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 and Sataro Goto’ Department 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 oxy- gen on aminoacyl-tRNA synthetases partially purified from rat liver. Treatment of leucyl-tRNA synthetase with Fe3+ -ascorbate resulted in the increased heat-la- bility of the enzyme. The inactivation was inhibited by radical scavengers such as mannitol and benzoate, sug- gesting that hydroxyl radicals are responsible for heat- labilization of the enzyme. On the other hand, a consid- erable part of tyrosyl-tRNA synthetase was converted to heat-labile forms without added iron and ascorbate under aerobic conditions but not under anaerobic con- ditions. These and other findings suggested that the heat-labilization of this enzyme is caused by active oxy- gens probably generated by the reaction of dioxygen and transition metal ions present in the enzyme prepa- rations. Heat-inactivation curves of the enzymes modi- fied as described above were similar to those observed for the enzymes from aged animals in that these en- zymes exhibited higher percentages of heat-labile forms than the unmodified enzymes from young ani- mals [Takahashi and Goto, 1987, Arch. Gerontol. Geri- atr. 6, 73-82; Takahashi and Goto, 1987, Arch. Bio- them. 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 appar- ently 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 experi- mental 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.

228

(3, 4) and the accumulation of such proteins is impli- cated 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 mole- cules of the elongation factor of translation (5) and ami- noacyl-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 in- cluding our own work, however, have indicated that the fidelities of decoding (13-17) and aminoacylation (18- 20) do not appear to change during aging. Other possibil- ities are post-translational modifications such as deami- dation, racemization, glycosylation, methylation, lim- ited 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 al- tered forms of enzymes using aminoacyl tRNA synthe- tases as a model. Similar studies have been conducted by Oliver et al. (22,23). Our findings are essentially consis- tent 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 Phar- macia. 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,OOOg and the supernatant was fractionated with polyethylene glycol 6000 and then by DEAE-cellulose column chromatography. The partially puri- fied 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

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HEAT-LABILIZATION OF AMINOACYL-tRNA SYNTHETASE BY OXIDATIVE DAMAGE 229

150

iL

E $pO 4

P

1 50

E

0

40 mMAsc.

100 NM Fe3 +40 mM Asc.

0 1 2 3 4 5

TIME (hr)

FIG. 1. Inactivation of leucyl-tRNA synthetase by FeCl, and ascor- 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 was linear under the experimen- tal conditions.

Oxidation of leucyl- and tyrosyl-tRNA synthetases. Leucyl-tRNA synthetase was incubated in buffer D (20 mM sodium phosphate, pH 7.2,25 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, and 25% glyc- erol) 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-inactiuation studies. Heat-stability of the enzymes was de- 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 mole- cule was estimated (6,7).

Determination of iron and copper contents. The contents of iron and copper in the partially purified enzyme preparations were deter- mined using a model FLA-100 atomic absorption spectrophotometer (Nippon Jarrell-Ash Co., Ltd., Kyoto Japan).

Detection of ferritin. Partially purified enzyme preparations exhib- 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 molec- ular 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 leu- cyl-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).

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). Ascor- bate 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 mo- lecular oxygen is involved in the inactivation of the en- zyme, the reaction was performed under nitrogen; under this condition, no significant decrease in the enzyme ac- tivity 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 un- der 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 effi- cient but significant. Superoxide dismutase and dithio- threitol had little effect, while EDTA stimulated the in- activation (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

Additions LeuRS TyrRS

[Fe 3+-ascorbate] [air oxidation]

None Inert gas (N,) o-phenanthroline (1 mM) EDTA (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)

100 100 14 10 38 32

174 39

93 53

102 101 27 71 99 100 34 73 49 77 79 57 87

Note. The partially purified enzyme preparations for leucyl and ty- rosy1 tRNA synthetases contained 1.2 and 0.3 mg/ml protein, respec- tively. The enzyme activity was determined at 30°C under the condi- tions described under Materials and Methods. Extent of the reduction of the activity in the standard conditions without additions was re- garded 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.

TAKAHASHI AND GOT0 230

100 a0

g 60

E 40

: ZlQ

20

10

Fe3’-Asc. (-1 6.5 h (0)

J 0 5 10 15 20

PRENCUl3ATON TME (rnkd at 46%

FIG. 2. Thermal inactivation of leucyl-tRNA synthetase preincu- bated in FeCl,-ascorbate. The enzyme was incubated in FeCls-ascor- bate 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 ac- tivity, restored the activity up to 60% of the original ac- tivity. 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 sig- nificant decrease of tyrosyl-tRNA synthetase activity at low concentrations (N 100 PM). The activity was not re- stored 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 con- siderable part of tyrosyl-tRNA synthetase was con- verted 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 en- zyme requires oxygen, the enzyme solution was incu- bated 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-phenanthro- line 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 contaminat- ing transition metal ions (see below). From these stud- ies, 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 FeC13- ascorbate (see under Discussion).

We next examined whether the enzyme is converted to heat-labile forms by the oxidative treatment.

Heat-Labilization of Aminoacyl-tRNA Synthetases by Oxidative Modification in Vitro.

Figure 2 shows typical thermal inactivation curves of partially inactivated leucyl-tRNA synthetase by the FeC&-ascorbate treatment. The enzyme exhibited ap- parent biphasic patterns of inactivation, suggesting the presence of enzyme molecules with different heat-stabil- ities. 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 treat- ment (Fig. 2). Without iron-ascorbate, however, the pro- portion 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 incu- bation without iron-ascorbate, although most of the en- zyme 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 incu- bation mixture is involved in the heat-labilization of the enzyme. To investigate this point, the enzyme was incu- bated under anaerobic conditions in nitrogen and then tested for heat-stability. The heat-inactivation curve of the enzyme as such was indistinguishable from that be- fore incubation (Fig. 3).

