastic tissues, this control mechanism has been altered as several reports have indicated that the glutathione total levels are significantly elevated (24, 25). These increased levels are thought to afford increased resistivity to chemotherapeutic agents and radiation (24,26, 27) since inhibition of glutathione synthesis resulted in sensitization of the neoplastic cells to antineoplastic treatment (24, 28). An interesting hypothesis arose out of these findings; if one could selectively alter tumor glutathione levels relative to normal cells, antineoplastic treatment would feasibly kill neoplastic tissue while leaving normal cells virtually unharmed (24,25,28). Glutathione reductase is central for maintaining the high ratio of reduced to oxidized glutathione intracellularly. This enzyme is responsible for the conversion of oxidized glutathione formed upon the action of glutathione peroxidase which converts hydrogen peroxide to water, thus decreasing the effects of several toxic agents. In addition, glutathione reductase has been illustrated to be an inducible enzyme when rat liver cells were treated with various compounds (29). This suggests that glutathione reductase is of strong importance to the protection of cells against toxic agents. The potential involvement of the oncofetal Ca’+-binding protein oncomodulin in the regulation of glutathione
performed of ONC (1 ONC (0) or (1 pM) (O),
reductase was prompted by the observation that the disulfide-linked dimer of ONC could be reduced to its monomer in the presence of GSSGredase. This is unprecedented as this enzyme has previously been demonstrated to have an absolute specificity for its natural substrate GSSG (15). An alternative explanation is that the reduction of ONC-d disulfide is not occurring at the active site but via disulfide exchange with the noncatalytic free thiols of the GSSGredase. This enzyme contains 10 Cys residues per monomer (29). Of these, Cys-58 and Cys-63 are at the active site and are reversibly oxidized to a disulfide bridge during the catalytic cycle (30). The native structure of the enzyme is thought to contain an intersubunit disulfide bridge (Cys-90). The reduction of this disulfide would result in the dissociation of the enzyme into its subunits and its concomitant inactivation as the active site is found at a subunit interface (14). The reduction of the Cys-90 intersubunit disulfide has been proposed as a potential locus for the self-regulation of the enzyme via negative feedback by its product (22). Cys-2 of GSSGredase which is found in a flexible segment of the protein has been shown to undergo disulfide exchange reactions with a variety of thiols (22). Conceivably, the ONC-d disulfide could be reduced by Cys-90 or Cys-2, or other noncatalytic thiols of GSSGredase. Although the mechanism by which the disulfide is re-
154
PALMER,
MACMANUS,
duced is unclear, the fact that this reaction takes place suggests that these two macromolecules must interact. However, the interaction of the disulfide-linked dimer with GSSGredase is not expected to occur intracellularly in view of the demonstrated lability of the ONC-d disulfide to reduction by GSH (Fig. 1, gel C). As a result, only the potential interactions between ONC (monomer) and GSSGredase were explored. In these studies, GSSGredase was shown to interact in a Ca2+-dependent manner with ONC that was covalently attached to Sepharose. What makes these findings even more interesting is the fact that under identical conditions, GSSGredase failed to bind to calmodulin-Sepharose. Additional evidence for the interaction of ONC with GSSGredase was obtained when the enzymes from bovine and murine sources were assayed in the presence of increasing amounts of ONC. Under these conditions, GSSGredases from rat liver and bovine intestinal mucosa were inhibited in an ONC dose-dependent manner with I(&,, of -5 /*mol/liter. Although this ICsOappears to be relatively high, it is very close to the levels of ONC expressed in rat Morris hepatomas (100 mg/kg or -10 pmol/liter) (31). The ONC-induced inhibition required Ca2+ as ONC had no effect on the enzyme activity when the titrations were performed in the presence of EGTA, a further indication that ONC interacts with GSSGredase in a Ca2+-dependent manner. The specificity of ONC/GSSGredase interactions was demonstrated by the fact that calmodulin and rabbit parvalbumin failed to mimic the negative modulatory effect of ONC. This is the first report of a Ca-dependent regulatory mechanism for oncomodulin which is not also a property of calmodulin. Since oncomodulin is oncofetal in origin, this would imply that this inhibition would occur upon neoplastic transformation. This feasibly could result in a different equilibrium point between reduced and oxidized glutathione compared to normal cells. GSSGredase is the main enzyme responsible for converting GSSG back to GSH. Therefore, in the presence of oncomodulin, this conversion would be decreased, resulting in decreased levels of GSH. GSH is known to inhibit yglutamylcysteinyl synthase, the first committed step of glutathione biosynthesis. This feedback inhibition would be abolished by decreased GSH levels, resulting in increased biosynthesis and possibly increased levels of total cellular glutathione. In fact, neoplastic tissues have been demonstrated to contain GSH levels up to lo-fold higher than normal ceils (24,25). These elevated levels are thought to be partially responsible for the multidrug resistivity observed in selective neoplasms (28,32). The implication of oncomodulin, an oncofetal protein, in the regulation of GSH levels via GSSGredase inhibition is potentially a significant control mechanism of neoplastic metabolism.
