Changes in rat liver mitochondria with aging
Lon protease-like activity and
N
e
-carboxymethyllysine accumulation in the matrix
Hilaire Bakala
1
, Evelyne Delaval
1
, Maud Hamelin
1
, Jeanne Bismuth
1
, Caroline Borot-Laloi
1
,
Bruno Corman
2
and Bertrand Friguet
1
1
Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite
´
Paris7-Denis Diderot, Paris, France;
2
Service de Biologie Cellulaire, Commissariat a
`
l’Energie Atomique/Saclay, Gif-sur-Yvette, France
Aging is accompanied by a gradual deterioration of cell
functions. Mitochondrial dysfunction and accumulation of
protein damage have been proposed to contribute to this

)1
) to 27 months
(17.81 ± 1.83 pmol CMLÆlgprotein
)1
). These results
indicate that the accumulation of deleterious oxidized and
carboxymethylated proteins in the matrix concomitant
with loss of the Lon protease activity may affect the ability
of aging mitochondria to respond to additional stress.
Keywords: aging; mitochondria; matrix; Lon protease;
carboxymethyllysine.
A striking characteristic of normal aging in long-lived
animals is the gradual decline in their physiological func-
tions. This decline is associated with an increase in reactive
oxygen species (ROS) production [1] and an accumulation
of macromolecules damaged by post-translational nonenzy-
matic modifications which alter the structure and function
of tissue and cellular proteins [2–5]. Under oxidative stress,
carbohydrates, lipids and proteins are the major targets of
reactive oxygen species. Proteins can be damaged either
directly or indirectly through the reactive carbonyl com-
pounds derived from the oxidation of carbohydrates and
lipids [6,7]. These carbonyl compounds react with protein
amino-groups to give glycoxidation products such as
N
e
-carboxymethyllysine (CML) [8]. This glycoxidation
process modifies cell proteins and the cumulative effects
lead to the tissue alterations and cell dysfunction typical of
aging and diabetes [9,10]. Recent data also indicate that

Cellulaire du Vieillissement, Universite
´
Paris7-Denis Diderot,
T23-33 1
er
e
´
tage CC 7128, 2 Place Jussieu, 75252 Paris, France.
Fax: + 33 1 44 27 82 34; Tel.: + 33 1 44 27 82 35;
E-mail:
Abbreviations: ROS, reactive oxygen species; AGE, advanced glycat-
ion end products; CML, carboxymethyllysine; ABTS, 2,2¢-azinobis
(3-ethylbenzo-6-thiazolinesulfonic acid); RCR, respiratory
control ratio.
(Received 22 January 2003, accepted 27 March 2003)
Eur. J. Biochem. 270, 2295–2302 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03598.x
Our results indicate that mitochondrial matrix proteins
undergo oxidative and glycoxidative modifications. These
damaged proteins accumulate with aging, in parallel with
a large decrease in Lon protease activity.
Materials and methods
Animals
Experiments were performed on male Wistar rats (WAG/
Rij) born and raised in the animal care facilities of the
Commissariat a
`
l’Energie Atomique (CEA Gif-sur-Yvette,
France). This strain remains lean even when fed ad libitum
and does not suffer from age-associated nephropathy [29].
The animals were fed a commercial diet (DO4; UAR,

3
in 1 mL of 0.2
M
sodium phosphate buffer (pH 7.4), and then extensively
dialyzed against phosphate-buffered saline (NaCl/P
i
).
The CML content of the modified BSA was measured by
amino acid analysis following hydrolysis of the modified
protein in 6
M
HCl, 0.2% phenol (Laboratoire de Micro-
sequenc¸ age des Prote
´
ines, Institut Pasteur, Paris, France).
There were 43.2 CML residues and 16.1 Lys residues in
BSA-modified protein, while native BSA contained 57.9 Lys
in a total of 692 residues. As the expected value in the
primary sequence is 59 Lys, the error in the CML–adduct
rate can be estimated to be as low as 2%. The CML content
was expressed as 0.644 nmol CML per lg BSA, and this
solution was used as the standard in a competitive ELISA.
Isolation of mitochondria
A 10% tissue homogenate was prepared in an ice-cold
medium containing 220 m
M
mannitol, 70 m
M
sucrose,
2m

M
succinate or 5 m
M
glutamate and 5 m
M
malate (state 3) and
when all the ADP has been consumed (state 4 or resting
state). Oxygen-consumption rates are expressed as ng atoms
of oxygen consumed per minute and per mg protein. The
rate of oxygen consumption in state 3 and in state 4,
respiratory control ratio (RCR) of oxygen consumptions in
states 3 and 4 and the ADP/O ratio were calculated. Oxygen
consumption in the presence of 40 l
M
of dinitrophenol
(uncoupled state) was also checked.
Enzymatic activities
ATP-stimulated Lon protease activity was determined using
casein-fluoresceine isothiocyanate (0.5 mgÆmL
)1
) as sub-
strate. Casein was incubated with mitochondrial matrix
extract (70 lgprotein)in70lL buffer (final concentration:
50 m
M
Tris/HCl, pH 7.9, 10 m
M
MgCl
2
, with or without