It is thus suggested that active oxygens cause heat- labilization of the two aminoacyl-tRNA synthetases in vitro, though tyrosyl-tRNA synthetase appears to be more sensitive.

N2 6h (0) A& 0 h 1.1

6.5 h (0)

101 I ,

0 5 10 15 20 PREWUBATKIN TIME (mh) at 46C

FIG. 3. Thermal inactivation of tyrosyl-tRNA synthetase preincu- bated under aerobic and anaerobic conditions. The enzyme prepara- tion (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 SYNTHETASE BY OXIDATIVE DAMAGE

Possible Involvement of Ferritin in the Heat-Labilization of Tyrosyl-tRNA Synthetase in Vitro Tyrosyl-tRNA synthetase was inactivated even with-

out added iron under aerobic conditions but was pro- tected 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 concentra- tions of 75 ng/mg protein and 19 ng/mg protein, respec- tively. Concentrations of contaminating iron and copper in the buffer were less than 0.2 PM and 0.06 PM, respec- tively, 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 abun- dant 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 approxi- mate molecular masses of 400 kDa for brownish materi- als. Analysis of a putative ferritin purified from the enzyme preparations by sodium dodecyl sulfate-poly- acrylamide 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 re- gions of the purified ferritin was similar to that of com- mercial rat ferritin (data not shown). These results strongly suggest that ferritin is in fact present in our en- zyme preparations.

We next examined whether the iron in ferritin present in the enzyme preparations is involved in the heat-labili- zation of tyrosyl-tRNA synthetase. Figure 4 shows thermal inactivation curves of tyrosyl tRNA synthetase after incubation in the presence or absence of o-phenan- throline under aerobic conditions. The proportion of heat-labile enzyme was about 20 and 45% in the pres- ence and absence of o-phenanthroline, respectively. Thus, the chelating agent prevented the heat-labiliza- tion of the enzyme. These results suggest that irons pres- ent in the ferritin are involved in the heat-labilization of tyrosyl tRNA synthetase by active oxygens in the ab- sence 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

o-phenanthroline (-1 (0) (+I (0)

231

0 5 10 15 20

PREHCUBATKIN TME (min) 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-phenanthro- line for 5.5 h under aerobic conditions. After o-phenanthroline was removed by Sephadex G-50 column chromatography, the heat-stabil- ity of the enzyme was determined. The remaining activity of the en- zyme after incubation in the presence and absence of o-phenanthro- line was 81 and 79%, respectively.

FeC13-ascorbate system to study the effect of active oxy- gens 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 pres- ence of both FeCl, and ascorbate under aerobic condi- tions 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 un- der 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 puri- fied enzyme preparations. Most of the iron appears to be present in ferritin in the partially purified enzyme prep- arations (see under Results) and caused inactivation and heat-labilization of the enzyme as discussed below.

Thomas et al. reported that ferritin promotes superox- ide-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 re- port by Farber and Levine (34) is of interest. They found that the inactivation of glutamine synthetase is a site- specific process in which Fe’+ is tightly bound to a metal- binding site on the enzyme and then reacts with hydro- gen peroxide to generate active oxygen species [namely, OH’, singlet oxygen, and ferry1 oxygen (21)], which oxi- dize histidinyl residue at the metal binding site. This leads to a loss of catalytic activity of the enzyme. Fur- thermore, Samuni et al. (30) reported that the inactiva-

232 TAKAHASHI AND GOT0

tion of purified penicillinase by radiolytically formed su- peroxide 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, yield- ing the reduced metal-protein complexes. The reduced complexes then react with hydrogen peroxide produced in the Fenton reaction to generate secondary OH’ radi- cals 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 pre- vent the inactivation while chelating agents did (30, 31, 34), observations similar to those for tyrosyl-tRNA syn- thetase in our experiments (Table I). In our case, it is possible that iron in contaminating ferritins is trans- ferred 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 activ- ity of malic enzyme in the liver of aged rats. The loss of the histidine residue is probably due to oxidative modi- fications of the enzyme since a similar change was ob- served in an in vitro model system of mixed function oxi- dation (24,32-34).

Heat-Labilization of Aminoacyl-tRNA Synthetase by Oxidation in Vitro

Post-translational modifications, notably oxidative damage, have been implicated in the aging process (2). Studies in several laboratories demonstrated that spe- cific activities of many enzymes decrease with the ad- vancing age of organisms from which the enzymes were derived (3,4).

We have reported that proportions of heat-labile mol- ecules of aminoacyl-tRNA synthetases increase in tis- sues 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 en- zymes from aged animals and enzymes oxidized in vitro both exhibited higher percentages of heat-labile forms compared to those from young animals and those unoxi- dized (6-8). Oliver et al. (22,23) reported that glucose-6- phosphate dehydrogenase treated with a mixed function oxidation system similar to the one used in the present experiment is more heat-labile than the untreated en- zyme.

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.

Several other post-translational modifications such as deamidation, racemization, and glycosylation have been reported to occur in proteins derived from senescent ani- mals (3, 4, 24). At present, we do not know whether al- tered aminoacyl-tRNA synthetases were generated in vivo by any of these mechanisms including oxidation.

ACKNOWLEDGMENT

We thank S. Iwasa (Department of Analytical Chemistry, Toho University) for analyzing the content of transition metals in the en- zyme preparations.

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