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Originally published In Press as doi:10.1074/jbc.M005536200 on August 7, 2000
J. Biol. Chem., Vol. 275, Issue 45, 34853-34857, November 10, 2000
The Role of  , -Dicarbonyl Compounds in the Toxicity of
Short Chain Sugars*
Ayako
Okado-Matsumoto and
Irwin
Fridovich
From the Department of Biochemistry, Duke University Medical
Center, Durham, North Carolina 27710
Received for publication, June 23, 2000, and in revised form, August 2, 2000
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ABSTRACT |
The extent to which sugars serve as targets for
superoxide was examined using glycolaldehyde as the simplest sugar and
using superoxide dismutase (SOD)-replete and SOD-null strains growing under aerobic and anaerobic conditions. Glycolaldehyde was more toxic
to the SOD-null strain than to its SOD-replete parent, and this
differential effect was oxygen-dependent. The product,
glyoxal, could be trapped in the medium by 1,2-diaminobenzene and
assayed as quinoxaline. The SOD-null strain produced more glyoxal and eliminated it more slowly than the SOD-replete parent strain. Glyoxal
was ~10 times more toxic than glycolaldehyde and was more toxic to
the SOD-null strain than to the parental strain. 1,2-Diaminobenzene protected against the toxicity of glycolaldehyde. These
Escherichia coli strains contained the
glutathione-dependent glyoxalases I and II, as well as the
glutathione-independent glyoxalase III. Of these enzymes, glyoxalase
III was most abundant, and it was inactivated within the aerobic
SOD-null strain and also in extracts when exposed to the flux of
superoxide and hydrogen peroxide imposed by the xanthine oxidase
reaction. Thus, it appears that short chain sugars are oxidized by
superoxide yielding toxic dicarbonyls. Moreover, the defensive
glyoxalase III is also inactivated by the oxidative stress imposed by
the lack of SOD, thereby exacerbating the deleterious effect of
sugar oxidation.
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INTRODUCTION |
Sugars, in which carbon chain backbone is too short
to permit conversion to cyclic hemiacetals, are prone to enolization
and then to air oxidation. The superoxide is a product of air oxidation (1-3). Because the superoxide can also initiate the oxidation of such enediolates, free radical chain oxidations are possible (4).
Fig. 1 presents a scheme for the
tautomerism of the open chain forms of aldoses (I) to the
corresponding enediols (II) and for the sequential
oxidations of the enediols to a monoradical (III) and then
to a very unstable diradical (IV), which rearranges to the
 , -dicarbonyl (V). The one-electron oxidation can be
caused slowly by dioxygen yielding superoxide or more rapidly by
superoxide yielding hydrogen peroxide. We have previously noted that
short chain sugars are toxic to Escherichia
coli, aerobically but not
anaerobically and that a scavenger of
dicarbonyls, such as aminoguanidine, protected (5). Because a
SOD1-null strain was more prone to this toxicity than the
parental strain, we concluded that superoxide was a factor in the
oxidation of the short chain sugars and that ,-dicarbonyls were
the proximate toxic products of that oxidation. We did not then
actually measure the ,-dicarbonyls that were supposed to be the
cause of the toxicity, nor did we consider the protective actions of
glyoxalases that convert ,-dicarbonyls to -hydroxy acids. The
data presented below fill those gaps and add to our understanding of
sugars as sources of superoxide and as targets for that radical. We
find that glyoxal is produced from glycolaldehyde more rapidly by a SOD-null strain than by the parental strain. It is also seen that the
parental strain eliminates glyoxal more rapidly than the SOD-null strain. One particular ,-diketone, i.e. methylglyoxal,
can be made from dihydroxyacetone phosphate by a specific synthase that is widespread in bacteria and that has been cloned, sequenced, and
overexpressed (6, 7). The ,-dicarbonyl compounds, whether made by
methylglyoxal synthase or as a result of autoxidation of short chain
sugars, are potentially toxic because of their propensity to covalently
modify both nucleic acids and proteins (8, 9). Our results suggest that
under conditions of oxidative stress, such as stress imposed by a lack
of SOD, the autoxidation of short chain sugars to dicarbonyls is more
of a problem than is the activity of methylglyoxal synthase. Our
results also indicate that the specificity of the defensive glyoxalases
is broad enough to encompass glyoxal.
