The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO₂ hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K_m of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (K_i = 1.22 × 10^?3M), arsenate (K_i = 1.17 × 10^?3M), and molybdate (K_i = 1.34 × 10^?7M), with these inhibitors having no effect on the CO₂ hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO₂ hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO₂ hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.     

Specific arginine modification at the phosphatase site of muscle carbonic anhydrase

Mammalian carbonic anhydrase III has previously been shown to catalyze the hydrolysis of p-nitrophenyl phosphate in addition to possessing the conventional CO2 hydratase and p-nitrophenylacetate esterase activities. Modification of pig muscle carbonic anhydrase III with the arginine reagent phenylglyoxal yielded two clearly distinctive results. Reaction of the enzyme with phenylglyoxal at concentrations equivalent to those of the enzyme yielded stoichiometric inactivation titration of the enzyme's phosphatase activity, approaching 100% loss of activity with the simultaneous modification of one arginine residue, the latter based on a 1:1 reaction of phenylglyoxal with arginine. At this low ratio of phenylglyoxal to enzyme, neither the CO2 hydratase activity nor the acetate esterase activity was affected. When the modification was performed with a significant excess of phenylglyoxal, CO2 hydratase and acetate esterase activities were diminished as well. That loss of activity was accompanied by the incorporation of an additional half dozen phenylglyoxals and, presumably, the modification of an equal number of arginine residues. The data in their entirety are interpreted to show that the p-nitrophenylphosphatase activity is a unique property of carbonic anhydrase III and that excessive amounts of the arginine-modifying reagent lead to unspecific structural changes of the enzyme as a result of which all of its enzymatic activities are inactivated.     

Carbonic anhydrase III: the phosphatase activity is extrinsic

The carbonic anhydrases reversibly hydrate carbon dioxide to yield bicarbonate and hydrogen ion. They have a variety of physiological functions, although the specific roles of each of the 10 known isozymes are unclear. Carbonic anhydrase isozyme III is particularly rich in skeletal muscle and adipocytes, and it is unique among the isozymes in also exhibiting phosphatase activity. Previously published studies provided evidence that the phosphatase activity was intrinsic to carbonic anhydrase III, that it had specificity for tyrosine phosphate, and that activity was regulated by reversible glutathionylation of cysteine186. To study the mechanism of this phosphatase, we cloned and expressed the rat liver carbonic anhydrase III. The purified recombinant had the same specific activity as the carbonic anhydrase purified from rat liver, but it had virtually no phosphatase activity. We attempted to identify an activator of the phosphatase in rat liver and found a protein of approximately 14 kDa, the amount of which correlated with the phosphatase activity of the carbonic anhydrase III fractions. It was identified as liver fatty acid binding protein, which was then purified to test for activity as an activator of the phosphatase and for protein-protein interaction, but neither binding nor activation could be demonstrated. Immunoprecipitation experiments established that carbonic anhydrase III could be separated from the phosphatase activity. Finally, adding additional purification steps completely separated the phosphatase activity from the carbonic anhydrase activity. We conclude that the phosphatase activity previously considered to be intrinsic to carbonic anhydrase III is actually extrinsic. Thus, this isozyme exhibits only the carbon dioxide hydratase and esterase activities characteristic of the other mammalian isozymes, and the phosphatase previously shown to be activated by glutathionylation is not carbonic anhydrase III.     

Carbonic anhydrase C in white-skeletal-muscle tissue.

We investigated the activity of carbonic anhydrase in blood-free perfused white skeletal muscles of the rabbit. Carbonic anhydrase activities were measured in supernatants and in Triton extracts of the particulate fractions of white-skeletal-muscle homogenate by using a rapid-reaction stopped-flow apparatus equipped with a pH electrode. An average carbonic anhydrase concentration of about 0.5 microM was determined for white skeletal muscle. This concentration is about 1% of that inside the erythrocyte. Some 85% of the muscle enzyme was found in the homogenate supernatant, and only 15% appeared to be associated with membranes and organelles. White-skeletal-muscle carbonic anhydrase was characterized in terms of its Michaelis constant and catalytic-centre activity (turnover number) for CO2 and its inhibition constant towards ethoxzolamide. These properties were identical with those of the rabbit erythrocyte carbonic anhydrase C, suggesting that a type-C enzyme is present in white skeletal muscle. Affinity chromatography of muscle supernatant and of lysed erythrocytes showed that, whereas rabbit erythrocytes contain about equal amounts of carbonic anhydrase isoenzymes B and C, the B isoenzyme is practically absent from white skeletal muscle. Similarly, ethoxzolamide-inhibition curves suggested that white skeletal muscle contains no carbonic anhydrase A. It is concluded that white skeletal muscle contains essentially one carbonic anhydrase isoenzyme, the C form, most of which is probably of cytosolic origin.     