AND
MUTUS
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29. Carlberg, I., DePierre, J. W., and Mannervik, B. (1981) Biochim. Biophys. Acta 677,140-145. 30. Krauth-Siegel, R. L., Blatterspiel, R., Saleh, M., Schultz, E., Schirmer, R. H., and Untucht-Grau, R. (1982) Eur. J. Biochem.
121,259-267. ACKNOWLEDGMENTS This work was supported by grants from the Natural Engineering Research Council of Canada, The Research versity of Windsor, and the J. P. Bickell Foundation.
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31. MacManus, J. P., and Brewer, mology, Vol. 139, pp. 156-168, 32. Kramer, 697.
R. A., Zakner,
L. M. (1987) in Methods Academic Press, Orlando,
J., and Kin,
G. (1988)
Science
in EnzyFL. 241,694-
Vol.
OF BIOCHEMISTRY
277, No. 1, February
Inhibition Elizabeth
AND
BIOPHYSICS
15, pp. 149-154,199O
of Glutathione
J. Palmer,
J. P. MacManus,”
Reductase and Bulent
by Oncomodulin Mutus?’
*Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada KIA OR6, and TDepartment of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4
Received
July
5,1989,
and in revised
form
September
30,1989
Evidence for a specific interaction between oncomodulin and glutathione reductase is presented. Glutathione reductase (EC 1.6.4.2) isolated from either the bovine intestinal mucosa or the rat liver was bound in a Ca2+-dependent manner to oncomodulin which was covalently attached to Sepharose. In addition, glutathione reductase was able to catalyze the reduction of the disulfide-linked dimer of oncomodulin. The interaction of these proteins could also be indirectly demonstrated by monitoring glutathione reductase activity since oncomodulin was shown to inhibit the enzyme in a dosedependent manner with an apparent I& of -5 PM. The kinetic analysis of the oncomodulin-dependent effects on glutathione reductase activity indicates that oncomodulin interacts at a site other than the active site as the oncomodulin-induced inhibition was of the noncompetitive type. The in uiuo inhibition of glutathione reductase appears to be an oncomodulin-specific effect as closely related members of the troponin C superfamily such as rabbit (~15.5) or carp (~14.25) parvalbumins, as well as calmodulin, failed to affect the activity of this enzyme. The present in vitro study indicating that oncomodulin can regulate the activity of glutathione reductase could be very significant with respect to the elucidation of a physiological role for oncomodulin. o isso Academic
Press,
Inc.
Oncomodulin is an oncofetal Ca’+-binding protein of the troponin C superfamily (1). Unlike the parvalbumins, oncomodulin can undergo a Ca2+-specific conformational change (2-4). Oncomodulin (ONC)2 is ex’ To whom correspondence should be addressed. * Abbreviations used: ONC, oncomodulin; ONC-d, oxidized dimer of ONC; GSSGredase, glutathione reductase; CaM, calmodulin; SAMONC, S-iodoacetamide-labeled oncomodulin; cPV, carp P-parvalbumin; EGTA, ethylene glycol bis(@aminoethyl ether)-N,N,N’,N’tetraacetate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; It&,, the inhibitor concentration at which the activity is inhibited by 50%; KBetlVltY, apparent dissociation constant = the con0003-9861/90
Copyright All
rights
$3.00 1990 by of reproduction
0
Academic in any
Press, Inc. form
reserved.
