Expression of hepatic mitochondrial carbonic anhydrase V.

We have raised specific (rabbit anti-rat) polyclonal antibodies to hepatic mitochondrial carbonic anhydrase V (CA V) and used them to assay the amounts of protein expressed in liver mitochondria isolated from term-foetal, control or diabetic adult rats and in perivenous and periportal rat hepatocytes. The levels of CA V expressed in mitochondria isolated from the livers of adult male and female rats are similar and increase (about 2-fold) in mitochondria from adult diabetic rats when compared to those isolated from the livers of control rats. The level of enzyme in adult liver was higher than in the livers of term-foetal rats. CA V is expressed in both perivenous and periportal hepatocytes, but the level of expression is greater (approx. 40%) in perivenous cells. The implications and significance of these findings are discussed with reference to the roles and properties of the other carbonic anhydrase isoenzymes and the metabolic function of the mitochondrial isoenzyme.

Immunocytochemical localization of mitochondrial carbonic anhydrase in rat tissues.

We used a monospecific polyclonal antiserum against mitochondrial carbonic anhydrase (CA V) from rat liver to study tissue localization of this new member of the carbonic anhydrase gene family. Strong granular immunostaining reaction of CA V was observed in hepatocytes, myocardium, and in certain populations of skeletal muscle fibers. This is the first time that mitochondrial carbonic anhydrase is described in cardiac tissue of rat or any other species. Different epithelial cells revealed very heterogeneous staining reaction, suggesting that mitochondria are a heterogeneous population with respect to their CA V content. Many cells in different glandular epithelia did not show any CA V, whereas some cells, such as gastric parietal cells, were intensely stained with CA V antibodies. No systematic co-expression of CA V with CA I, CA II, or CA III was observed, although the distribution of CA V in skeletal muscle was somewhat similar to that of CA III. Connective tissue cells such as fibroblasts, chondroblasts, and osteoblasts were negative.

The value of inherited deficiencies of human carbonic anhydrase isozymes in understanding their cellular roles.

Very little light has been shed on the role of the low-activity CA I isozyme in humans by studies on CA I-deficient individuals. On the other hand, CA II-deficient individuals exhibit abnormalities of bone, kidney and brain, implicating a functional role for the high-activity CA II isozyme in cells from these tissues and organs. It also appears that the CA II-deficient red cell is capable of normal respiratory function under unstressed conditions. In addition, there is some preliminary evidence that those organs such as the eye which primarily contain the CA II isozyme, may be able to function effectively in the absence of CA II.

Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined.

The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol.

Carbonic anhydrase: inhibition results in decreased urea production by hepatocytes.

The amount of urea produced in 60 min, [urea]t = 60, from intact guinea pig hepatocytes incubated in NH4Cl, oleate, lactate, NaHCO3, and ornithine at 37 degrees C at pH 7.1 is decreased by ethoxzolamide (EZ): Ki,EZ [urea]t = 60 +/- SD at 37 degrees C, pH 7.1 is 0.14 +/- 0.11 mM (10 Dixon plots). This value is in the same range as Ki,EZ for carbonic anhydrase (CA) activity of disrupted hepatocytes at 37 degrees C: 0.08 +/- 0.06 mM (2). [Urea]t = 60 is pH dependent whether external CO2 is supplied (25 mM HCO-3, 95% O2-5% CO2 and 5 mM HCO-3, 99% O2-1% CO2) or not [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 100% O2]. Ki,EZ [urea]t = 60 is independent of both external pH and external total CO2. Ki,EZ (CA) of disrupted mitochondria at 37 degrees C is 0.033 +/- 0.013 microM (2). This value was approximately 3,000-fold lower than the Ki,EZ [urea]t = 60 for intact hepatocytes or Ki,EZ (CA) for disrupted hepatocytes. These results support the general hypothesis that mitochondrial CA is involved in urea synthesis by intact hepatocytes and that cytosolic components raise the experimentally determined Ki,EZ [urea]t = 60. We also conclude that the value of Ki,EZ [urea]t = 60 is independent of the availability of the substrate HCO-3 from external sources.

Carbonic anhydrases in cytosol, nucleus, and membranes of rat liver.