Identification of pseudoisoenzymic subforms of muscle carbonic anhydrase.

Rabbit muscle carbonic anhydrase III, a recently discovered third isoenzyme (possibly muscle specific) of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) (Register, A.M., Koester, M.K. and Noltmann, E.A. (1978) J. Biol. Chem. 253, 4143--4152) has been subjected to isoelectric focusing. When monomer samples, shown to be homogeneous by both ion-exchange and molecular sieve chromatography, were analyzed by this technique, three subspecies were produced, which were similar in amino acid composition and specific CO2 hydratase activity. In addition to having either monomer or dimer status, the subspecies differed in the extent of oxidation of their sulhydryl groups and in their isoelectric pH values (9.3, 8.8, and 8.4, respectively). Also, the presence of dithiothreitol will affect their relative concentrations. These subforms are therefore designated as pseudoisoenzymes and are considered to be neither genetically nor functionally separate enzyme species.     

Immunohistochemical localization of carbonic anhydrase isoenzymes II and III in quail kidney

The immunohistochemical localization of carbonic anhydrase isoenzymes has never been investigated in avian renal tissue previously. Enzyme activity has largely been documented by histochemical and physiological reports. In this investigation, specific antisera were used to study the distribution of the cytosolic carbonic anhydrase II and III isoenzymes in the quail kidney. Comparison between the present findings and the corresponding histochemical patterns, previously obtained in the same species by a cobalt phosphate precipitation method, resulted in the bulk of renal carbonic anhydrase activity being attributed to the carbonic anhydrase II isoenzyme. Conversely, moderate carbonic anhydrase III immunostaining appeared to be confined to the smooth muscle cells of ureteral and arteriolar walls. Indirect evidence of the occurrence, in the quail kidney, of a membrane-associated carbonic anhydrase form, antigenically distinct from the II and III isoforms, was inferred.     

Carbonic anhydrase in guinea pig skeletal muscle mitochondria

The presence of carbonic anhydrase activity was demonstrated in guinea pig skeletal muscle mitochondria purified by Percoll gradient centrifugation such that contamination by sarcoplasmic reticulum vesicles was less than 5%. Assay of purified heavy sarcoplasmic reticulum vesicles for carbonic anhydrase activity showed these to have somewhat less activity than the mitochondria, so that any contribution by sarcoplasmic reticulum vesicles to mitochondrial activity would be negligible. In agreement with this observation, rabbit skeletal muscle mitochondria prepared by the Percoll method had no detectable activity. Assay of the guinea pig muscle mitochondrial enzyme activity in the presence of Triton X-100 showed a sixfold greater activity than in its absence, indicating a matrix location for the carbonic anhydrase. The enzyme is highly sensitive to the sulfonamide inhibitor ethoxzolamide, with Ki = 8.7 nM. The activation energy obtained from the rate constant for CO2 hydration, kenz with units (mg/ml)-1 s-1, over the range 4 to 37 degrees C was 12.8 kcal/mol. These properties are those expected for a carbonic anhydrase of the CA II class of isozymes, rather than for CA I, CA III, and the liver mitochondrial enzyme CA V.     

Basic muscle protein,a third genetic locus isoenzyme of carbonic anhydrase?

