ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 277, No. 2, March, pp. 228-233, 1990
Alteration of Aminoacyl-tRNA Synthetase with Age:
Heat-Labilization of the Enzyme by Oxidative Damage
Ryoya Takahashi
Department
and Sataro Goto’
of Biochemistry,
School of Pharmaceutical
Science, Toho University,
Miyama 2-2-1, Funabashi,
Chiba 274, Japan
Received June 5,1989, and in revised form October 14, 1989
Active oxygens have been suggested to be involved
in
age-related
alterations
of organelles
and molecules.
In
this study we investigated
the influence
of active oxygen on aminoacyl-tRNA
synthetases
partially
purified
from rat liver.
Treatment
of leucyl-tRNA
synthetase
with Fe3+ -ascorbate
resulted
in the increased
heat-lability of the enzyme. The inactivation
was inhibited
by
radical scavengers
such as mannitol
and benzoate, suggesting that hydroxyl
radicals are responsible
for heatlabilization
of the enzyme. On the other hand, a considerable part of tyrosyl-tRNA
synthetase
was converted
to heat-labile
forms without
added iron and ascorbate
under aerobic conditions
but not under anaerobic
conditions.
These and other findings
suggested
that the
heat-labilization
of this enzyme is caused by active oxygens probably
generated
by the reaction
of dioxygen
and transition
metal ions present in the enzyme preparations. Heat-inactivation
curves of the enzymes modified as described
above were similar
to those observed
for the enzymes from aged animals
in that these enzymes exhibited
higher
percentages
of heat-labile
forms than the unmodified
enzymes from young animals [Takahashi
and Goto, 1987, Arch. Gerontol.
Geriatr. 6, 73-82;
Takahashi
and Goto, 1987, Arch. Biothem. Biophys.
257, 200-2061.
The present
findings
are consistent
with the theory that active oxygens are
involved
in the age-related
alterations
of enzymes.
0 1990
Academic
Press,
Inc.
While animals with widely varying life spans apparently age similarly (l), the mechanism of aging is little
understood. The free radical theory of aging is one that
has been supported by a considerable amount of experimental evidence since Harman first proposed it [for a
review, see Ref. (2)].
On the other hand, functionally
altered proteins are
reported to be present in various organs of aged animals
’ To whom correspondence
should be addressed.
(3, 4) and the accumulation
of such proteins is implicated in the mechanism of age-associated deterioration
of the physiological
functions of tissues. We have also
found an increase in the proportion of heat-labile molecules of the elongation factor of translation (5) and aminoacyl-tRNA
synthetases (6-8) in aged mice and rats. A
possible mechanism of the alteration is a reduction in
the fidelity of translation
(9-12). Extensive studies including our own work, however, have indicated that the
fidelities of decoding (13-17) and aminoacylation
(1820) do not appear to change during aging. Other possibilities are post-translational
modifications such as deamidation, racemization,
glycosylation,
methylation,
limited degradation and oxidation [for reviews, see Refs. (3,
4, WI.
In the present work we examined in vitro the possible
involvement
of active oxygens in the generation of altered forms of enzymes using aminoacyl tRNA synthetases as a model. Similar studies have been conducted by
Oliver et al. (22,23). Our findings are essentially consistent with the conclusions reached by these investigators
(see under Discussion).
MATERIALS
AND
METHODS
Animals.
Male Fischer F-344/DuCrj
rats were purchased from
Charles River Japan (Crj) Inc. and maintained as reported previously
(7). In the present study 3- to 8-month-old animals were used.
Chemicals.
L-[3H]Tyrosine
(51 Ci/mmol)
was purchased from
Amersham. L-[3H]Leucine
(50 Ci/mmol) was from ICN. ATP was
from Boehringer-Mannheim
and Sephadex G-50 was from Pharmacia. All other reagents were of ultrapure grade.
Preparation
of aminoacyl-tRNA
synthetase.
Aminoacyl-tRNA
synthetase was partially purified from the liver as described previously
(67). Briefly, the tissue homogenate was centrifuged at 100,OOOgand
the supernatant was fractionated
with polyethylene
glycol 6000 and
then by DEAE-cellulose
column chromatography.
The partially purified enzyme preparation has activities for all the ten aminoacyl-tRNA
synthetases tested (18).
Assay. Enzyme activity was determined by measuring the binding
activity of a specific radioactive amino acid to unfractionated
rat liver
tRNA as described previously (6, 7). The reaction was carried out for
5 or 10 min at 25°C. The relation between the activity and the amount
228
All
0003.9861/90
$3.00
Copyright
0 1990 by Academic
Press, Inc.
rights
of reproduction
in any form reserved.