The relative contribution of each functional carbonic anhydrase (CA) isozyme to liver CA activity of fed or starved adult male rats has been determined. The functional isozymes are CA II, CA III, CA IV, and CA V. Total CA, CA III, CA II, CA IV, and CA V activities (in mumol CO2 converted.min-1.liver-1), as measured by mass spectrometric assay using NaHC18O16O in aqueous solution at pH 7.4 and 37 degrees C, were 94,867, 38,621, 37,000, 14,515, and < 5,000 in fed rats and 40,630, 10,498, 9,137, 18,338, and < 2,600 in starved rats, respectively. CA II was unevenly distributed throughout the liver. In perivenous and periportal cytosols, as determined by the digitonin-pulse perfusion technique, CA II activity was (in mg cytosolic protein-1) 325 and 69 in fed rats and 167 and 33 in starved rats, respectively. CA III was more evenly distributed and less affected by starvation: CA III activity in perivenous and periportal cytosols was (in mg cytosolic protein-1) 84 and 55 in fed rats and 113 and 52 in starved rats, respectively. Evidence that CA III was concentrated in the nucleus was obtained histochemically by the Ridderstråle cobalt-precipitation technique in 2-microns-thick glutaraldehyde-fixed sections from adult fed rats. Liver CA activity was higher in the perivenous hepatocytes in cytosols and nuclei, whereas CA IV was homogeneously distributed. Incubation of the 2-microns sections with 1 microM acetazolamide resulted in inhibition of all membrane-associated CA, 50% of cytosolic CA, and no nuclear CA.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbonic anhydrase activity of intact carbonic anhydrase II-deficient human erythrocytes.

Intact erythrocytes from subjects with deficiency of blood carbonic anhydrase (CA) II and from normal subjects were assayed for enzyme activity by use of an 18O exchange technique in a solution containing 25 mM (CO2 + NaHCO3) plus 125 mM NaCl. At 25 degrees C and pH 7.4, the catalyzed reaction velocity was 0.32 +/- 0.04 M/s for the CA II-deficient and 1.60 +/- 0.12 M/s for the normal cells, a ratio of 1:5. Under the same conditions at 37 degrees C the relative difference between the CA II-deficient and normal cells was much less: the velocity for the CA II-deficient cells was 0.84 +/- 0.07 M/s and for the normal cells 1.60 +/- 0.32 M/s, a ratio of 1:1.9. Results were comparable for the hemolysates with the NaHCO3 reduced to 85 mM (the corresponding intracellular concentration): at 25 degrees C CA II-deficient cells had a velocity of 0.36 +/- 0.01 M/s compared with 1.12 +/- 0.04 M/s for the normal cells, a ratio of 1:3.1. At 37 degrees C again the relative difference between hemolysates from CA II normal and deficient cells was much less: the CA II-deficient cells had a reaction velocity of 1.17 +/- 0.22 M/s vs. 2.60 +/- 0.36 M/s for the normal cells, a ratio of 1:2.2. The greater fractional reduction of enzyme velocity of CA II-deficient cells at 25 degrees C compared with 37 degrees C appears to be explained by a greater chloride inhibition of the presumed CA I at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of 18O exchange and pH stop-flow assays for carbonic anhydrase.

The hydration velocity of CO2 (0.002 M) catalyzed by bovine carbonic anhydrase (BCA) was measured at 25 degrees C and pH 7.4 by three different techniques: two initial-rate (steady-state) stop-flow methods, one using a glass pH electrode (in Hannover, method 1) and one using spectrophotometric measurements of a pH indicator (in Philadelphia, method 2), and an exchange method in which the disappearance of C18O16O from a bicarbonate solution was determined at equilibrium (in Philadelphia, method 3). The Michaelis-Menten constant (Km) and the inhibition constants for chloride (Ki,Cl) and ethoxzolamide (Ki,ez) were the same for methods 1, 2, and 3. The turnover numbers were 270,000, 400,000, and 555,000 s-1 by methods 1, 2, and 3, respectively. Values for CO2 hydration velocity measured by methods 2 and 3 on the same solution of BCA at the same time were the same. Km, maximal reaction velocity (Vmax), Ki,ez, and Ki,Cl obtained from normal human hemolysate at 37 degrees C and pH 7.2 by methods 2 and 3 were the same. Km and Vmax of the carbonic anhydrase isozyme CA III of homogenate from rabbit soleus were also identical by methods 1 and 3. According to Michaelis-Menten theory, the values of Km and Vmax obtained by method 3 should have been significantly smaller than those obtained by methods 1 and 2. We conclude that the catalytic step itself is apparently not rate limiting under physiological conditions and that method 3 can be used to obtain Michaelis-Menten characteristics of carbonic anhydrase.

Rat lung carbonic anhydrase: activity, localization, and isozymes.