Rabbit muscle cytosol extract contains a basic protein which represents about 2% of the total cytosol protein. It contains zinc in a 1:1 stoichiometric ratio, based on a molecular weight of 30,000, and it catalyzes the hydration of CO₂. It is immunochemically distinct from the high and low activity forms of rabbit blood carbonic anhydrase. It has comparatively poor activity as an esterase, and about 20% of the CO₂ hydratase activity of the rabbit blood low activity carbonic anhydrase. This CO₂ hydratase activity is not inhibited by acetazolamide at concentrations which totally inhibit the activity of the blood carbonic anhydrases. The evidence obtained to date, though circumstantial, suggests that this basic metalloprotein is a carbonic anhydrase derived from a third genetic locus with properties considerably different from those of the mammalian carbonic anhydrases heretofore identified.     

Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.     

Some properties of site-specific mutants of human carbonic anhydrase II having active-site residues characterizing carbonic anhydrase III

Four amino acid residues, His64, Asn67, Leu198 and Val207, in the active site of human carbonic anhydrase II, have been replaced by Lys64, Arg67, Phe198 and Ile207, which are characteristic for the muscle-specific, low-activity isoenzyme form, carbonic anhydrase III. The aim of the investigation has been to test if any of these residues, or a combination of them, is important for the low CO2 hydration activity, low esterase activity, low pKa for the pH/rate profile and low affinity for sulfonamide inhibitors characterizing carbonic anhydrases III. However, no evidence for such critical roles was found. A combination of Lys64 and Arg67 appears to result in a decrease in CO2 hydration activity, but even the quadruple mutant having all four changes is only eight times less active (kcat/Km) than unmodified isoenzyme II, in contrast to isoenzyme III which is nearly 300 times less active than isoenzyme II. The 4-nitrophenyl acetate hydrolase activity of the quadruple mutant is sevenfold lower than that of unmodified isoenzyme II, while the active site of isoenzyme III hardly catalyzes the hydrolysis of this ester at all. The pKa controlling the esterase activity of the quadruple mutant is 6.2, which should be compared to a value of 6.8 for unmodified isoenzyme II, and about 5 for isoenzyme III. While isoenzyme III binds sulfonamide inhibitors 10(3)-10(4) times less strongly than isoenzyme II, only [Asn-67----Arg]isoenzyme II shows a weaker binding of the investigated sulfonamide, dansylamide, but only by a factor of two. Some of the other mutants show enhanced affinities, up to nearly fourfold for the double mutant with Phe198 and Ile207. It is speculated that additional differences between the active sites of isoenzyme II and III might be important for the precise orientations and interactions of the side chains of isoenzyme-III-specific amino acid residues.     

Purification and localization of human carbonic anhydrase

Summary Three different isoenzymes of human carbonic anhydrase are now well characterized. Carbonic anhydrase I and II have been known for several years and are located in high amounts in red blood cells as well as in many other tissues.Carbonic anhydrase III, a protein showing CO₂ hydratase and p-nitrophenylphosphatase activity was isolated from skeletal muscle some years ago. Earlier observations based on enzyme activity and radioimmunoassay studies have suggested that this protein is present in greater quantities in red skeletal muscles than in white ones. We have purified CA III from human soleus muscle and using obtained monospecific polyclonal antibody localized this protein in the same muscle fibers which show acid resistant ATPase activity. Using this protein as a marker for type I muscle fibers, fiber classification into type I and II could now be done also from paraffin embedded sections.This study is supported by the Research Council of Physical Education and Sport, Ministry of Education, Finland     

Purification and characterization of human salivary carbonic anhydrase

A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.     

Partial amino acid sequence of erythrocyte carbonic anhydrase from tiger shark

1. A partial primary structure (197 residues) of carbonic anhydrase from tiger shark (Galeocerdo cuvieri) erythrocytes has been determined. 2. The amino acid sequence of the enzyme is identical to those of human cytoplasmic carbonic anhydrases (CA I-III) by as much as 52-60%. 3. It is shown that tiger shark CA most closely resembles the CA II isoenzyme of amniotes. 4. Isoelectric focusing and inhibition studies on carbonic anhydrase from dogfish (Squalus acanthias) blood and muscle indicate the presence of the same isoenzyme in shark blood and muscle.     

Hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle

We report three experiments which show that the hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle occurs at a site on the enzyme different than the active site for CO2 hydration. This is in contrast with isozymes I and II of carbonic anhydrase for which the sites of 4-nitrophenyl acetate hydrolysis and CO2 hydration are the same. The pH profile of kcat/Km for hydrolysis of 4-nitrophenyl acetate was roughly described by the ionization of a group with pKa 6.5, whereas kcat/Km for CO2 hydration catalyzed by isozyme III was independent of pH in the range of pH 6.0-8.5. The apoenzyme of carbonic anhydrase III, which is inactive in the catalytic hydration of CO2, was found to be as active in the hydrolysis of 4-nitrophenyl acetate as native isozyme III. Concentrations of N-3 and OCN- and the sulfonamides methazolamide and chlorzolamide which inhibited CO2 hydration did not affect catalytic hydrolysis of 4-nitrophenyl acetate by carbonic anhydrase III.     

Catalytic enhancement of human carbonic anhydrase III by replacement of phenylalanine-198 with leucine

Carbonic anhydrase III, a cytosolic enzyme found predominantly in skeletal muscle, has a turnover rate for CO2 hydration 500-fold lower and a KI for inhibition by acetazolamide 700-fold higher (at pH 7.2) than those of red cell carbonic anhydrase II. Mutants of human carbonic anhydrase III were made by replacing three residues near the active site with amino acids known to be at the corresponding positions in isozyme II (Lys-64----His, Arg-67----Asn, and Phe-198----Leu). Catalytic properties were measured by stopped-flow spectrophotometry and 18O exchange between CO2 and water using mass spectrometry. The triple mutant of isozyme III had a turnover rate for CO2 hydration 500-fold higher than wild-type carbonic anhydrase III. The binding constants, KI, for sulfonamide inhibitors of the mutants containing Leu-198 were comparable to those of carbonic anhydrase II. The mutations at residues 64, 67, and 198 were catalytically independent; the lowered energy barrier for the triple mutant was the sum of the energy changes for each of the single mutants. Moreover, the triple mutant of isozyme III catalyzed the hydrolysis of 4-nitrophenyl acetate with a specific activity and pH dependence similar to those of isozyme II. Phe-198 is thus a major contributor to the low CO2 hydration activity, the weak binding of acetazolamide, and the low pKa of the zinc-bound water in carbonic anhydrase III. Intramolecular proton transfer involving His-64 was necessary for maximal turnover.     

Purification and Properties of Three Pig Erythrocyte Carronic Anhydrases

Three distinct forms of the zinc containing enzyme carbonic anhydrase were isolated from pig erythrocytes. One low activity type enzyme and two genetic variants of the high activity type enzyme with identical CO2 hydratase activities which were 8 times as high were isolated from Danish Black and White Swine. In the isolation procedure described, the hemoglobin was eliminated by precipitation with chloroform-ethanol, and the isoenzymes were separated by DEAE-Sephadex chromatography. A number of enzymatically active minor components were separated. They were apparently all genetically linked to one of the three major components. The three purified isoenzymes behaved as homogeneous components during isoelectric focusing and electrophoresis at different pH values. They were characterized in terms of molecular weight, isoelectric pH, zinc content, amino acid composition, and enzymatic activity against CO2, p-nitrophenyl acetate, and β-napthyl acetate. The circular dichroism of the enzymes in the ultraviolet region was studied. The properties of the enzymes were similar to those of carbonic anhydrases of corresponding types isolated from other mammalian species. Sulphur containing amino acid residues were absent in the low activity type enzyme. The amino acid composition of the two high activity mutants deviated only in that an arginine residue in the most widespread genetic variant was replaced by a histidine residue in the less frequent variant. Otherwise the two mutants showed identical properties.     

Acylation and carbamylation of equine muscle carbonic anhydrase (CA-III) upon reaction with p-nitrophenyl esters and carbamoyl phosphate

Equine muscle carbonic anhydrase (CA-III) behaves like ubiquitin in undergoing extensive acylation of N epsilon-lysine residues upon reacting with p-nitrophenyl esters. The enzyme undergoes extensive carbamoylation of lysine residues when reacted with carbamoyl phosphate. The modification of from 6 to 7 lysine residues results in the production of a series of more anodic electrophoretic components. The derivatization of the lysine residues leads to a marked decrease in the enzyme's ability to hydrate CO2. The equine CA-III possesses both acid and alkaline phosphatase activities in contrast to the rabbit which possesses only the former type.     