pressed early in development and upon neoplastic transformation. It has been detected in tumors from rats, mice, guinea pigs, and humans and also in the rat placenta (1, 2,5, 6). ONC shows extensive homology to the /3-parvalbumin subclass of the troponin C superfamily, and is known to activate several calmodulin-dependent enzymes, for example, bovine heart phosphodiesterase (7) and bovine brain calcineurin (8). It is also able to stimulate DNA synthesis in Ca2+-deprived nonneoplastic cells with higher potency than calmodulin (9). Recently, our laboratory has demonstrated that in vitro ONC can undergo intermolecular disulfide bond formation. The oxidized dimer of ONC (ONC-d) appears to be the molecular species responsible for CaM-like behavior as it was shown to interact with high affinity with the amphiphilic peptide melittin, and activated two CaMdependent enzymes with Kactivity 5 to 20-fold higher than those observed for CaM (8). Despite this line of evidence, it is unlikely that in viuo this protein shares the regulation of the same processes as CaM. This is in part due to the lower affinity displayed, relative to CaM, by ONC-d for the CaM-dependent enzymes (8), but also to the fact that the disulfide bridge of ONC-d is not expected to survive in the highly reducing intracellular environment (10,ll). Therefore, elucidation of a biological role for oncomodulin must await the discovery of processesthat demonstrate absolute specificity for this protein. In this paper, we present evidence for the in vitro ONC-specific modulation of glutathione reductase, which catalyzes the NADPH-dependent reduction of glutathione disulfide (GSSG), thereby maintaining a high (300/l) intracellular [GSH]/[GSSG] ratio (12). Glutathione reductase has been extensively studied (13, 14). It is a homodimer with a subunit M, of 52.4 kDa. Each subunit contains 478 amino acids and one FAD molecule. Its catalytic mechanism and amino acid se-
centration of an activator rPV; rabbit parvalbumin.
which
gives rise to half-maximal
activation;
149
150
PALMER,
quence, as well as its three-dimensional been determined (13,15,16). MATERIALS
AND
structure,
MACMANUS,
AND
have
MUTUS
12
123456
123
METHODS
Oncomodulin was isolated from rat Morris hepatoma 5123tc (17). Bovine brain calmodulin was purified according to the method of Sharma and Wang (18). Glutathione reductase was purified from rat liver by the method of Carlberg and Mannervik (19). Carp fl-parvalbumin (~14.25) from skeletal muscle was a gift from Dr. L. Lee, University of Windsor. Glutathione reductase (EC 1.6.4.2) from bovine intestinal mucosa and rabbit skeletal muscle parvalbumin were purchased from Sigma as were all other reagents and biochemicals. Oncomodulin dimer was prepared by incubating oncomodulin in buffer A (Tris HCl, 20 mM; KCl, 150 mM, pH 7.5) containing 1 mM Ca*+ in the absence of reducing agents. The dimer was then separated from reduced oncomodulin by organomercurial-Sepharose chromatography. After application of the protein, the column was washed with buffer A to remove the disulfide-linked dimer. The monomer was then eluted with buffer A containing 10 mM 2-mercaptoethanol. The purity of all proteins was assessed by 10% SDS-PAGE on a Bethesda Research Laboratories V12 vertical slab apparatus according to the method of Laemmli (20). Protein determination was performed according to Bradford (21). Oncomodulin-Sepharose was prepared as follows. Sepharose 4B (5 ml) was activated by CNBr treatment and incubated with oncomodulin (-1 mg) overnight at 4°C. Glycine was then added to react with any remaining active groups on the Sepharose. Before use, the column was washed with 100 mM Tris-HCl, pH 7.0, containing 10 mM 2-mercaptoethanol to ensure that all the bound oncomodulin was in the monomeric form. The column was then equilibrated in 100 mM Tris-HCl, 1 mM calcium chloride, pH 7.0. The glutathione reductase (containing 1 mM calcium chloride) was applied to the column and the column was washed with equilibrating buffer. The bound glutathione reductase was then eluted with 100 mM Tris-HCl containing 2 mM EGTA. Calmodulin-Sepharose chromatography of GSSGredase was performed in the same manner. Glutathione reductase was assayed according to previously published procedures (19). The reaction mixture (1 ml) contained 40 mM Tris-HCl, pH 7, 0.1 mM NADPH, GSSG, and modulator proteins as indicated. The reaction was initiated by the addition of GSSGredase (-2 nM) and allowed to proceed for 60 s. The reaction was then terminated by the addition of 50 ~1 10% SDS. The assays were performed at 30°C in either 1 mM EDTA or 1 mM Ca2+ as indicated. The change in absorbance was monitored at 340 nm on a Shimadzu UV-160 spectrophotometer. To ensure that the reduced oncomodulin used for the titrations and kinetics was indeed reduced, it was separated from excess reducing agent by Sephadex G-25 chromatography immediately prior to use. In addition, a sample was taken at the end of the titration for SDSPAGE analysis to determine whether oxidation had occurred during the course of the experiment. Samples for SDS-PAGE to study the interaction of oncomodulin dimer with glutathione reductase were prepared as follows. Oxidized oncomodulin (15 pg) was incubated at 30°C in the presence of GSH (10 mM) f glutathione reductase (10 nM) and NADPH (0.1 mM). Control samples of oxidized oncomodulin in the presence and absence of NADPH were also incubated without enzyme. Aliquots were removed at various time intervals and the reaction was terminated by decreasing the pH to -4.0. The samples were then run on 10% SDS-PAGE under nonreducing conditions (20).
