From the
Oxidative modification of DNA, lipids, and proteins occurs as a
consequence of reaction with free radicals and activated oxygen.
Oxidative modification of total cellular proteins has been described
under many pathologic and experimental conditions, but no specific
proteins have been identified as in vivo targets for oxidative
modification. Utilizing an immunochemical method for detection of
oxidatively modified proteins, we identified a protein in rat liver
that was highly oxidized. It was purified to homogeneity and identified
as carbonic anhydrase, isozyme III. Its characteristics match those
previously described for a protein that was lost during aging of the
rat, senescence marker protein-1. Carbonic anhydrase III was purified
from rats aged 2, 10, and 18 months, and the proteins were
characterized. All three preparations were highly oxidatively modified
as assessed by their carbonyl content. The enzyme has three known
catalytic activities, and the specific activities for carbon dioxide
hydration and for ester hydrolysis decreased during aging by
There now exists evidence for a large array of biological
processes in which free radicals are produced(1, 2) . As
a consequence of exposure to these reactive species, nucleic acids,
lipids, and proteins may undergo oxidative modification(3) .
Oxidative attack on proteins can covalently modify residues and can
cause peptide bond cleavage leading to a variety of
products(4) . Introduction of carbonyl groups (aldehydes and
ketones) into amino acid residues is a frequent consequence of protein
oxidation, and these carbonyl groups are accepted as a marker of
oxidative modification(5, 6, 7) . Several
sensitive methods for the determination of protein-bound carbonyl
groups have been developed(7, 8, 9) , with
derivatization by 2,4-dinitrophenylhydrazine being the most widely used
method. Using this assay, substantial increases in oxidatively modified
proteins have been demonstrated in tissues after a variety of
stresses(4) , including hyperoxia(10, 11) ,
reperfusion after
ischemia(12, 13, 14, 15) , and
neutrophil activation(16, 17, 18) . A doubling
or tripling of the steady-state level of oxidized proteins has been
demonstrated to occur during the last third of the lifespan of flies,
rodents, and
humans(11, 19, 20, 21, 22) . For
reviews and additional references, see Ref. 6. All in vivo studies published so far have measured the extent of oxidative
modification of homogenates, without identification of specific
proteins that are especially modified.
An immunologic method for
detection of oxidatively modified proteins was reported
recently(23) . The technique uses Western blotting with
anti-dinitrophenyl antibodies to detect protein-bound carbonyl groups
that have been derivatized with 2,4-dinitrophenylhydrazine. We used
this technique to identify oxidatively modified proteins in rat liver
homogenates. One protein band was especially prominent, being the most
oxidatively modified protein thus far found in vivo. It was
purified to homogeneity, guided by the immunochemical assay. The
protein was identified as carbonic anhydrase, isozyme III.
Immunodetection was performed as published (7) except that reaction with 2,4-dinitrophenylhydrazine was
carried out after transferring the proteins to nitrocellulose. This
modification was necessary because the strong acid and high SDS
concentration were not compatible with isoelectric focusing. Although
samples for SDS-PAGE could have been derivatized before
electrophoresis, they were also derivatized after transfer to
nitrocellulose in order to simplify comparison with samples analyzed by
isoelectric focusing. Derivatization on the membranes was performed
simply by immersing them in 0.2% 2,4-dinitrophenylhydrazine (in 2 M HCl) for 10 min followed by a wash in 2 M HCl for another
10 min. Immunodetection was carried out as described (26) using
the Tropix kit. Two different anti-DNP antibodies were used with
equivalent results. These were a monoclonal mouse IgE (Sigma D-8406)
and a polyclonal rabbit preparation (Dako V401, Carpenteria, CA).
Depending on the choice of primary antibody, the secondary antibody was
either rat anti-mouse IgE conjugated with horseradish peroxidase
(Southern Biotechnology Associates, Birmingham, AL) or goat anti-rabbit
conjugated with alkaline phosphatase (Tropix). Biotinylated secondary
antibodies were not used because they also reacted with protein-bound
biotin present in the homogenates.