HEAT-LABILIZATION
OF AMINOACYL-tRNA
150
40 mMAsc.
iL
E
$pO
4
P
1
50
100 NM Fe3 +40 mM Asc.
E
0
0
1
2
TIME
3
4
5
(hr)
of leucyl-tRNA
synthetase by FeCl, and ascorFIG. 1. Inactivation
bate. Partially purified enzyme (0.8 mg/ml) was incubated at 30°C for
the time indicated and the remaining activity was determined under
standard assay conditions (see under Materials and Methods).
of protein in the enzyme preparation
tal conditions.
was linear under the experimen-
Leucyl-tRNA
Oxidation of leucyl- and tyrosyl-tRNA
synthetases.
synthetase was incubated in buffer D (20 mM sodium phosphate, pH
7.2,25 mM KCl, 0.2 mM phenylmethylsulfonyl
fluoride, and 25% glycerol) containing 100 FM FeCl, and 40 mM sodium ascorbate at 30°C.
The mixture was then passed through a column of Sephadex G-50 to
remove materials of low molecular weight including the active oxygen
generating systems. Tyrosyl-tRNA
synthetase was incubated in buffer
D without FeC& and ascorbate at 30°C and then the heat-stability
of
the enzyme was determined (see below).
Heat-stability
of the enzymes was deHeat-inactiuation
studies.
termined by the method reported previously with slight modification
(6,7). The enzyme preparation was heated for 2 to 20 min in buffer D
containing 1 mg/ml bovine serum albumin and chilled in an ice bath.
Then, the remaining enzyme activity was determined under standard
assay conditions and the proportion
of the heat-labile enzyme molecule was estimated (6,7).
The contents of iron
Determination
of iron and copper contents.
and copper in the partially purified enzyme preparations were determined using a model FLA-100 atomic absorption spectrophotometer
(Nippon Jarrell-Ash Co., Ltd., Kyoto Japan).
Partially purified enzyme preparations exhibDetection of ferritin.
ited a readily visible brownish color. So we examined a possibility of
contamination
of ferritin since iron in the enzyme preparations
may
affect results of the experiments (see under Results and Discussion).
Ferritin was purified from the enzyme preparation by the method of
Thomas et al. (39). The ferritin was identified by comparing the molecular weight and absorption spectrum with those of purified ferritin
from rat liver (Sigma). Molecular weight was determined by sodium
dodecyl sulfate-polyacrylamide
gel electrophoresis
and Sepharose 6B
(Pharmacia) gel filtration.
Visible and ultraviolet
absorption spectra
were recorded with a model U-3210 spectrophotometer
(Hitachi).
RESULTS
Inactivation
of Leucyl- and Tyrosyl-tRNA
Synthetases
We investigated the effects of FeCl,-ascorbate on leucyl-tRNA
synthetase partially purified from rat liver
since this system has often been used to generate active
oxygens under mild conditions in vitro (24,25).
SYNTHETASE
BY OXIDATIVE
229
DAMAGE
While the exposure of the enzyme to either ascorbate
or FeC& alone did not cause inactivation
of the enzyme,
incubation with ascorbate and FeC& together resulted in
a gradual loss of the activity with time (Fig. 1). Ascorbate alone appeared to activate the enzyme. We don’t
know the reason for this but other investigators reported
similar observations (36). To examine whether the molecular oxygen is involved in the inactivation
of the enzyme, the reaction was performed under nitrogen; under
this condition, no significant decrease in the enzyme activity was observed (Table I). We next investigated the
effect of various radical scavengers to know what types
of active oxygens are responsible for the inactivation under aerobic conditions. Addition of o-phenanthroline
to
the reaction mixture prevented the inactivation
most
efficiently. Prevention of the inactivation
by catalase,
mannitol, sodium benzoate, and histidine was less efficient but significant. Superoxide dismutase and dithiothreitol had little effect, while EDTA stimulated the inactivation
(Table I). The effect of EDTA has been
interpreted as iron-EDTA
complexes being catalysts of
the Fenton reaction which generate hydroxyl radicals
(24,25). Addition of dithiothreitol
after FeCl,-ascorbate
TABLE
1
Effect of Additions on the Inactivation of Leucyl-tRNA
Synthetase (LeuRS) and Tyrosyl-tRNA Synthetase (TyrRS)
% Inactivation
LeuRS
[Fe 3+-ascorbate]
Additions
None
Inert gas (N,)
o-phenanthroline
EDTA (1 mM)
(1 mM)
Superoxide dismutase
(660 units/ml)
Heat-inactivated
superoxide dismutase”
Catalase (1,100 units/ml)
Heat-inactivated
catalase”
Histidine (10 mM)
D-mannitol(250
mM)
Sodium henzoate (10 mM)
Dithiothreitol
(5 mM)
TyrRS
[air oxidation]
100
14
38
174
100
10
32
39
93
53
102
27
99
34
49
79
87
101
71
100
73
77
57
Note. The partially purified enzyme preparations for leucyl and tyrosy1 tRNA synthetases contained 1.2 and 0.3 mg/ml protein, respectively. The enzyme activity was determined at 30°C under the conditions described under Materials and Methods. Extent of the reduction
of the activity in the standard conditions without additions was regarded as 100%. The losses of the enzyme activity under the respective
standard conditions, i.e., FeC&-ascorbate
system and air oxidation
system, were 50 and 40% for leucyl and tyrosyl tRNA synthetases,
respectively.