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Fig. 1.
Oxidation of sugars to
,-dicarbonyl
compounds. Compound I is an aldose in the open chain
configuration. Tautomerism yields the enediol (II).
Subsequent univalent oxidations by O2 or by O 2
yield the intermediates III and IV.
The diradical IV rapidly collapses to the dicarbonyl
(V).
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EXPERIMENTAL PROCEDURES |
Materials--
Methylglyoxal, glyoxal, glycolaldehyde,
DL-glyceraldehyde, 1,2-diaminobenzene, aminoguanidine,
S-D-lactoylglutathione, glutathione, ATP,
and NADP were obtained from Sigma. D-Erythrose was from
Fluka. 2,3-Dimethylquinoxaline was from Aldrich. Xanthine oxidase,
catalase, and glucose-6-phosphate dehydrogenase were from Roche
Molecular Biochemicals. Yeast extract, Bacto-peptone, and casamino
acids were from Difco.
Cell Culture--
The E. coli strain used was AB1157,
which was the parental strain for JI132 that was the  sodA/sodB
mutant (10). Starter cultures were grown overnight in aerobic LB
medium at 37 °C and were then diluted to 2 × 106
cells/ml in M9CA medium. LB and M9CA media are as described previously (11). The anaerobic condition was achieved in a BBL Gas Pak anaerobic system (Becton Dickinson). When needed, extracts were prepared from 6-h cultures by centrifugation (washing the cells two
times in 50 mM potassium phosphate, pH 7.8); then the
cells, which had been resuspended in this buffer, were disrupted with a
French press. The lysate was clarified by centrifugation.
Glyoxal and Methylglyoxal Assay--
Glyoxal and methylglyoxal
were assayed in the medium by using 1,2-diaminobenzene as derivatizing
reagent by a modification of the protocol of Cordeiro and Ponces Freire
(12). To a 1-ml sample containing glyoxal and/or methylglyoxal, we
added 0.2 ml of 5 M HClO4, 0.2 ml of
2,3-dimethylquinoxaline as an internal standard, 0.2 ml of 10 mM 1,2-diaminobenzene, and water to a 2-ml final
volume. After 1 h at 25 °C, high pressure liquid
chromatography (HPLC) analysis was performed in a LKB-Bromma
chromatograph. The column was a 5-µm, 250 × 4-mm RP-18
(Merck LiChrospher). The mobile phase was 40% (v/v) 25 mM ammonium formate buffer, pH 3.4, and 60% (v/v)
methanol. A volume of 150 µl was injected. The flow rate was 1.6 ml/min and quinoxalines were detected at 315 nm.
Enzymatic Assays--
Glyoxalase I was assayed by following an
increase in A240 because of
S-D-lactoylglutathione formation (13). One unit
of glyoxalase I is defined as the amount of enzyme required to form 1 µmol of S-D-lactoylglutathione/min. Glyoxalase
II was assayed by monitoring the decrease in
A240, accompanying the conversion of
S-D-lactoylglutathione to lactate plus GSH (14).
One unit of glyoxalase II is defined as the amount of enzyme that
hydrolyzes 1 µmol of
S-D-lactoylglutathione/min. Glyoxalase III was
assayed by a modification of the method of Misra et al. (15)
using the HPLC assay for glyoxal and methylglyoxal as described above.
One unit of glyoxalase III is defined as the amount of enzyme required to utilize 1 µmol of methylglyoxal or to form 1 µmol of
D-lactate/min. 50 µM xanthine, 2 nM xanthine oxidase, 1 µg of Cu,Zn-SOD, 1 µg of
catalase, or 25 mM mannitol were added to the cell extracts to explore the effects of reactive oxygen species.