The aromatic amino acid content was determined by analysis of the
protein's ultraviolet spectrum by second derivative spectroscopy,
a technique that typically gives results within 5% of the actual value
(27). A spectrum was obtained from a sample
Searching a protein sequence data
bank(28) , we retrieved entries of molecular weight
28,000-35,000. This range was chosen to allow for error in the
mass determination and because entries would have higher molecular
weights if they are unprocessed precursors. This list was then narrowed
to those proteins that contained between 5 and 15 Tyr and Trp residues.
Two proteins in this refined list had equal numbers of Tyr and Trp
residues, a rat pancreatic tissue kallikrein precursor and carbonic
anhydrase. The kallikrein precursor has 8 Phe, 8 Tyr, and 8 Trp, while
rat muscle carbonic anhydrase isozyme III has 11 Phe, 8 Tyr, and 8 Trp
and an unmodified molecular weight of 29,270.
The carbonyl content of the purified
protein was quantitated by reaction with 2,4-dinitrophenylhydrazine in
SDS followed by separation of excess reagent by HPLC gel filtration as
described(7) . S-glutathiolation of carbonic anhydrase
III was determined by reverse phase chromatography as described for
peptide purification, except that the gradient ran from 30 to 45%
acetonitrile over 30 min. Peaks were collected for determination of
their molecular weight by mass spectrometry. S-thiolation and
dethiolation were also assessed by isoelectric focusing(32) .
Protein concentrations were determined by the procedure of Bradford (25) using bovine serum albumin as standard.
The molecular weight and
aromatic amino acid composition of carbonic anhydrase III fit well with
measured values for the purified, oxidatively modified protein. The
purified preparation was then analyzed for carbonic anhydrase activity,
and it was found to be catalytically active with a specific activity of
110 units/mg protein. Isozyme III is unique among the seven known
isozymes in being relatively resistant to inhibition by
acetazolamide(33) . The other 6 isozymes are inhibited by
concentrations of acetazolamide in the range of 10
The
carbonic anhydrases are generally acetylated at their amino terminus,
preventing Edman degradation(28) . When the purified protein was
subjected to automated Edman degradation, no cleavage occurred.
Cyanogen bromide peptides were therefore prepared and purified as
described under ``Experimental Procedures.'' One purified
peptide was subjected to 10 cycles of Edman degradation, and the
sequence matched exactly with that reported for carbonic anhydrase III
from Ala
The reduction of oxygen to water occurs with great efficiency
in cells(43) . However, this four-electron process is not
perfectly efficient, and activated oxygen species are generated as a
consequence. These molecules (e.g. superoxide, hydrogen
peroxide, hydroxyl, and ferryl) have the potential to damage cellular
components. Cells have thus evolved an antioxidant defense system,
which includes molecules such as glutathione, ascorbate, and
tocopherols along with enzymatic systems such as peroxidases, catalase,
superoxide dismutase, and thiol-specific antioxidant(44) . This
defense system, while effective, is also imperfect, and cellular
macromolecules are damaged by the oxidants that are not scavenged. This
has long been appreciated for lipids and DNA, with Ames (45) estimating that up to 100,000 bases are oxidized in the
average cell per day. Recently cellular proteins have been studied as
targets of oxidative stress. Using the introduction of carbonyl groups
to provide a minimal value, one can estimate that at least 10% of
cellular proteins carry an oxidative
modification(6, 8) . This burden increases dramatically
in certain disease states and during aging, as noted in the
Introduction.
Using an immunoassay for detection of protein-bound
carbonyl groups, we identified a protein in liver that had a
particularly high level of oxidative modification. The protein was
purified and shown to be carbonic anhydrase III. We also purified
carbonic anhydrase III from the muscle of 2-month-old male rats and
found its carbonyl content to be comparable with that for the protein
purified from liver (not shown). Thus, the high extent of oxidative
modification of carbonic anhydrase III is not limited to liver.
The
seven carbonic anhydrases are found in many organs, being important in
a variety of biological activities including acid-base balance,
CO
Chatterjee et al.(52) described a protein that they referred to as
senescence marker protein-1 (SMP-1). This unidentified protein was
present in high amounts in liver, decreased during aging, was
androgen-dependent, and had a molecular weight of about 30,000. From
these characteristics it appears very likely that SMP-1 is carbonic
anhydrase III.
The sequence and three-dimensional structure of
isozyme III are very similar to those of the other, well-studied
isozymes of carbonic anhydrase, notably isozyme II(53) .