Esterase activity of carbonic anhydrases serves as surrogate for selecting antibodies blocking hydratase activity

Abstract

Carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase’s. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases.     

Extracellular carbonic anhydrase of skeletal muscle associated with the sarcolemma

We report here 1) the synthesis and properties of a new macromolecular carbonic anhydrase inhibitor, Prontosil-dextran, 2) its application to determine the localization of a previously described extracellular carbonic anhydrase in skeletal muscle, and 3) the application of a recently published histochemical technique using dansylsulfonamide to the same problem. Stable macromolecular inhibitors of molecular weights of 5,000, 100,000 and 1,000,000 were produced by covalently coupling the sulfonamide Prontosil to dextrans. Their inhibition constants towards bovine carbonic anhydrase II are 1-2 X 10(-7) M. The Prontosil-dextrans, PD 5,000, PD 100,000, and PD 1,000,000, were used in studies of the washout of H14CO3-) from the perfused rabbit hindlimb. This washout is slow due to the presence of an extracellular carbonic anhydrase and can be markedly accelerated by PD 5,000 but not by PD 100,000 and PD 1,000,000. Since PD 5,000 is accessible to the entire extracellular space and PD 100,000 and PD 1,000,000 are confined to the intravascular space, we conclude that the extracellular carbonic anhydrase of skeletal muscle is located in the interstitium. The histochemical studies show a strong staining of the sarcolemma of the muscle fibers with high oxidative capacity. It appears likely, therefore, that the extracellular carbonic anhydrase of skeletal muscle is associated with muscle plasma membranes with its active site directed toward the interstitial space.     

Rat kidney mitochondrial carbonic anhydrase

Mitochondrial carbonic anhydrase has previously been quantitated in liver mitochondria; it was not detected in guinea pig kidney cortical mitochondria. Evidence of this enzyme in rat kidney cortical mitochondria is reported. Electron microscopy showed that intact mitochondria were free of other intracellular organelles. When intact kidney mitochondria were added to isotonic 3'-(N'-morpholino) propanesulfonic acid buffer with 25 mM KHCO3 (1% labeled with 18O) the rate of disappearance of C18O16O was biphasic; this indicates that there is carbonic anhydrase within the inner mitochondrial membrane. Intact rat kidney mitochondria were assayed for carbonic anhydrase activity at 4 degrees C by the changing pH technique. The rate of CO2 hydration in the presence and absence of intact mitochondria was identical; this rate increased when Triton X-100 was added which indicates that all carbonic anhydrase is inside the inner mitochondrial membrane. Carbonic anhydrase activity was quantitated as kenz (units, ml.s-1 mg-1 mitochondrial protein) at 37 degrees C, pH 7.4, in 25 mM NaHCO3 (1% labeled with 18O) by following the rate of disappearance of C18O16O from solutions before and after addition of disrupted mitochondria. Values of Kenz for liver and kidney mitochondria from rats given free access to normal rat chow and water at neutral pH were 0.06 and 0.08 (respectively). Values of kenz for liver and kidney mitochondria from rats fed as above and with free access to water adjusted to pH 2.5 with HCl were 0.04 and 0.16, respectively. Values of kenz for rats starved for 48 h were 0.06 and 0.12 (respectively). The values of kenz remained 0.11-0.14 in liver mitochondria from guinea pigs fed normally, given dilute acid, or starved and the value was always at zero in guinea pig kidney mitochondria. Values of Kenz were measured with disrupted mitochondria by the 18O technique as a function of pH at 25 degrees C, 25 to 75 mM NaHCO3, ionic strength 0.3. From pH 7.0 to 8.0 kenz increased threefold for mitochondria from rat liver, fed rat kidney, and acid rat kidney, and increased eightfold for mitochondria from guinea pig liver. kenz was decreased similarly by increasing HCO3- in mitochondria from rat liver, fed kidney, and acid kidney; it is concluded that carbonic anhydrase in rat liver mitochondria is probably the same isozyme as in rat kidney mitochondria. The published observation that rat kidney cortices are up to 10 times as gluconeogenic from pyruvate as guinea pig kidney cortices can be explained by the presence of mitochondrial carbonic anhydrase in rat but not guinea pig mitochondria.