ATP-dependent phosphorylation of glucose by glucokinase was
assayed in cell extracts by monitoring the formation of NADPH and by
using excess glucose-6-phosphate dehydrogenase according to the
protocol of Fraenkel and Horecker (16), except that a higher ATP
concentration (7.5 mM) was used (17). One unit of enzyme
activity is defined as the amount of glucokinase that catalyzes the
formation of 1 µmol of glucose 6-phosphate/min.
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RESULTS |
Glycolaldehyde and Glyoxal: Effect of Superoxide, SOD, and
1,2-Diaminobenzene--
Glycolaldehyde did not inhibit aerobic growth
of the SOD-replete AB1157 strain until its concentration exceeded 4.0 mM (Fig. 2A, line
1). In contrast, the growth of the SOD-null JI132 strain was
suppressed in the lower range of 0-4.0 mM (Fig. 2A,
line 2). This protective effect of endogenous SOD on the
sensitivity to glycolaldehyde was not seen under anaerobic conditions
(Fig. 2A, lines 3 and 4). When glyoxal was
examined (Fig. 2B), a similar pattern was seen but at
10-fold lower concentrations. Thus, the anaerobic growth suppression
became pronounced above 0.3 mM for both the JI132 and
AB1157 strains (Fig. 2B, lines 3 and 4), and aerobically JI132 was more sensitive than AB1157 (Fig. 2B, lines 1 and 2). 1,2-Diaminobenzene converts
,-dicarbonyls to quinoxalines (12, 18), and if glyoxal is the
cause of glycolaldehyde toxicity, 1,2-diaminobenzene should protect
JI132. Comparison of lines 1 and 2 in
Fig. 2C demonstrates that 2.0 mM glycolaldehyde
slowed the aerobic growth of the JI132 strain, whereas lines
3 and 4 in Fig. 2C show that 1.0 mM 1,2-diaminobenzene significantly lessened the
effect of glycolaldehyde. It should be noted that 1,2-diaminobenzene at
1.0 mM did not itself affect the growth of the JI132 strain in the absence of glycolaldehyde, although it was a growth inhibitor at
a higher concentration (data not shown). It should also be recalled
that 2.0 mM glycolaldehyde or 0.2 mM
glyoxal were without effect on the anaerobic growth rates of AB1157 or
JI132 (Fig. 2, A and B, lines 3 and
4). These data support the conclusions that glyoxal may be a
cause of the aerobic toxicity of glycolaldehyde, superoxide plays a
role in the conversion of the latter into the former, and somehow
superoxide also increases the toxicity of glyoxal.
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Fig. 2.
Superoxide dependence of the toxicity of
short chain sugars and the effect of 1,2-diaminobenzene.
A, toxicity of glycolaldehyde to aerobic and anaerobic
E. coli. Line 1, AB1157 under aerobic conditions;
line 2, JI132 under aerobic conditions; line 3,
AB1157 under anaerobic conditions; line 4, JI132 under
anaerobic conditions. B, toxicity of glyoxal to aerobic and
anaerobic E. coli. Line 1, AB1157 under aerobic
conditions; line 2, JI132 under aerobic conditions;
line 3, AB1157 under anaerobic conditions; line
4, JI132 under anaerobic conditions. C, glycolaldehyde
toxicity and protection by 1,2-diaminobenzene. Line 1,
AB1157 + 2 mM glycolaldehyde; line 2, JI132 + 2 mM glycolaldehyde; line 3, AB1157 + 2 mM glycolaldehyde + 1 mM 1,2-diaminobenzene;
line 4, JI132 + 2 mM glycolaldehyde + 1 mM 1,2-diaminobenzene. Overnight cultures of AB1157 and
JI132 in LB medium were diluted to 2 × 106 cells/ml
in M9CA medium. Cell growth was monitored at 600 nm after 8 h
(A, B) of incubation at 37 °C.