However, isozyme III has several characteristics that distinguish it
from the other isozymes. The first is its low specific activity as a
carbon dioxide hydratase, leaving unsettled its actual physiologic
function. Isozyme III's activity is only about 1% that of isozyme
II(54) , although it retains a well defined binding site for
bicarbonate(53) . This binding site may predispose the protein
to oxidative modification. Recent studies make clear that the
bicarbonate/carbonate system dramatically accelerates oxidation of
amino acids, peptides, and proteins when compared with other buffer
systems(55, 56, 57, 58, 59) .
The stoichiometry and kinetics of oxidation of amino acids have been
described in detail by Stadtman et al.(58) , and
bicarbonate is part of the oxidizing complex. Thus, the reactions that
are catalyzed by carbonic anhydrase III may lead to its own oxidative
modification. Even in young animals, carbonic anhydrase III bears
covalent modifications characteristic of proteins exposed to an
oxidizing environment, that is S-glutathiolation and carbonyl
groups. We speculate that the protein has a physiologic role that
either places it in such an environment or creates the environment as a
consequence of the reactions it catalyzes.
A second distinguishing
characteristic of isozyme III is that it has inherent phosphatase
activity(31) . The domain responsible for phosphatase activity
has not been defined, but it has been distinguished from the regions
responsible for hydratase and esterase
activity(31, 53) . While the absolute amount of isozyme
III in the male liver decreased during aging, the specific activities
for carbon dioxide hydratase and esterase changed only modestly (Fig. 4). In contrast, the phosphatase activity unique to isozyme
III was almost completely lost by 18 months of age.
From a detailed
study of the protein-tyrosine phosphatase of Yersinia, Zhang et al.(60) established that the sequence
Cys-X
Inspection of the sequence of carbonic anhydrase III from rat,
mouse, cow, horse, and human (28, 53) demonstrated the
Cys-X
The average liver protein has a
carbonyl content of
We thank Dr. Henry M. Fales of the National Heart,
Lung, and Blood Institute for the mass spectrometric measurements
reported in this paper.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
30%.
However, the third activity, that of a phosphatase, was virtually lost
during aging. While the physiologic role of carbonic anhydrase III is
unknown, we suggest that it functions in an oxidizing environment,
which leads to its own oxidative modification.
Materials
Acetazolamide and
2,4-dinitrophenylhydrazine were from Sigma, cyanogen bromide was from
Aldrich, and phenylmethanesulfonyl fluoride was from Fluka (Buchs,
Switzerland). HPLC(
)grade acetonitrile was
supplied by Baker (Phillipsburg, NJ), and trifluoroacetic acid was
supplied by Pierce. Carbon dioxide was supplied by MG Scientific
(Buffalo Grove, IL). Precast gels for electrophoresis were purchased
from Novex (San Diego, CA), and the chemiluminescent detection kit was
from Tropix (Bedford, MA). Nitrocellulose membranes were from
Schleicher & Schuell. N-glycosidase F was item 710S from
New England Biolabs (Beverly, MA).
Preparation of Cell Extracts from Rat Liver
Livers
from Fischer 344 rats of varying ages were used. Animals were obtained
from the National Institute of Aging colonies maintained at Harlan
Sprague-Dawley, Inc. (Indianapolis, IN). The animals were fed the
NIH-31, Teklad Premier diet and maintained on a cycle of light (6:00
a.m. to 6:00 p.m.)/dark (6:00 p.m. to 6:00 a.m.). Rats were
anesthetized by intraperitoneal injection of sodium pentobarbital (1
mg/kg), following which the abdomen was opened and the liver excised,
rinsed, weighed, and stored at -70 °C. To prepare extracts,
livers were washed and then homogenized with a Potter-Elvehjem device
in 5 volumes of 50 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 1 mM EDTA, and 0.25 mM phenylmethanesulfonyl fluoride. The homogenate was centrifuged at
100,000 g for 40 min, and the supernatant fraction was
retained for study.
Immunodetection of Protein-bound
2,4-Dinitrophenylhydrazones (Western Blot)
SDS-polyacrylamide
gel electrophoresis was performed according to Laemmli(24) ,
using precast polyacrylamide gels (8-16% acrylamide) followed by
staining with Coomassie Blue(25) . Isoelectric focusing was
performed in precast gels (pH 3-10, 5% acrylamide) run for 130
min at a constant 2-watt current. Gels were fixed with 11%
trichloroacetic acid, 5% sulfosalicylic acid for 30 min and stained
with Coomassie Blue.