a Superoxide dismutase and catalase were heated for 10 min at 90°C
in buffer D.
230
TAKAHASHI
Fe3’-Asc.
100
(-1
6.5 h
GOT0
We next examined whether the enzyme is converted
to heat-labile forms by the oxidative treatment.
(0)
a0
g
AND
60
Heat-Labilization
of Aminoacyl-tRNA
Oxidative Modification in Vitro.
E 40
:
ZlQ
20
10
0
5
PRENCUl3ATON
15
10
TME
(rnkd
20
J
at 46%
FIG. 2. Thermal inactivation
of leucyl-tRNA
synthetase preincubated in FeCl,-ascorbate.
The enzyme was incubated in FeCls-ascorbate for 0 (A), 4.5 (a), and 6.5 (0) h or for 6.5 h in its absence (0) under
aerobic conditions. The reaction mixture contained 2.5 mg/ml protein
of partially purified enzyme. The remaining activity of the enzyme
after incubation in FeCl,-ascorbate
for 4.5 and 6.5 h was 65 and 35%,
respectively. In the absence of Feels-ascorbate,
85% of the enzyme
activity remained.
treatment for 6.5 h, which results in 70% loss of the activity, restored the activity up to 60% of the original activity. This finding suggests that oxidative modification
of sulfhydryl groups in the enzyme is responsible for the
inactivation and it is reversible.
In contrast, FeCl,, when present alone, caused significant decrease of tyrosyl-tRNA
synthetase activity at
low concentrations
(N 100 PM). The activity was not restored by gel filtration on Sephadex G-50 or by addition
of chelating agents such as EDTA or o-phenanthroline
(1 mM) to remove FeC& from the reaction mixture (data
not shown). It thus appeared that iron is irreversibly
bound to the active site of the enzyme.
In the course of these studies, we noticed that a considerable part of tyrosyl-tRNA
synthetase was converted readily to a heat-labile form even in the absence
of iron and ascorbate under aerobic conditions, although
the activity of the enzyme did not decline greatly (see
below). To determine whether the inactivation of the enzyme requires oxygen, the enzyme solution was incubated under nitrogen; under this condition, however, the
enzyme was not appreciably
inactivated
(Table I).
Effects of radical scavengers are also shown in the table.
Addition of chelating agents EDTA and o-phenanthroline prevented the inactivation.
Superoxide dismutase
and sodium benzoate had some preventive effect, but
catalase and mannitol had little effect. These results
suggest that the inactivation
is due to active oxygens
generated by the reaction of dioxygen and contaminating transition metal ions (see below). From these studies, it is conceivable that the mechanism of inactivation
of tyrosyl-tRNA
synthetase in the air is somewhat
different from that of leucyl-tRNA
synthetase in FeC13ascorbate (see under Discussion).
Synthetases by
Figure 2 shows typical thermal inactivation
curves of
partially
inactivated
leucyl-tRNA
synthetase by the
FeC&-ascorbate treatment. The enzyme exhibited apparent biphasic patterns of inactivation,
suggesting the
presence of enzyme molecules with different heat-stabilities. The proportion of the heat-labile component was
determined by extrapolating the slope of the more stable
component to zero heating time (6, 7). The proportion
of heat-labile enzyme was increased to about 55% after
6.5 h from the value of less than 10% before the treatment (Fig. 2). Without iron-ascorbate, however, the proportion converted to heat-labile forms was much smaller
(Fig. 2).
On the other hand, tyrosyl tRNA synthetase exhibited
a clear biphasic pattern of heat-inactivation
after incubation without iron-ascorbate,
although most of the enzyme activity (about 80%) remained. The proportion of
heat-labile enzyme was estimated to be about 50% after
6.5 h (Fig. 3). It was conceivable that oxygen in the incubation mixture is involved in the heat-labilization
of the
enzyme. To investigate this point, the enzyme was incubated under anaerobic conditions in nitrogen and then
tested for heat-stability.
