(Received for publication, January 17, 1995; and in revised form, June 5, 1995)
From the
S-nitro-N-acetyl-DL-penicillamine
(SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione
peroxidase (GPx) in a dose- and time-dependent manner. The IC of SNAP for GPx was 2 µM at 1 h of incubation and
was 20% of the IC
for another thiol enzyme,
glyceraldehyde-3-phosphate dehydrogenase, in which a specific cysteine
residue is known to be nitrosylated. Incubation of the inactivated GPx
with 5 mM dithiothreitol within 1 h restored about 50% of
activity of the start of the SNAP incubation. A longer exposure to NO
donors, however, irreversibly inactivated the enzyme. The similarity of
the inactivation with SNAP and reactivation with dithiothreitol of GPx
to that of glyceraldehyde-3-phosphate dehydrogenase, suggested that NO
released from SNAP modified a cysteine-like essential residue on GPx.
When U937 cells were incubated with 100 µM SNAP for 1 h, a
significant decrease in GPx activity was observed although the change
was less dramatic than that with the purified enzyme, and intracellular
peroxide levels increased as judged by flow cytometric analysis using a
peroxide-sensitive dye. Other major antioxidative enzymes, copper/zinc
superoxide dismutase, manganese superoxide dismutase, and catalase,
were not affected by SNAP, which suggested that the increased
accumulation of peroxides in SNAP-treated cells was due to inhibition
of GPx activity by NO. Moreover, stimulation with lipopolysaccharide
significantly decreased intracellular GPx activity in RAW 264.7 cells,
and this effect was blocked by NO synthase inhibitor N
-methyl-L-arginine. This indicated
that GPx was also inactivated by endogenous NO. This mechanism may at
least in part explain the cytotoxic effects of NO on cells and
NO-induced apoptotic cell death.
Nitric oxide (NO) ()is a messenger molecule with
multiple biological functions including smooth muscle relaxation,
neurotransmission, and macrophage-mediated cytotoxicity (1) .
NO is highly reactive with molecular oxygen, superoxide anion, and heme
as well as non-heme iron. NO or its derivatives also interacts with the
thiol groups of proteins and glutathione to form nitrosothiols (2) . By this mechanism, NO can be stabilized and its function
prolonged (3) . Nitrosylation of enzymes such as GAPDH and
protein kinase C blocks their catalytic
activity(4, 5, 6, 7) .
Glutathione peroxidase (GPx) is an antioxidative enzyme that
scavenges various peroxides. Three isozymes, cellular GPx,
extracellular GPx, and phospholipid hydroperoxide GPx, are known, and
each contains a seleno-cysteine in its catalytic center(8) .
Cellular GPx, the most characterized form, can react with hydrogen
peroxide and organic peroxides but not lipid hydroperoxide. Catalase is
found in many types of cells and scavenges hydrogen peroxide as its
sole substrate. The content of catalase is lower than the content of
GPx in most cells, except for hepatocytes and erythrocytes, and the Kvalue of catalase for hydrogen peroxide
is higher than that of GPx, implying the primary importance of GPx in
most tissues. Selenium has been shown to regulate the level of cellular
GPx at both the transcriptional and translational stages(9) .
Selenium deficiency in cells causes a decrease in GPx mRNA and protein,
resulting in increased susceptibility of cells to oxidative damage.
Moreover, recent reports demonstrated that oxidative stress is one of
the direct causes of apoptotic cell death and that GPx as well as bcl2,
a proto-oncogene that blocks apoptotic death in multiple
contexts(10) , can prevent apoptosis(11) . Reactive
oxygen species also participate in many cellular events including
signal transduction and antibacterial defense. Hence, the maintenance
of a balance between oxidants and antioxidants is of significance for
cellular homeostasis.
We described here the inactivation of GPx by NO in a rather specific manner compared with another NO-sensitive enzyme, GAPDH, and discussed its physiological relevance.
Figure 1:
Dose dependence (A) and time
course (B) of inactivation of bovine GPx and bovine GAPDH.
Purified bovine GPx (,
) or GAPDH (
,
) at a
concentration of 1 mg/ml was preincubated with various concentrations
of SNAP for 1 h (
,
) or 2 h (
,
) (A)
or with 10 µM SNAP for various times (B) at 37
°C. Activities are given as the percentage of the control
value.
Figure 2:
Reduction of the SNAP-induced inactivation
of GPx by Carboxy PTIO. GPx (1 mg/ml) was incubated with 10 µM SNAP in the presence () or absence (
) of 1 mM carboxy PTIO, and GPx activities were measured at each time
point.
Figure 3:
Reversal of the SNAP-induced inactivation
of GPx and GAPDH with DTT. GPx (1 mg/ml) was reduced by preincubation
with 5 mM DTT for 1 h. After incubation of the reduced GPx
with (,
) or without (
,
) 100 µM SNAP for 1 h, DTT was added (arrows) to 5 mM (
,
), and GPx activities were measured at each time
point.