Purification and Identification of the Oxidized Protein
from Rat Liver
All steps were carried out at 4 °C except
HPLC steps, which were at room temperature. Liver extract from young
male rats was prepared as described above, and solid ammonium sulfate
was added with continuous stirring to give a 50% concentration. After
centrifugation at 20,000 g for 15 min, the supernatant
was brought to 70% ammonium sulfate and again centrifuged at 20,000
g for 15 min. The pellet was resuspended in 50
mM Tris-HCl, pH 7.0, and dialyzed overnight against the same
buffer. The solution was applied to a DEAE-5PW preparative HPLC column
(2.15
15 cm, TosoHaas, Philadelphia, PA) equilibrated with the
same buffer running at 3 ml/min. The oxidized protein did not bind to
the column, and the run-through fraction was applied to a low pressure
Cibachrome Blue column (2.5
20 cm, Bio-Rad) in the same buffer.
The oxidized protein was again recovered in the flow-through, which was
concentrated on an Amicon PM10 ultrafilter (Beverly, MA) and then made
1 M in ammonium sulfate. The solution was applied to a
Phenyl-5-PW hydrophobic interaction column (2.15
15 cm,
TosoHaas, Philadelphia, PA) equilibrated with 50 mM Tris-HCl,
pH 7.5, 1 M ammonium sulfate (buffer A). Buffer B was 50
mM Tris-HCl, pH 7.5. A gradient from 0 to 30% B was developed
over 60 min at a flow rate of 3 ml/min. The oxidized protein eluted at
about 0.8 M ammonium sulfate in a single symmetrical peak,
well separated from other components. The sample was dialyzed overnight
against 50 mM Tris-HCl, pH 7.0. Analysis by SDS-PAGE gave a
single band, while isoelectric focusing gave two bands with estimated
pI of 6.9 and 7.4. The solutions were stored at -70 °C.
0.15 mg/ml protein,
determined by Coomassie Blue binding. From the molecular weight
determined by MALDI, the estimated protein concentration was thus 5
µM. The measured concentrations of aromatic amino acids
were 44.4 µM Phe, 32.8 µM Tyr, and 30.7
µM Trp, giving a Phe:Tyr:Trp ratio of 1.35:1.00:0.94.
Thus, the number of tyrosine residues equaled the number of tryptophan
residues. Assuming that the true protein concentration was between 2
and 10 µM, the actual ratio of residues could be 15:11:11,
11:8:8, or 8:6:6.
Catalytic Activities of Carbonic
Anhydrase
Carbonic anhydrase III possesses three enzymatic
activities: carbonic anhydrase, esterase, and phosphatase. Each assay
was performed with and without enzyme, and the blank rate was
subtracted to calculate the catalyzed rate. Carbonic anhydrase activity
was measured by CO hydration, according the electrometric
method of Wilbur and Anderson (29). In this assay one determines the
time required for a saturated CO
solution to lower the pH
of 0.02 M Tris-HCl buffer from 8.3 to 6.3 at 4 °C. The p-nitrophenylacetate esterase activity was measured by the
method of Verpoorte et al.(30) . Activity was followed
spectrophotometrically at 348 nm, the isosbestic point of p-nitrophenol and the p-nitrophenolate ion. The p-nitrophenyl phosphatase activity was determined
spectrophotometrically as described(31) .
Other Analyses
Mass spectrometry was kindly
performed by H. Fales at the National Institutes of Health using
matrix-assisted laser desorption ionization (Kompact MALDI-III with a
337-nm nitrogen laser; Kratos, Manchester, United Kingdom) and
electrospray ionization and detection with a Finnigan TSQ700 mass
spectrometer (San Jose, CA). Automated sequence analyses were performed
at the core facility of Michigan State University (Lansing, MI) under
the direction of J. Leykam. No sequence was obtained on the intact
protein, implying a blocked amino terminus. The protein was thus
digested with cyanogen bromide in 70% formic acid for 15 h at room
temperature and then quenched by the addition of excess methionine.