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Oxidation of Glycolaldehyde into Glyoxal--
Glycolaldehyde
autoxidizes into glyoxal, and the extent of this autoxidation must be
known so that corrections for it can be applied to results obtained
with E. coli. Line 1 in Fig.
3A presents the accumulation
of glyoxal from 2.0 mM glycolaldehyde in M9 medium. The
rate was slower in M9CA medium (Fig. 3A, line 2)
presumably because of consumption of glyoxal by reaction with the amino
acids in the casein hydrolysate. Aminoguanidine completely eliminated
the accumulation of glyoxal (Fig. 3A, line 3) by
coupling with it to form an asymmetrical triazine (19). In accordance with these results, when the persistence of glyoxal was examined (Fig.
3B), glyoxal was seen to be stable in M9 medium (Fig.
3B, line 1) but less stable in M9CA medium (Fig.
3B, line 2). Arginine caused rapid
consumption of glyoxal (Fig. 3B, line 4), whereas the
scavenging of glyoxal by aminoguanidine was most pronounced (Fig.
3B, line 3).
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Fig. 3.
Glyoxal contents in the medium without
E. coli. A, glyoxal production by
glycolaldehyde. Line 1, M9 + 2 mM
glycolaldehyde; line 2, M9CA + 2 mM
glycolaldehyde; line 3, M9CA + 2 mM
glycolaldehyde + 20 mM aminoguanidine. B,
effects of aminoguanidine and arginine on glyoxal. Line 1,
M9 + 200 µM glyoxal; line 2, M9CA + 200 µM glyoxal; line 3, M9CA + 200 µM glyoxal + 20 mM aminoguanidine; line
4, M9CA + 200 µM glyoxal + 20 mM
arginine. M9 medium was enriched with 0.2% glucose plus 3 mg/ml
pantothenic acid and thiamine. M9CA medium contained 0.2% casamino
acids plus M9 medium. All media were incubated at 37 °C under
aerobic conditions. Glyoxal contents of the medium were measured by
HPLC as quinoxaline. 2,3-dimethylquinoxaline was used as an internal
standard.
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The effect of 2.0 mM glycolaldehyde on the growth of
E. coli and the concomitant accumulation and consumption of
glyoxal were examined. Fig. 4A
shows that both the SOD-replete AB1157 and the SOD-null JI132 strains
grew at the same rates anaerobically (lines 2 and
4), whereas aerobically AB1157 grew much faster (line
1) than did JI132 (line 3). The glyoxal content of the
medium was also followed as shown in Fig. 4B. Line
1 shows that aerobic AB1157 accumulated glyoxal to a maximum of 40 µM during the first 4 h of growth and then consumed
it during subsequent growth. In contrast, aerobic JI132 continued to
accumulate glyoxal to a maximum of 100 µM during the
10 h of observation (line 3). Under anaerobic conditions, there was no accumulation of glyoxal by either strain (lines 2 and 4). Line 5 shows the
production of glyoxal by autoxidation in the aerobic M9CA medium
without cells, whereas line 6 depicts an
anaerobic control for the effect of medium alone. It again appears that
JI132 produces glyoxal from glycolaldehyde more rapidly and eliminates
it more slowly than AB1157. This is probably why its growth was
more strongly inhibited by glycolaldehyde.
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Fig. 4.
Cell growth and glyoxal production in the
medium. A, growth of E. coli treated with 2 mM glycolaldehyde. B, glyoxal accumulated in the
medium. Line 1, AB1157 under aerobic conditions; line
2, AB1157 under anaerobic conditions; line 3, JI132
under aerobic conditions; line 4, JI132 under anaerobic
conditions; line 5, M9CA + 2 mM glycolaldehyde
under aerobic conditions; line 6, M9CA + 2 mM
glycolaldehyde under anaerobic conditions.
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Glyoxalase Activities--
E. coli is known to contain
glyoxalases I, II, and III, although one report states that glyoxalase
II was not detected (15). Because glyoxalases I and II cooperate in
performing the GSH-dependent conversion of
,-dicarbonyls to -hydroxy acids, it would be expected that
glyoxalase II would be present if glyoxalase I was present. Fig.