RESULTS
Intimate contact between ONC and bovine intestinal mucosa GSSGredase was first suspected during at-
C FIG. 1.
Electrophoretic analysis of ONC-d under a variety of conditions. Polyacrylamide (10%) electrophoresis was performed under denaturating conditions (0.1% SDS) in the absence of thiol reducing agents. The gels were stained with Coomasie brillant blue (R-250; 0.5%). (A) ONC-d (15 rg), lane 1; molecular weight standards, lane 2. (B) Molecular weight standards, lane 1; ONC-d, lane 5; ONC-d (15 fig) plus NADPH (0.1 mM), lane 3; bovine intestinal mucosa GSSGredase (15 fig), lane 4; ONC-d (15 rg) plus GSSGredase incubated at 30°C for 2 min, with (lane 6) or without (lane 2) NADPH. (C) Molecular weight standards, lane 1; ONC-d (15 rg) incubatedwith GSH (10 mM) at 30°C for 1 min, lane 2; 5 min, lane 3. Molecular weight standards: bovine serum albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,100), lactalbumin (14,200).
tempts at determining the stability of ONC-disulfide linked dimer (ONC-d) to reduction by physiologically relevant concentrations of GSH. In these experiments GSH, GSSGredase, and NADPH had been incubated with ONC-d for various time intervals. GSSGredase had been included in the incubation mixture to prevent the oxidation of GSH, in the event that the ONC-d disulfide was not readily accessible, and therefore required long incubation times for the reduction. The relative amounts of ONC and ONC-d in the aliquots removed at various time intervals were determined by nonreducing SDS-PAGE electrophoresis (Figure 1). From these experiments, it is apparent that the ONC-d (Fig. 1, gel A) would not survive in the intracellular environment as all of the ONC-d was converted to the monomer within 5 min in the presence of GSH (10 mM) (Fig. 1, gel Clanes 2 and 3). Surprisingly, ONC-d was also reduced when incubated only with GSSGredase (plus or minus NADPH) (Fig. 1, gel B-lanes 2 and 6). The reduction in the absence of GSH was dependent on GSSGredase as NADPH alone could not affect the conversion of ONC-d to ONC (Fig. 1, gel C-lane 3). These results suggest that GSSGredase is catalyzing the reduction of ONC-d. However, the fact that NADPH is not required for the reduction of ONC-d (Fig. 1, gel B-lane 2) would suggest that this reduction is not occuring at the active site of the enzyme. An alternate explanation would be that the ONC-d disulfide is being reduced via disulfide exchange with one of the noncatalytic thiols of GSSGredase (22). In addition, small-molecular-weight thiol contamination of the enzyme samples is not likely to be the source
GLUTATHIONE
0.000
REDUCTASE
INHIBITION
BY
0.000 0
5
10
15
20
25
5
0
10
VOLUME (ml)
0.000
151
ONCOMODULIN
15
20
25
VOLUME (ml)
0
5
V&ME
;L)
20
FIG. 2. Elution profiles of bovine intestinal mucosa GSSGredase from oncomodulinand 280 nm. (A) GSSGredase in buffer A containing 1 mM Cazf was applied to ONC-Sepharose enzyme was eluted by the application of buffer A containing 2 mM EGTA. (B) GSSGredase to ONC-Sepharose (in the same buffer). Buffer A containing 2 mM Ca2+ and 2 M magnesium any bound protein. (C) GSSGredase was applied to calmodulin-Sepharose in buffer A, 1 mM of buffer A containing 2 mM EGTA.