Peptides were purified on a C reverse phase column (Vydac
218TP54, Hesperia, CA) using a linear gradient of 0-50%
acetonitrile containing 0.05% trifluoroacetic acid. The gradient change
was 1%/min, and the flow rate was 1 ml/min. Peptides were monitored by
their absorbance at 210 nm with a Hewlett Packard model 1050 diode
array detector. To avoid the amino-terminally blocked peptide of
residues 1-77, second derivative spectroscopy of the purified
peptides was again utilized(27) . We selected the peptide peak
presumed to contain residues 222-259 with 2 Phe, no Tyr, and 1
Trp. It was dried and rerun in the same system at a gradient change of
0.5%/min for final purification. This peptide was then subjected to 10
cycles of Edman degradation.
Immunodetection and Identification of a 30-kDa Protein
with High Carbonyl Content
Proteins in the soluble fraction of a
liver homogenate from a 2-month-old male rat were separated by both SDS
gel electrophoresis and isoelectric focusing gels. Oxidized proteins
were visualized on the Western blots (Fig. 1). One prominently
oxidized band was seen on the SDS gel with a molecular weight of about
30,000. Two bands were relatively prominent on the isoelectric focusing
gel, with pI of 7.4 and 6.9, consistent with a difference of one
titratable group. When these two bands were rerun on SDS gel, both had
the same apparent molecular weight of 30,000, and both were strongly
positive on the Western blot (data not shown). Control blots from which
2,4-dinitrophenylhydrazine was omitted had no detectable staining. As
reported previously, free 2,4-dinitrophenylhydrazine reagent spotted
directly on the membrane gave no signal when carried through the
immunoanalysis(23) .
Figure 1:
Detection of oxidatively modified
proteins in liver extracts. Liver extracts (25 µg of protein) from
2- and 24-month-old male rats were analyzed by SDS-PAGE (A)
and isoelectric focusing (B). Coomassie Blue protein staining
and anti-dinitrophenyl immunostaining are shown. Molecular weight and
isolectric point markers are shown on the left of the Proteinstainpanels. An oxidized protein of
30,000 molecular weight is prominent in extracts from the young
animal.
We tentatively assumed that the two
bands visualized on isoelectric focusing were isoforms of a protein
with a molecular weight of about 30,000. We purified the protein by
standard chromatographic methods as described under ``Experimental
Procedures.'' The purification was guided by Western blot analysis
using SDS gel electrophoresis. The protein was pure as judged by the
presence of a single band on the SDS gel. Two bands were again observed
on isoelectric focusing, with pI of 7.4 and 6.9. The molecular weight
estimated from the SDS gel was confirmed by mass spectroscopy (MALDI)
which gave a value of 29,520. The aromatic amino acid composition was
determined spectrophotometrically as described under
``Experimental Procedures.''
to 10
M, while isozyme III is
resistant to even micromolar concentrations. The purified preparation
had this resistance, with measured activity being unaffected by 7
µM acetazolamide. Thus, the protein was identified as
carbonic anhydrase III (E.C. 4.2.1.1, carbonic dehydratase).
to Ala
, again confirming the
identification made initially from consideration of molecular weight
and aromatic amino acid content.
Carbonyl Content of Purified Carbonic Anhydrase
III
Protein staining of SDS gels demonstrated that the prominent
carbonic anhydrase III was present in mature male rat liver (Fig. 1), but not in mature female liver nor in prepubertal males
(not shown). This sex-specific expression is well described for
carbonic anhydrase III(34) . We also found that the level of
protein decreased markedly during aging of male rats, decreasing from
5% of total protein at 2 months to virtually undetectable at
20-24 months (Fig. 2A). A similar pattern was seen
in the Western blots for detection of oxidatively modified proteins (Fig. 2B).
Figure 2:
Oxidatively modified proteins during aging
of the rat. Shown are SDS-PAGE analyses of extracts (20 µg of
protein) from liver of male rats aged 2-28 months. A,
Coomassie Blue protein stain; B, anti-dinitrophenyl Western
blot.
We purified the enzyme from livers of male
rats aged 2, 10, and 18 months in order to study possible age-related
changes in the protein. Spectrophotometric quantitation of the carbonyl
content of carbonic anhydrase III purified from two-month-old rats
confirmed the very high level detected on Western blot ().