5 presents the glyoxalase activities in
the AB1157 and JI132 strains grown under different conditions. The
first point to be made is that glyoxalases I, II, and III are all
present, although glyoxalase III > glyoxalase I > glyoxalase II > 0. Hence most of the glyoxalase activity in
E. coli is because of the GSH-independent glyoxalase III. A
comparison of Fig. 5, A and D, makes it clear that the AB1157 and JI132 strains have comparable glyoxalase activities when grown anaerobically but that JI132 has less glyoxalase III under
aerobic conditions. There was no induction of glyoxalase III by growth
in the presence of methylglyoxal (Fig. 5B). Paraquat, which
can increase the aerobic production of superoxide, suppressed glyoxalase III in JI132 (Fig. 5C).
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Fig. 5.
Glyoxalases I, II, and III activities.
A, glyoxalase activities of AB1157 and JI132. B,
glyoxalase activities of AB1157 and JI132 treated with 200 µM methylglyoxal. C, glyoxalase activities of
AB1157 and JI132 treated with 200 µM methylglyoxal and 1 µM paraquat. D, glyoxalase activities of
AB1157 and JI132 under anaerobic conditions. Overnight cultures of
AB1157 and JI132 in LB medium were diluted to 2 × 106
cells/ml in M9CA medium ± 200 µM methylglyoxal and
1 µM paraquat. Anaerobic condition was achieved in a BBL
Gas Pak jar. Cell extracts were prepared from 6-h cultures. All
enzyme activities were determined seven times, and the mean ± S.D. is shown.
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Inactivation of the abundant glyoxalase III by superoxide or by
reactive species derived therefrom could explain the lower glyoxalase
activity in the aerobic JI132 cells than in the AB1157 strain and could
also explain the effect of paraquat; it could further clarify why
glyoxal was more toxic to JI132 than to AB1157 and why this
differential toxicity was oxygen-dependent. Hence, this
possibility was explored.
Inactivation of Glyoxalase III--
The effect of a flux of
superoxide, produced by the xanthine oxidase reaction (20) on the
glyoxalase III activity in extracts of E. coli, was
examined. Fig. 6A shows that
glyoxalase III activity was diminished in the case of AB1157 by
exposure to the xanthine oxidase reaction, whereas Fig. 6B
shows that the effect on JI132 extracts was greater. Because the SOD
endogenous to AB1157 and present in the extract could have accounted
for this difference, SOD was added to the extracts and was found to
protect completely. Indeed, the added SOD raised the glyoxalase III
activity in JI132 extracts to a level greater than that seen in
extracts not exposed to the xanthine oxidase reaction. We suppose that
this is explained by the inactivation of some glyoxalases III by
endogenous superoxide production in the JI132 extracts before the
sampling for assay. Superoxide can release Fe(II) from the [4Fe-4S]
clusters of dehydratases, and that Fe(II) can reduce hydrogen peroxide
to yield the hydroxyl radical (21-23). If hydroxyl radical produced in
this way was the cause of the inactivation of glyoxalase III, then
catalase should protect by lowering hydrogen peroxide, and mannitol
should protect by scavenging the hydroxyl radical. Fig. 6 illustrates
the protective effects of catalase and mannitol. It follows that
glyoxalase III, the major glyoxalase in E. coli, is
sensitive to inactivation by the pro-oxidant conditions created by the
lack of SOD and that Fenton chemistry generates the proximate
inactivator.
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Fig. 6.
Effect of superoxide, hydrogen peroxide, and
hydroxyl radical on glyoxalase III. Overnight cultures of AB1157
and JI132 in LB medium were diluted to 2 × 106
cells/ml in M9CA medium. Cell extracts were prepared from 6-h cultures.
All enzyme activities were determined seven times, and the mean ± S.D. is shown.