of the reducing potentialas the GSSGredase samples were subjected to Sephadex G-25 chromatography prior to these experiments. Although this observation is interesting, it is thought not to have any physiological relevance, in light of the demonstrated unstability of ONCd to reduction by GSH (Fig. 1, gel C). In an extension of these studies, the interaction between ONC (monomer) and GSSGredase was explored. This was attempted by subjecting the enzyme to chromatography on a column of Sepharose to which ONC was covalently linked. The ONC-Sepharose elution profiles are presented in Fig. 2. When the column was equilibrated with buffer which contained Ca2+ (1.0 mmol/liter) -$ of the GSSGredase was recovered in the breakthrough. The remainder of the enzyme was bound to the column in a Ca2+-dependent manner as it could be eluted in the presence of the Ca2+ chelator, EGTA (Fig. 2A). There are several possible explanations for the partial binding of GSSGredase to ONC-Sepharose. Of these, the most likely is that the affinity of the enzyme for immobilized ONC is probably low. Alternatively, partial binding could indicate that the enzyme population is not homogeneous. Such heterogeneity could result from either different allosteric forms of GSSGredase or, more likely, a mixture of native and denatured forms of the enzyme. The requirement of Ca2+ in the
25
calmodulin-Sepharose. Protein was monitored at (preequilibrated in buffer A with 1 mM Ca*+). The (in buffer A containing 1 mM EGTA) was applied chloride were then applied in an attempt to elute Ca’+. Bound protein was eluted by the application
binding of GSSGredase to ONC-Sepharose was further suggested by the observation that ONC-Sepharose, which was preequilibrated with buffer containing EGTA, failed to bind any GSSGredase (Fig. 2B). Furthermore, CaM does not appear to interact with GSSGredase as this enzyme failed to bind to a column of CaMSepharose (Fig. 2C). The functional consequences of ONC/GSSGredase interactions were probed by monitoring the catalytic activity of the enzyme as a function of [ONC] (Fig. 3). GSSGredase from the bovine intestinal mucosa as well as that from the rat liver was inhibited by ONC in a dosedependent manner with an estimated I&, of -5 pmol/ liter (Fig. 3B). This relatively low affinity interaction could account for the partial binding of the enzyme to ONC-Sepharose. In addition, ONC did not completely inhibit the activity of the enzymes from both tissue sources. At the highest [ONC] employed (-0.6 mM) the bovine intestinal mucosa and the rat liver enzymes retained 37 and 30% of their activity, respectively. This observation as well as the partial binding of GSSGredase to ONC-Sepharose (Fig. 2), could arise as a consequence of the heterogeneity of the enzyme samples. Further evidence for the requirement of Ca2+ in the ONC/GSSGredase interaction could be demonstrated by the fact that the ONC-induced inhibition was not ob-
152
PALMER,
c? 0 $
60.. 70.. 60.. 60..
c 0 c-l
40.. 30.. 20..
MACMANUS,
Log~%%n]
(mol/L)-4.w0
110 x
loo
.-> 2
.
BI
EGTA
90 60.. 70..
MUTUS
the absence of ONC (Fig. 4A). However, in the presence of Ca2+, increasing amounts of ONC increased both the slope and y intercept of the double-reciprocal plots of the data while not affecting the x intercept, a behavior typical of a noncompetitive inhibitor (Fig. 4B). Under identical conditions (buffer containing Ca2+) CaM or PV had no effect as the kinetic data were identical to those obtained in their absence (Fig. 4C). The inability of closely related proteins to effect the same changes on the catalytic properties of the enzyme strongly suggest that this effect is specific to the oncodevelopmental protein oncomodulin.
-6.500 X
AND
\
Reduced glutathione is present in millimolar levels in virtually all cells and is responsible for maintaining intracellular reducing conditions. Glutathione has many functions, one of which is the control of its own biosynthesis through feedback inhibition of y-glutamylcysteinyl synthase, the first committed step of the glutathi-
-x
60.. 50.. 40..
, f
30.. 20 . . to-3.500
DISCUSSION
-3.000
TABLE
FIG. 3.
Dose-dependent effects of oncomodulin, calmodulin, and parvalbumin on GSSGredase activity. The reaction mixture (30°C) contained GSSG (3 mM), NADPH (0.1 mM), and GSSGredase (2 nM). (A) Increasing amounts of calmodulin (a) or rabbit parvalbumin (0) were added in the presence of Ca*+ (1.0 mM). (B) increasing amounts of ONC were added in the presence of Ca2+ (1.0 mM) (A) or EGTA (0).