It remained equally high in protein purified from the older animals.
The observed carbonyl content of 13 nmol/mg protein (0.4 mol/mol
protein) is more than 5 times that observed for total liver protein
from young rats (19) and approximates the levels achieved after
extensive in vitro oxidation of proteins by irradiation or
metal-catalyzed oxidation(23) .
Glutathiolation of Carbonic Anhydrase III during
Aging
Carbonic anhydrase III is known to form a disulfide link
between a cysteine residue and glutathione, a process termed S-glutathiolation(32) . When our preparation was
analyzed by electrospray mass spectrometry, two components were
observed. Their masses were 29,651 and 29,345, a difference of 306 mass
units. A difference of 305 would be expected upon addition of one
glutathione. Isoelectric focusing separates the unmodified protein from
the S-thiolated form(32) , accounting for the two bands
observed in our preparations (Fig. 1). The more acidic band
differs by one titratable group, as expected upon addition of a
glutathione. A second cysteine group in carbonic anhydrase III can also
be S-thiolated, accounting for a third, more acidic band that
is sometimes visualized. Treatment with dithiothreitol converts the
glutathiolated forms to the unmodified form (Fig. 3, inset). S-glutathiolation of proteins has been reported to occur when
cells and tissues are exposed to oxidative
stress(35, 36, 37, 38, 39, 40) .
With the recognition that carbonic anhydrase has a high carbonyl
content, which is sustained during aging, it was of interest to
determine the extent of S-glutathiolation during aging. The
native and modified forms were separated by reverse phase
chromatography to facilitate quantitation of each form (Fig. 3).
The fraction of glutathiolated enzyme doubled from 2 to 18 months of
age ().
Figure 3:
S-glutathiolation of carbonic
anhydrase III. Purified carbonic anhydrase III from 2-, 10-, and
18-month-old male rats was analyzed by isoelectric focusing and reverse
phase chromatography as described under ``Experimental
Procedures.'' A chromatogram of 25 µg of carbonic anhydrase
III from a 2-month-old rat without (solidline) and
with (dashedline) exposure to 30 mM dithiothreitol for 10 min at room temperature. The two major peaks
from the unexposed preparation were collected and analyzed by
electrospray mass spectrometry. The mass of the later eluting peak was
29,345 Da, in reasonable agreement with the 29,312 Da expected from the
sequence of the muscle protein with amino-terminal acetylation. (The
full sequence of the liver protein has not been reported. Partial
sequencing showed that the liver and muscle proteins are very similar,
but differences do exist (32).) The earlier eluting peak had a mass of
29,651 Da. This is 306 Da greater than the later eluting peak,
confirming that the earlier eluting protein was monoglutathiolated. The
small peak eluting just after 36 min was not further characterized, but
its mass was the same as that of the reduced form. Inset,
effect of increasing concentrations of dithiothreitol (DTT) on
the isoelectric focusing of the carbonic anhydrase
III.
Changes in Catalytic Activity during Aging
Enzymes
purified from older animals may exhibit lower specific activity than
those purified from younger animals(6) . It has been
hypothesized that many of the biological changes associated with aging
are a consequence of chronic oxidative stress, including altered
specific activities as a result of oxidative
modifications(6, 41) . Since carbonic anhydrase III
shows changes consistent with exposure to oxidizing environments, it
was of interest to determine specific activities during aging. Carbonic
anhydrase III has three known enzymatic activities: 1) carbon dioxide
hydration, 2) esterase, and 3) phosphatase. The specific activities
were determined on purified proteins from rats aged 2, 10, and 18
months. The results were the same for two separate preparations (Fig. 4). Carbon dioxide hydratase and esterase activity showed a
modest loss of 30% from 2 to 18 months of age. However, p-nitrophenyl phosphatase activity was almost completely lost
in the protein purified from the 18-month-old rats.
Figure 4:
Changes
in carbonic anhydrase III activities during aging. CO hydratase, p-nitrophenyl esterase, and p-nitrophenyl phosphatase activities were determined in
preparations purified from 2-, 10-, and 18-month-old male rats. The
specific activities of the 2-month-old rats were set to 100%. They were
110 units/mg for CO
hydratase, 3.5 nmol/min/mg protein for
the p-nitrophenyl esterase, and 0.14 nmol/min/mg protein for
the p-nitrophenyl phosphatase. Solidbars, 2
month old; shadedbars, 10 month old; openbars, 18 month old.