Figure 4:
Inactivation of GPx in U937 cells by
incubation with SNAP. U937 cells were incubated with 100 µM SNAP at each time and disrupted by sonication for 10 min. After
centrifugation at 10,000 g for 10 min, the GPx
activity of the supernatant was measured. Data are presented as means
± S.D. of triplicate experiments.
Figure 5:
Inactivation of GPx in RAW 264.7 cells
treated with LPS. RAW 264.7 cells were incubated with 10 ng/ml LPS in
the presence or absence of 1 mMN-methyl-L-arginine for 18 h.
Nitrite levels in the medium were measured(15) . The GPx
activities of these cells were assayed as described in the legend to Fig. 4. Data are presented as means ± S.D. of triplicate
experiments.
Figure 6: Flow cytometric analysis of intracellular peroxides in U937 cells treated with SNAP. Cells were preincubated with (blackarea) or without (whitearea) 100 µM SNAP for 0, 1, 3, and 6 h and were subjected to flow cytometric analysis using a peroxide-sensitive dye, DCFH-DA.
The molecular basis of the cytocidal and cytostatic effects of NO is unclear and could be rooted in any of the numerous functions of this small molecule. Several enzymes that catalyze reactions essential to metabolism are known to be inactivated or modified by NO(22) . The modifications of these enzymes are classified into two groups. (i) In proteins containing heme or non-heme irons as their cofactors, such as a soluble guanylate cyclase(1) , aconitase(22) , cytochrome c oxidase(23) , and cyclooxygenase(24) , NO binds to the iron molecule of these cofactors. (ii) In enzymes that contain a catalytically essential sulfhydryl group, such as GAPDH(4, 5, 6) , the N-methyl-D-aspartic acid subtype of glutamate receptor(25) , and protein kinase C(7) , NO interacts with the thiol group to form a nitrosocompound, thereby inactivating the enzyme. This inactivation can be reversed by incubation with reductants such as DTT.
Here we have shown that GPx was also
inactivated by NO, with 5 times higher sensitivity than GAPDH (Fig. 1). This inactivation would be induced by direct binding
of NO to an amino acid residue in the molecule, because the NO
scavenger carboxy PTIO alleviated the inactivation by SNAP (Fig. 2). The inactivation of GPx was reversed by reduction with
DTT (Fig. 3), which suggested that the modified amino acid
residue in GPx was similar to the essential cysteine residue in GAPDH.
Since GPx is a seleno-enzyme in which seleno-cysteine functions as the
catalytic center of the peroxidase reaction, the seleno-cysteine
residue is a likely candidate for modification with NO. Although we
used SNAP as an NO donor in this study(26) , SNAP can also
produce nitrosonium ion (NO) as other nitroso
compounds(27) . If inactivation is due to S- or Se-nitrosylation, it is likely that SNAP is acting as an
NO
donor. In spite of extensive trials to identify a
specific residue bound NO using
N NMR by essentially the
same method as used for bovine serum albumin(28) , we failed to
detect any definite signals characteristic to a chemical shift in GPx
incubated with acidified Na
NO
(data not
shown). The following could be explanations for this. (i) Because GPx
was not soluble at pH 1.0, which is the pH necessary for efficient
production of [
N]NO from
[
N]NaNO
, we had to incubate GPx with
NaNO
at pH 4.0. Although we prolonged the incubation time,
this might not have supplied enough [
N]NO for
modification of GPx. (ii) As is well known, GPx is easily autooxidized
in the absence of reducing compounds. GPx
NO complex would
therefore be unstable and undergo further oxidation, resulting in
release of [
N]NO, as hypothesized for protein
kinase C (7) and N-methyl-D-aspartic acid
subtype of glutamate receptor(25) .
NO can bind
spontaneously to thiol groups in various compounds such as glutathione
that are rich in cells. Nevertheless, treatment of cells with SNAP
decreased GPx activity without changing the enzyme level, although to a
lesser extent than in the experiment using the purified enzyme ( Fig. 1and Fig. 4). A significant decrease in
intracellular GPx activity was also observed in LPS-treated RAW 264.7
cells (Fig. 5), presumably due to induction of NO synthase by
LPS because GPx activity was protected in the presence of N-methyl-L-arginine. Inactivation of
GPx may, therefore, occur in certain cells that produce NO or in
surrounding cells. Because other antioxidative enzymes were not
affected by NO (Table 1), the increased accumulation of peroxides
within cells after treatment with an NO donor (Fig. 4) or
induction of NO synthase (Fig. 5) was likely a consequence of
inactivation of GPx by NO. However, we can't neglect the
possibility that production of peroxides in the cells was increased by
SNAP treatment. Recent reports showed that, in addition to having a
cytostatic effect, NO induced apoptotic cell death in several types of
cells(29, 30) . Because redox regulation of cells and
GPx activity are closely tied to apoptosis(31, 32) ,
inactivation of GPx by NO may be one of the causes of apoptotic cell
death in these cells.