Kinetic
EDTA
(1
Ca2+ (1
mM)
(1
ONC
(1 PM)
ONC
(10
CaM
(1
rPV
(1
50.1 2.35
pM)
in
EDTA
(1
mM)
wM)
PM) pM)
B. Bovine EDTA
(1
Ca2+ (1
mM)
ONC
(1
ONC
(10
CaM
(1
rPV
(1
n Unless
GSSGredase
mM)
ONC
intestinal
mucosa
mM)
PM) FM)
FM) pM)
stated,
Reductase
K m,app(PM) V max,app (nmol/min/md
Condition A. Rat liver
served in the absence of Ca2+ (EGTA 0.1 mmol/liter) (Fig. 3B). The inhibition of GSSGredase appears to be a property specific to ONC as closely related Ca2+-binding proteins such as rabbit parvalbumin and calmodulin did not alter enzyme activity (Fig. 3A). In the next stage of these studies, the effect of ONC on the steady-state kinetic behavior of GSSGredase was examined with a view of gaining information on the ONC binding site (Table I; Fig. 4). The steady-state kinetics were performed in the presence of EDTA or Ca’+, as a function of [GSSG] with constant [NADPH] (0.1 mmol/liter). The Km,*,,* and Vmax,appvalues estimated in the absence of ONC with the liver enzymes and intestinal mucosa are summarized in Table I. The GSSGredase assay is routinely performed in the presence of EDTA (19). Since this study concerns the interaction of Ca2+binding proteins with this enzyme, its catalytic parameters were initially estimated in the presence of Ca’+. While the Km,app values were identical, within experimental error, in the presence of EDTA or Ca2+, the V max,app estimated for the enzymes from the rat liver and bovine intestinal mucosa was 2.3- and 1.67-fold lower, respectively, in Ca2+than in EDTA (Table I). When the kinetic study was repeated in the presence of ONC, with buffer containing EDTA, the double-reciprocal plots of the data were nearly superimposable to that obtained in
I
Parameters of Glutathione Utilization of GSSG”
all kinetics
were performed
f +
2.9 0.04
67.1 + 1.01 +
4.0 0.03
57.0 2.37 62.1 0.72 62.1 0.55 68.0 1.35 70.6 1.03
2.4 0.09 5.9 0.04 5.5 0.04 2.3 0.04 1.0 0.12
+ f f f f k f ic + *
GSSGredase 85.9 5.08+90.3 3.04 88.9 1.91+ 95.1 1.40
+14
94.1 3.11 98.9 3.16
f & +f
in 1
mM
0.05 f 7 k 0.02 2~ 15 0.16 +15 + 0.16
05 0.01 10 0.02 Cd%.
GLUTATHIONE
REDUCTASE
INHIBITION
BY
=iiN
1 /[GSSG]
(M-’
-02E4 -8
-iN
0
l/[GSSG]
)
-1E4
0
lE4
I/[GSSG]
153
ONCOMODULIN
2E4
lE4
2E4
I
SE4
(M-l)
?
(M-l)
FIG. 4. Kinetic analysis of GSSGredase activity in the presence of oncomodulin, calmodulin, and parvalbumin. All kinetics were - 2 nM). (A) Kinetics performed in EDTA (1.0 mM) in the presence (0) and absence (0) at 30°C (NADPH, 0.1 mM; GSSGredase FM). (B) Kinetics performed in buffer containing CaCl, (1.0 mM) and EDTA (0.01 mM) in the presence of 1 pM ONC (0) or 10 pM in the absence of ONC (0). (C) Kinetics performed in Ca” (1.0 mM) in the presence of calmodulin (1 pM) (V), rabbit parvalbumin or Ca*+ alone (0).
