©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Carbonic Anhydrase III
OXIDATIVE MODIFICATION IN VIVO AND LOSS OF PHOSPHATASE ACTIVITY DURING AGING (*)

Elisa Cabiscol (§) , Rodney L. Levine (¶)

From the (1)Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-0320

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

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.

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.

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.

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 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.

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.

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.

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.


RESULTS

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.''

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 to 10M, 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).

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 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.




DISCUSSION

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 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).

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-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.

Inspection of the sequence of carbonic anhydrase III from rat, mouse, cow, horse, and human (28, 53) demonstrated the Cys-X-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

The average liver protein has a carbonyl content of 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%.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the Lleida Council, Lleida, Spain.

To whom correspondence should be addressed: NIH, 3 Center Dr., MSC 0320, Bethesda, MD 20892-0320. Tel.: 301-496-2310; Fax: 301-496-0599; E-mail: rlevine@nih.gov.

The abbreviations used are: HPLC, high pressure liquid chromatography; MALDI, matrix-assisted laser desorption ionization; PAGE, polyacrylamide gel electrophoresis; SMP-1, senescence marker protein-1.


ACKNOWLEDGEMENTS

We thank Dr. Henry M. Fales of the National Heart, Lung, and Blood Institute for the mass spectrometric measurements reported in this paper.


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