)in50m
M
sodium carbonate buffer,
2296 H. Bakala et al.(Eur. J. Biochem. 270) Ó FEBS 2003
pH 9.6 by incubation overnight at 4 °C. The wells were
washed three times with NaCl/P
i
containing 0.05% (v/v)
Tween 20 (buffer A) and free binding sites were blocked by
incubation for 1 h at room temperature with 100 lLNaCl/
P
i
containing 6% (w/v) skimmed milk. The wells were then
washed with buffer A, 50 lL of competing antigen (test
samples at 0.100 mgÆmL
)1
or serial dilutions of standard
CML-BSA from 0.64 m
M
to 128 m
M
) was added, followed
by 50 lL monoclonal antibody clone 6D12 (diluted
1 : 1000 in NaCl/P
i
). The plate was incubated for 2 h at
room temperature, washed and then incubated with 50 lL
horseradish peroxidase-conjugated anti-mouse IgG (50 lL
per well, second antibody diluted 1 : 1000) for 2 h at room
temperature. The wells were washed, 100 lLofsubstrate

containing 0.2% (v/v) Tween 20 (wash buffer).
The membrane was then incubated for 2 h at room
temperature with anti-CML mAb clone 6D12 (diluted
1 : 1000 in NaCl/P
i
/0.1% Tween 20), washed four times
with wash buffer, incubated for 1 h with anti-mouse IgG
coupled to horseradish peroxidase (1 : 2500 dilution) and
given a final wash. The proteins were revealed with an
ECL reagent (Amersham-Pharmacia Biotech).
Western blot of carbonylated proteins. Carbonylated
proteins were analyzed using the oxyblot kit according to
the manufacturer’s instructions (Oxyblot Detection, Inter-
gen). Briefly, samples (10 lg protein per lane) were treated
with 10 m
M
2,4-dinitrophenolhydrazine in 2
M
HCl, incu-
bated at room temperature and neutralized. The derivatized
proteins were separated by SDS/PAGE, transferred to a
nitrocellulose membrane and treated as previous Western
blotting (see below). The primary antibody used was against
2,4-dinitrophenol, and detection was performed using the
ECL reagent.
Western blot of Lon protease. A polyclonal antibody
against Lon protease was raised in rabbit against a synthetic
peptide corresponding to amino acids 208–221 of rat Lon.
This antibody mainly recognized one protein band with an
estimated molecular mass of 100 kDa corresponding to the

As shown in Table 1, there was no difference in mito-
chondrial oxygen consumptions between 10-month- and
27-month-old-rats, whatever the substrate used. The
Table 1. Biochemical respiratory parameters in rat liver mitochondria from 10- and 27-month-old rats. Data represent mean values ± SEM
(n, number of animals). Oxygen consumption rates were measured polarographically in the presence of 10 m
M
succinate or 5 m
M
glutamate plus
5m
M
malate. State 3 respiration was determined after adding 310 nmoles ADP.
Glutamate (n ¼ 4) Succinate (n ¼ 7)
10 months 27 months 10 months 27 months
State 4 (ng atom OÆmin
)1
Æmg
)1
) 30.3 ± 0.3 26.5 ± 3.5 46.0 ± 3.5 47.1 ± 7.6
State 3 (ng atom OÆmin
)1
Æmg
)1
) 120.8 ± 7.4 117.8 ± 9.4 189.7 ± 15.8 166.9 ± 14.1
RCR 3.98 ± 0.26 4.52 ± 0.32 4.15 ± 0.20 3.79 ± 0.36
P/O 2.85 ± 0.13 2.94 ± 0.13 2.05 ± 0.08 1.91 ± 0.06
Ó FEBS 2003 Protein alteration in mitochondrial matrix with aging (Eur. J. Biochem. 270) 2297
ADP/O ratios obtained with succinate or glutamate indicate
no change in coupling efficiency. The yield of mitochondrial
preparation as well as the respiratory control ratio (state 3/