one biosynthetic pathway (23). In neoplastic tissues, this control mechanism has been altered as several reports have indicated that the glutathione total levels are significantly elevated (24, 25). These increased levels are thought to afford increased resistivity to chemotherapeutic agents and radiation (24,26, 27) since inhibition of glutathione synthesis resulted in sensitization of the neoplastic cells to antineoplastic treatment (24, 28). An interesting hypothesis arose out of these findings; if one could selectively alter tumor glutathione levels relative to normal cells, antineoplastic treatment would feasibly kill neoplastic tissue while leaving normal cells virtually unharmed (24,25,28). Glutathione reductase is central for maintaining the high ratio of reduced to oxidized glutathione intracellularly. This enzyme is responsible for the conversion of oxidized glutathione formed upon the action of glutathione peroxidase which converts hydrogen peroxide to water, thus decreasing the effects of several toxic agents. In addition, glutathione reductase has been illustrated to be an inducible enzyme when rat liver cells were treated with various compounds (29). This suggests that glutathione reductase is of strong importance to the protection of cells against toxic agents. The potential involvement of the oncofetal Ca’+-binding protein oncomodulin in the regulation of glutathione
performed of ONC (1 ONC (0) or (1 pM) (O),
reductase was prompted by the observation that the disulfide-linked dimer of ONC could be reduced to its monomer in the presence of GSSGredase. This is unprecedented as this enzyme has previously been demonstrated to have an absolute specificity for its natural substrate GSSG (15). An alternative explanation is that the reduction of ONC-d disulfide is not occurring at the active site but via disulfide exchange with the noncatalytic free thiols of the GSSGredase. This enzyme contains 10 Cys residues per monomer (29). Of these, Cys-58 and Cys-63 are at the active site and are reversibly oxidized to a disulfide bridge during the catalytic cycle (30). The native structure of the enzyme is thought to contain an intersubunit disulfide bridge (Cys-90). The reduction of this disulfide would result in the dissociation of the enzyme into its subunits and its concomitant inactivation as the active site is found at a subunit interface (14). The reduction of the Cys-90 intersubunit disulfide has been proposed as a potential locus for the self-regulation of the enzyme via negative feedback by its product (22). Cys-2 of GSSGredase which is found in a flexible segment of the protein has been shown to undergo disulfide exchange reactions with a variety of thiols (22). Conceivably, the ONC-d disulfide could be reduced by Cys-90 or Cys-2, or other noncatalytic thiols of GSSGredase. Although the mechanism by which the disulfide is re-
154
PALMER,
MACMANUS,
duced is unclear, the fact that this reaction takes place suggests that these two macromolecules must interact. However, the interaction of the disulfide-linked dimer with GSSGredase is not expected to occur intracellularly in view of the demonstrated lability of the ONC-d disulfide to reduction by GSH (Fig. 1, gel C). As a result, only the potential interactions between ONC (monomer) and GSSGredase were explored. In these studies, GSSGredase was shown to interact in a Ca2+-dependent manner with ONC that was covalently attached to Sepharose. What makes these findings even more interesting is the fact that under identical conditions, GSSGredase failed to bind to calmodulin-Sepharose. Additional evidence for the interaction of ONC with GSSGredase was obtained when the enzymes from bovine and murine sources were assayed in the presence of increasing amounts of ONC. Under these conditions, GSSGredases from rat liver and bovine intestinal mucosa were inhibited in an ONC dose-dependent manner with I(&,, of -5 /*mol/liter. Although this ICsOappears to be relatively high, it is very close to the levels of ONC expressed in rat Morris hepatomas (100 mg/kg or -10 pmol/liter) (31). The ONC-induced inhibition required Ca2+ as ONC had no effect on the enzyme activity when the titrations were performed in the presence of EGTA, a further indication that ONC interacts with GSSGredase in a Ca2+-dependent manner. The specificity of ONC/GSSGredase interactions was demonstrated by the fact that calmodulin and rabbit parvalbumin failed to mimic the negative modulatory effect of ONC. This is the first report of a Ca-dependent regulatory mechanism for oncomodulin which is not also a property of calmodulin. Since oncomodulin is oncofetal in origin, this would imply that this inhibition would occur upon neoplastic transformation. This feasibly could result in a different equilibrium point between reduced and oxidized glutathione compared to normal cells. GSSGredase is the main enzyme responsible for converting GSSG back to GSH. Therefore, in the presence of oncomodulin, this conversion would be decreased, resulting in decreased levels of GSH. GSH is known to inhibit yglutamylcysteinyl synthase, the first committed step of glutathione biosynthesis. This feedback inhibition would be abolished by decreased GSH levels, resulting in increased biosynthesis and possibly increased levels of total cellular glutathione. In fact, neoplastic tissues have been demonstrated to contain GSH levels up to lo-fold higher than normal ceils (24,25). These elevated levels are thought to be partially responsible for the multidrug resistivity observed in selective neoplasms (28,32). The implication of oncomodulin, an oncofetal protein, in the regulation of GSH levels via GSSGredase inhibition is potentially a significant control mechanism of neoplastic metabolism.
AND
MUTUS
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