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Glucokinase was examined in a similar way to see whether the
sensitivity of glyoxalase III to oxidation was unusual. Fig. 7 shows that glucokinase was not
inactivated by the superoxide and hydrogen peroxide produced by the
xanthine oxidase reaction. Thus, the sensitivity of glyoxalase III was
special and might relate to the thiol group that is essential for its
activity and possibly to the binding of iron adjacent to the active
site thiol.
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Fig. 7.
Glucokinase activities of AB1157 and
JI132. Overnight cultures of AB1157 and JI132 in LB medium were
diluted to 2 × 106 cells/ml in M9CA medium. Cell
extracts were prepared from 6-h cultures. All enzyme activities were
determined seven times, and the mean ± S.D. is shown.
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Methylglyoxal--
While assaying for glyoxal in terms of the
quinoxaline produced from the reaction with 1,2-diaminobenzene, we also
measured methylglyoxal in terms of 2-methylquinoxaline. The primary
reason for doing so was to gauge the extent to which methylglyoxal
synthase was contributing to dicarbonyl production by its non-oxidative pathway. Fig. 8 indicates that the
contribution of the methylglyoxal synthase to the total dicarbonyl
production in cells exposed to glycolaldehyde was very small indeed.
Thus, M9CA medium conditioned by the growth of JI132 underwent reaction
with 1,2-diaminobenzene and then was subjected to HPLC in which
quinoxaline, derived from glyoxal, eluted at 3 min, 35 s, and in
which 2-methylquinoxaline, derived from methylglyoxal, eluted at 4 min,
20 s. There was no detectable methylglyoxal formed from the
glucose present in this medium and in only traces of other dicarbonyls
(Fig. 8A). When 200 µM glyoxal had been added
to the culture (Fig. 8B), it was detected as quinoxaline at
zero time eluted at 3 min, 35 s and was progressively consumed at
longer times of incubation. Similarly, enriching the medium with 200 µM methylglyoxal gave 2-methylquinoxaline eluting at 4 min, 20 s at zero time and progressively less in samples drawn at
longer times of incubation (Fig. 8C). The consumption of
these dicarbonyls by JI132 largely reflects the activity of glyoxalases. When cultures enriched with glycolaldehyde (Fig. 8D) or glyceraldehyde (Fig. 8E) were examined,
the major dicarbonyls detected were glyoxal and methylglyoxal,
respectively. However, in the case of glycolaldehyde, the glyoxal was
first generated and then consumed during the 24 h of incubation.
In contrast, the glyceraldehyde was contaminated with methylglyoxal in
the zero time sample. Erythrose (Fig. 8F) was contaminated
by polar dicarbonyls, presumably erythrosone, in which quinoxaline
products eluted at 2 min, 35 s. It also contained lesser amounts
of glyoxal and methylglyoxal.
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Fig. 8.
HPLC analysis of quinoxalines.
A, quinoxalines in the control medium; B,
quinoxalines in the medium enriched with 200 µM glyoxal;
C, quinoxalines in the medium enriched with 200 µM methylglyoxal; D, quinoxalines in the
medium enriched with 2 mM glycolaldehyde; E,
quinoxalines in the medium enriched with 2 mM
glyceraldehyde; F, quinoxalines in the medium enriched with
2 mM erythrose. All media were incubated with JI132. Ratios
of analyte peak height to internal standard peak height were used for
quantitative analysis. 2,3-Dimethylquinoxaline was added as an internal
standard (retention time is 5 min, 3 s).
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DISCUSSION |
Short chain sugars, in which carbonyl functions cannot be blocked
by the formation of furanose or pyranose rings, are prone to
enolization followed by oxidation to toxic dicarbonyls. Thus, they
express in an exaggerated way what can also occur with glucose by the
slower process of non-enzymatic glycation and oxidations (24, 25).