The heat-inactivation
curve of
the enzyme as such was indistinguishable
from that before incubation (Fig. 3).
It is thus suggested that active oxygens cause heatlabilization
of the two aminoacyl-tRNA
synthetases in
vitro, though tyrosyl-tRNA
synthetase appears to be
more sensitive.
N2
A&
101
0
I
5
PREWUBATKIN
,
10
TIME
15
(mh)
6h
(0)
0 h 1.1
6.5 h (0)
20
at 46C
FIG. 3. Thermal inactivation
of tyrosyl-tRNA
synthetase preincubated under aerobic and anaerobic conditions. The enzyme preparation (2.0 mg/ml protein) was incubated for 0 (A) and 6.5 h (0) in the
air or for 6 h (0) in NZ. The remaining activity of the enzyme after
incubation under aerobic and anaerobic conditions was 80 and 98%,
respectively.
HEAT-LABILIZATION
OF AMINOACYLtRNA
Possible Involvement of Ferritin in the Heat-Labilization
of Tyrosyl-tRNA
Synthetase in Vitro
Tyrosyl-tRNA
synthetase was inactivated even without added iron under aerobic conditions but was protected by chelators (Table I). It thus appeared that metal
ions are involved in the inactivation
although no metal
ions were added. Atomic absorption spectrochemical
analysis showed that iron and copper were present in the
partially
purified enzyme preparations
at concentrations of 75 ng/mg protein and 19 ng/mg protein, respectively. Concentrations of contaminating
iron and copper
in the buffer were less than 0.2 PM and 0.06 PM, respectively, values far below the levels that may explain the
concentrations found in the enzyme preparations. Dialysis
experiments suggest that most of these metals (>95%) are
bound to proteins since they are not dialyzable.
Presence of ferritin was suspected since the enzyme
preparations appeared brownish and ferritin is an abundant iron-storage protein present in large amount in the
liver (27). It seemed possible that ferritin contaminated
our partially purified liver aminoacyl-tRNA
synthetase
preparations since ferritin would be eluted with similar
concentrations of salt as the enzymes in DEAE-cellulose
column chromatography
(28). So, we examined whether
ferritin is present in the enzyme preparations or not. Gel
filtration of the enzyme preparations revealed approximate molecular masses of 400 kDa for brownish materials. Analysis of a putative ferritin purified from the
enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that it consists
of two polypeptides with molecular masses of 19 and 21
kDa (data not shown), values fully consistent with the
reported molecular masses of the subunits of ferritin
(40). Absorption spectrum of visible and ultraviolet regions of the purified ferritin was similar to that of commercial rat ferritin
(data not shown). These results
strongly suggest that ferritin is in fact present in our enzyme preparations.
We next examined whether the iron in ferritin present
in the enzyme preparations is involved in the heat-labilization of tyrosyl-tRNA
synthetase. Figure 4 shows
thermal inactivation
curves of tyrosyl tRNA synthetase
after incubation in the presence or absence of o-phenanthroline under aerobic conditions. The proportion
of
heat-labile enzyme was about 20 and 45% in the presence and absence of o-phenanthroline,
respectively.
Thus, the chelating agent prevented the heat-labilization of the enzyme. These results suggest that irons present in the ferritin are involved in the heat-labilization
of
tyrosyl tRNA synthetase by active oxygens in the absence of added iron in vitro.
DISCUSSION
Oxidative Inactivation of Leucyl- and
Tyrosyl-tRNA Synthetases
There are many methods of generating active oxygens
in vitro [for review, see Refs. (24, 26)]. We employed the
SYNTHETASE
BY OXIDATIVE
o-phenanthroline
0
5
10
PREHCUBATKIN
231
DAMAGE
TME
15
(min)
(-1
(+I
(0)
(0)
20
at 46%
FIG. 4. Effect of o-phenanthroline on the thermal inactivation
of
tyrosyl-tRNA
synthetase. The enzyme preparation
(2.2 mg/ml) was
incubated in the presence (0) or absence (0) of 2 mM o-phenanthroline for 5.5 h under aerobic conditions. After o-phenanthroline
was
removed by Sephadex G-50 column chromatography,
the heat-stability of the enzyme was determined. The remaining activity of the enzyme after incubation in the presence and absence of o-phenanthroline was 81 and 79%, respectively.
FeC13-ascorbate system to study the effect of active oxygens on aminoacyl-tRNA
synthetases since this system
is well-characterized
and may mimic an in vivo situation
(21,31,35).