Thermal Denaturation of Carbonic Anhydrase III from Young
and Old Rats
Changes in the rate of heat denaturation have been
described in enzymes purified from young and old animals. Such
differences provide additional evidence for the appearance of modified
forms of the protein during aging(42) . The thermal inactivation
pattern of carbonic anhydrase III from young and old rats is shown in Fig. 5. The patterns of inactivation differ between hydratase and
esterase activity and between young and old animals. The difference
between the two activities indicates that different domains of the
protein catalyze the two reactions. Focusing on the hydratase activity,
enzyme from the young animal was inactivated in a first-order process,
consistent with the presence of one detectable form of the protein. The
protein from the older animal was distinctly less stable to heat, a
pattern described for other enzymes purified from young and old animals
(42). Moreover, one can reproducibly demonstrate a biphasic pattern of
denaturation for the hydratase activity from the old animal, suggesting
a molecular heterogeneity.
Figure 5:
Thermal denaturation patterns of carbonic
anhydrase III. Enzyme purified from 2- and 18-month-old male rats was
incubated at 56 °C. CO hydratase and p-nitrophenyl esterase activities were measured at the plotted
times by dilution into assay medium.
--
,
hydratase from 2-month-old rats;
--
, hydratase
from 18-month-old rats;
- - -
,
esterase from 2-month-old rats;
- - -
,
esterase from 18-month-old rats. The dashedline extends the regression fit line for the 5-30 min points of
the hydratase from the 18-month-old rat. The results shown are the
average of two separate purifications for each age. Individual time
points for the two preparations were within 10% of each
other.
transfer, and ion exchange. Inherited deficiency of
isozyme II causes a syndrome of osteopetrosis, renal tubular acidosis,
and cerebral calcification(46) . However, the functional roles
of isozyme III are unknown. It is abundant in red skeletal muscle,
where it comprises about 8% of the cytosolic protein in both males and
females(47) . In adipocytes it constitutes almost a quarter of
the total cytosolic protein(48, 49) . It is a major
soluble protein in rat liver from sexually mature males but is almost
absent in females. Administration of androgens to females induces
expression in their liver, while administration of growth hormone to
males represses synthesis to the female level(50) . This sexual
dimorphism is mediated at the mRNA level(51) . It is specific to
liver, with both male and female muscle having comparable levels. The
initially high level of liver carbonic anhydrase III decreases during
aging of the male (Ref. 34 and Fig. 2).
-Arg is the signature for the catalytic motif
in phosphoester hydrolysis. They pointed out that this motif is also
observed in protein-serine-threonine phosphatases and in acid
phosphatases. Also, Barnea etal.(61) noted
that a family of receptor tyrosine phosphatases has a carbonic
anhydrase-like domain in an extracellular region of the protein.
-Arg phosphatase signature in each protein.
It is not present in carbonic anhydrase I or II. In the rat protein,
the Cys is residue 181 and the Arg is 187: Cys-Leu-Phe-Pro-Ala-Cys-Arg. S-glutathiolation of either Cys-181 or Cys-186 could certainly
disrupt catalytic function(60, 62) . Arginine residues
are known to be susceptible to metal-catalyzed oxidation(63) ,
and modification of Arg-187 will disrupt catalysis (60). While
experimental investigation is required to test the idea, it is possible
that the loss of phosphatase activity is a consequence of increased
susceptibility to oxidative stress during aging(6) . If carbonic
anhydrase III actually functions as a protein phosphatase, loss of
catalytic competence during aging could have pleiotropic effects.
Table: Carbonyl content and S-glutathiolation of
carbonic anhydrase III during aging
2 nmol/mg protein (19). The assays summarized
here were from four separate purifications at 2 months, one at 10
months, and two at 18 months. Reproducibility was good, with all
coefficients of variation being less than 10%. Taking the 2 month
assays as an example, the standard deviation of the carbonyl content
was 1.2 with a coefficient of variation of 3.4%, while the standard
deviation of the glutathiolation was 2.5% with a coefficient of
variation of 8%.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.