Fig. 1. Assessment of the purity of mitochondrial preparations. Electron
micrograph of isolated liver mitochondria from (A) 10-month-old and
(B) 27-month-old rats. All the recognizable organelles are mito-
chondria (arrow) showing different degrees of matrix density (star) and
dilatation of the cristae (arrowhead). Disrupted mitochondria are
visible (double arrow). Magnification, 30 000.
Fig. 2. Lon protease quantification and activity in liver mitochondrial
matrix from 10- and 27-month-old rats. Activitywasdeterminedinthe
absence of ATP or in the presence of 8 m
M
ATP (A, white and grey
columns respectively). Matrix proteins (20 lg) were subjected to
Western blotting using a polyclonal antibody against Lon protease (B)
and quantified by densitometric scanning (C), results being expressed
in arbitrary units. Values are the mean ± SEM for five independent
determinations. The P-value for enzymatic activity was significant
(**P < 0.01 for 10-month-old rats).
2298 H. Bakala et al.(Eur. J. Biochem. 270) Ó FEBS 2003
CML adduct content in mitochondrial matrix proteins
We used the monoclonal antibody clone 6D12 in competi-
tive ELISA to evaluate the CML-protein content (Fig. 3).
The CML content increased significantly by 52% from
11.71 ± 0.61 pmol CMLÆlgprotein
)1
(n ¼ 8) in 10-
month-old rats to 17.81 ± 1.83 pmol CMLÆlgprotein
)1
(n ¼ 9) in 27-month-old rats (P ¼ 0.007). These data
indicate an age-associated accumulation of CML adducts
in mitochondrial matrix.

Discussion
We used isolated rat liver mitochondria to analyse the
matrix defects that occur with aging. Mitochondria, similar
to the cytosol, contain a proteolytic system that controls the
metabolic stability of mitochondrial proteins and ensures
the elimination of damaged proteins [28]. This continuous
Fig. 3. Determination of CML-protein content in liver mitochondrial
matrix with aging. The CML content in the matrix proteins from
10- and 27-month-old rats was measured by competitive ELISA using
mAb 6D12. Results are expressed as pmol CMLÆlgprotein
)1
(n, number of animals). ***P ¼ 0.007 vs. 10-month-old rats.
Fig. 4. Western blot analyses of liver mitochondrial matrix proteins from 10-month- and 27-month-old rats. Samples (10 lg) were subjected to SDS/
PAGE under reducing conditions. The gel was stained with Coomassie blue (A) or subjected to Western blotting using either 6D12 monoclonal
antibody to detect CML-modified proteins (B) or oxyblot kit to reveal oxidized proteins (C). Samples from 10-month-old (lane 1) and from
27-month-old rats (lane 2). Standard molecular masses (lane 3). The arrow and arrowhead show the disappearance and appearance of bands,
respectively.
Ó FEBS 2003 Protein alteration in mitochondrial matrix with aging (Eur. J. Biochem. 270) 2299
protein turnover appears to be important for their main-
tenance and function, and consequently for cell integrity.
Lon protease plays a pivotal role because it is responsible for
breaking down abnormal matrix proteins.
In our study, we have shown a large decline in ATP-
stimulated Lon protease activity in the mitochondrial
matrix with aging. This activity in 27-month-old rats is
only 51% of that of adult (10-months) rats. Such a decrease
in matrix enzyme activity cannot result from the disruption
sensitivity of mitochondria as we observed roughly the same
pool of disrupted organelles in preparations from both old
and adult rats, as well as the same level of respiratory chain

study demonstrated for the first time that matrix proteins
undergo carboxymethylation. The level of CML-modified
protein increased with aging but affected particular proteins,
suggesting that some matrix proteins are more susceptible to
glycoxidative stress.
Although the source of the CM moiety is not defined,
several findings have shown that CML can originate from
both glycoxidation and lipoperoxidation reactions [6,35,36].
Recent studies on cultured vascular endothelial cells
revealed that hyperglyceamia caused overproduction of
mitochondrial ROS, which in turn initiate intracellular
advanced glycation end products (AGE) formation primar-
ily, if not exclusively, by increasing the concentration of
AGE-forming methylglyoxal [37–39]. In addition, the inner
mitochondrial membranes have a high percentage of
cardiolipin phospholipid, which contains a very high level
of the polyunsaturated fatty acid linoleic acid [40,41]. They
are prime target of ROS due to their location near to the site
of ROS production [42] and could be the source of carbonyl
adducts. Other recent reports have shown that there is
intracellular lipid glycoxidation that affects the phospha-
tidylethanolamine from membranes of these organelles [11].
In the present investigation, we detected CML proteins
using the anti-AGE mAb 6D12, which recognizes CML-like
structures, as well as carboxyethyllysine and several
unidentified AGE epitopes [43]. The oxidized proteins that
accumulated with aging in the range 95–120 kDa could
include the Lon protease, with an apparent molecular mass
of 100 kDa. The Lon protease may be structurally modified
by oxidation, which in turn could reduce its activity. In

mitochondrial integrity. All together, these age-dependent
alterations may contribute to disadvantage aged mitochon-
dria to respond to conditions of stress and compromise cell
viability.
Acknowledgements
The financial support of the Fondation pour la Recherche Me
´
dicale
and the French Ministry of Research is gratefully acknowledged.
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