Leucyl-tRNA
synthetase was inactivated in the presence of both FeCl, and ascorbate under aerobic conditions but not when either was lacking. As expected for a
reaction involving OH’ radicals, the inactivation
of the
enzyme was prevented by hydroxy radical scavengers
(see under Results). Tyrosyl-tRNA
synthetase, on the
other hand, was inactivated even without added iron under aerobic conditions but was protected by chelators
(Table I). It thus appeared that metal ions are involved
in the inactivation
although no metal ions were added.
Atomic absorption
spectrochemical
analysis showed
that iron and copper were present in the partially purified enzyme preparations. Most of the iron appears to be
present in ferritin in the partially purified enzyme preparations (see under Results) and caused inactivation and
heat-labilization
of the enzyme as discussed below.
Thomas et al. reported that ferritin promotes superoxide-dependent lipid peroxidation (29). It is possible that
the inactivation
of tyrosyl-tRNA
synthetase is also due
to active oxygens generated by the reaction of O2 and
irons present in ferritins (24, 29). In this regard, the report by Farber and Levine (34) is of interest. They found
that the inactivation
of glutamine synthetase is a sitespecific process in which Fe’+ is tightly bound to a metalbinding site on the enzyme and then reacts with hydrogen peroxide to generate active oxygen species [namely,
OH’, singlet oxygen, and ferry1 oxygen (21)], which oxidize histidinyl
residue at the metal binding site. This
leads to a loss of catalytic activity of the enzyme. Furthermore, Samuni et al. (30) reported that the inactiva-
232
TAKAHASHI
tion of purified penicillinase by radiolytically
formed superoxide radicals is greatly enhanced in the presence of
trace amounts of copper (II) ion. The metal ions bound
to the enzyme are reduced by superoxide radicals, yielding the reduced metal-protein
complexes. The reduced
complexes then react with hydrogen peroxide produced
in the Fenton reaction to generate secondary OH’ radicals locally, which react with the enzyme on that site,
resulting in the inactivation
of the enzyme. Shinar et al.
also showed that acetylcholine
esterase is inactivated
similarly in the presence of copper (II) ion and ascorbate
(31). In these instances, radical scavengers did not prevent the inactivation
while chelating agents did (30, 31,
34), observations similar to those for tyrosyl-tRNA
synthetase in our experiments (Table I). In our case, it is
possible that iron in contaminating
ferritins is transferred to the enzyme and inactivates it by a mechanism
similar to that suggested by the above investigators
(37,38).
With respect to the alteration
of enzymes during
aging, Gordillo et al. (32) reported that loss of a single
histidine residue appears to cause reduction of the activity of malic enzyme in the liver of aged rats. The loss of
the histidine residue is probably due to oxidative modifications of the enzyme since a similar change was observed in an in vitro model system of mixed function oxidation (24,32-34).
Heat-Labilization
of Aminoacyl-tRNA
Oxidation in Vitro
Synthetase by
Post-translational
modifications,
notably oxidative
damage, have been implicated in the aging process (2).
Studies in several laboratories demonstrated that specific activities of many enzymes decrease with the advancing age of organisms from which the enzymes were
derived (3,4).
We have reported that proportions of heat-labile molecules of aminoacyl-tRNA
synthetases increase in tissues of senescent mice (6-8); but the mechanism of the
heat-labilization
is not clear. The studies reported here
indicate that the aminoacyl-tRNA
synthetase can be
converted to heat-labile forms by oxidation (Figs. 2-4).
The thermal inactivation
profiles were similar to those
observed for the enzymes from aged animals in that enzymes from aged animals and enzymes oxidized in vitro
both exhibited higher percentages of heat-labile forms
compared to those from young animals and those unoxidized (6-8). Oliver et al. (22,23) reported that glucose-6phosphate dehydrogenase treated with a mixed function
oxidation system similar to the one used in the present
experiment is more heat-labile than the untreated enzyme.
Thus, our findings and those of other investigators
suggest that at least one mechanism for the inactivation
and/or the production of heat-labile forms of enzymes
during aging is oxidative modification.
AND
GOT0
Several other post-translational
modifications such as
deamidation, racemization, and glycosylation have been
reported to occur in proteins derived from senescent animals (3, 4, 24). At present, we do not know whether altered aminoacyl-tRNA
synthetases were generated in
vivo by any of these mechanisms including oxidation.
ACKNOWLEDGMENT
We thank S. Iwasa (Department
of Analytical
University)
for analyzing the content of transition
zyme preparations.
Chemistry, Toho
metals in the en-
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