Glycolaldehyde, the simplest sugar, is more toxic to a SOD-null strain
of E. coli (JI132) than to its SOD-replete parent (AB1157),
and this extra toxicity is oxygen-dependent. The
corresponding dicarbonyl, glyoxal, was ~10 times more toxic than
glycolaldehyde, and JI132 was again more sensitive than AB1157 in an
oxygen-dependent way. 1,2-Diaminobenzene protected JI132 against the toxicity of glycolaldehyde, presumably by converting glyoxal to the less toxic quinoxaline. We may infer that superoxide is
an important cause of the oxidation of glycolaldehyde, and we may
explain the greater and oxygen-dependent sensitivity of JI132 to glyoxal on the basis of an oxidative inactivation of glyoxalases. Glycolaldehyde itself becomes toxic at high
concentrations, even anaerobically, probably by converting essential
amino compounds to carbinolamines and to Schiff base salts.
When the SOD-replete AB1157 strain grew aerobically in the presence of
glycolaldehyde, glyoxal first accumulated in the medium and was
subsequently consumed. The SOD-null JI132, in contrast, accumulated
glyoxal during the entire 10 h of incubation. Thus, it appeared
that JI132 both converted glycolaldehyde to glyoxal more rapidly and
disposed of it more slowly than did AB1157. Glyoxalases I, II, and III
were all present in these strains, but the GSH-independent glyoxalase
III was the most abundant and suppressed in the JI132 strain grown
aerobically; it was further suppressed when JI132 grew in the presence
of 1 µM paraquat. Thus, it appears that glyoxalase III
was inactivated by the oxidative stress imposed by lack of SOD activity
and also by the presence of paraquat.
Exposure of bacterial extracts to the superoxide and hydrogen peroxide
produced by the xanthine oxidase reaction caused a loss of glyoxalase
III activity that was greater in the SOD-null extracts. This
inactivation was prevented by SOD, catalase, or mannitol. Glucokinase
in the extracts was not inactivated by the xanthine oxidase reaction.
Glyoxalase III may be selectively inactivated by a flux of superoxide
and hydrogen peroxide because it binds the Fe(II) released from the
[4Fe-4S] clusters of dehydratases oxidized by superoxide. This bound
Fe(II) would then react with hydrogen peroxide to yield Fe(II)-O,
Fe(III)-OH, or hydroxyl radical, and these strong oxidants would
preferentially attack the nearest target, which in this case is
glyoxalase III.
When glucose was the carbon source, no dicarbonyls could be trapped by
1,2-diaminobenzene. This negative result is a measure of the degree of
protection provided by blocking sugar carbonyls by hemiacetal ring
closure. Moreover, the steady state concentrations of the
triosephosphate intermediates of glycolysis must be very low because
the equilibrium constant of the aldolase reaction greatly favors
fructose 1,6-diphosphate. We conclude that dicarbonyl production from
dihydroxyacetone phosphate, by the action of methylglyoxal synthase,
must be insignificant and in full accord with the observation that
900-fold overexpression of this synthase did not cause observable detrimental effects (7). MacLean et al. (26) reported that glyoxalase III was the most abundant glyoxalase in E. coli
but nevertheless concluded that glyoxalases I plus II were the most important route of methylglyoxal detoxification. This conclusion was based on the heightened sensitivity to methylglyoxal exhibited by a
glyoxalase I-null mutant. However, this can be explained by the
protective effect of lowering cytoplasmic pH (27) because of the
activation of potassium efflux by the product of the glyoxalase I
reaction, S-lactoylglutathione (26). Lowering the pH would slow the reaction of dicarbonyls with target amino or thiol compounds.
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FOOTNOTES |
*
This work was supported in part by grants from the
Amyotrophic Lateral Sclerosis Association, the North Carolina
Biotechnology Center, and Incara Pharmaceuticals Inc.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 919-684-5122;
Fax: 919-684-8885; E-mail: fridovich@biochem.duke.edu.
Published, JBC Papers in Press, August 7, 2000, DOI 10.1074/jbc.M005536200
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ABBREVIATIONS |
The abbreviations used are:
SOD, superoxide
dismutase;
HPLC, high pressure liquid chromatography.
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