Carbonic anhydrase activity in rat lungs perfused free of blood was localized by homogenization of the tissue followed by differential centrifugation. Four fractions were obtained from the homogenate, a cell debris pellet with a mitochondrial pellet and a microsomal pellet with a clear cytosol supernatant. The last named fraction contained 67% of the total enzyme activity; the cell debris contained 18%, and the mitochondrial and microsomal contained 8 and 7%, respectively. Of the 33% of enzyme activity associated with the pellet fraction, 25% could be experimentally defined as membrane associated by its solubilization with 0.3 M tris-(hydroxymethyl) aminoethane sulfate buffer. The remainder was defined as membrane bound. Purification of the soluble carbonic anhydrase from the lung yielded two isozymes with electrophoretic and inhibitor sensitivities apparently identical with the blood isozymes. Hemoglobin analysis showed that the lung isozymes could not have included more than 0.03% enzyme from blood contamination. The carbonic anhydrase activity present in the whole rat lung would give an average acceleration of the CO2 hydration reaction under physiological conditions over the uncatalyzed rate of 122, sufficient to maintain equilibration between CO2 and plasma HCO3- during blood transit of the lung. If the membrane-associated activity is mostly on the plasma membrane of the endothelial cells and available to the capillary blood, it would be sufficient to give this acceleration. We suggest that the possible source of this membrane-associated activity might be adsorption from the blood of carbonic anhydrase liberated by erythrocyte lysis.

Failure of acetazolamide to decrease intraocular pressure in patients with carbonic anhydrase II deficiency.

The effect of the carbonic anhydrase inhibitor acetazolamide on intraocular pressure was studied in two patients with carbonic anhydrase II deficiency and in six control subjects. The deficient patients had the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. A dose of 125 mg of intravenous acetazolamide caused a significant (P less than .01) decrease in intraocular pressure from baseline (15.0 +/- 1.5 mm Hg) in the control subjects one hour (11.3 +/- 1.5 mm Hg) and four hours (13.8 +/- 1.2 mm Hg) after drug administration. In contrast, the patients with carbonic anhydrase deficiency showed no such decrease in intraocular pressure; baseline intraocular pressure (19.2 +/- 0.2 mm Hg) was significantly unchanged (P greater than .5) at one hour (20.0 +/- 0.1 mm Hg) and four hours (19.2 +/- 0.2 mm Hg).

Mitochondrial carbonic anhydrase is involved in rat renal glucose synthesis.

At 37 degrees C, pH 7.4, carbonic anhydrase activity (kenz) of disrupted rat renal proximal tubules and cortical mitochondria was 2.5 +/- 0.8 (n = 3) and 0.15 +/- 0.40 (n = 3) ml.mg-1.s-1, respectively. Turnover number for renal mitochondrial carbonic anhydrase (CA V) was 24,000 s-1. CA V activity of intact mitochondria was completely inhibited by 0.15 microM ethoxzolamide (EZ). Intact proximal tubules, prepared from 48-h starved male rats, were incubated at 37 degrees C in 10 mM pyruvate in Krebs-Henseleit bicarbonate saline buffer, 5% CO2-95% O2. The rate of glucose synthesis over 60 min was reduced 50% by including 0.6 microM EZ in the incubation solution. The concentration of NaHCO3 was doubled to 50 mM (with a corresponding decrease in NaCl) and the solution gassed with 10% CO2-90% O2; 2.4 microM EZ no longer decreased glucose synthesis. It was concluded that inhibition of glucose synthesis by EZ was directly a result of inhibiting the carbonic anhydrases. The rate of glucose production was subsequently determined with tubules incubating in a HCO3(-)-free N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) buffer; this rate was decreased 50% by 0.6 microM EZ. These data support the hypotheses that CA V provides HCO3- for pyruvate carboxylase and that CO2 can be provided by tubular metabolism. Intact tubules were incubated in from 5 to 20 mM pyruvate in either 25 or 50 mM HCO3-; in either buffer, the rate of glucose synthesis was similar, increasing with increasing pyruvate concentration. At no pyruvate concentration was there a change in the rate of glucose production when tubules were incubated in 50 mM HCO3- buffer with 1.6 microM EZ. These data also support the hypothesis that CA V provides the HCO3- substrate for pyruvate carboxylation when there is a high rate of intracellular CO2 production and external CO2 is low. It is further concluded that the cytosolic carbonic anhydrase (CA II) and the membrane-bound carbonic anhydrase (CA IV) are not involved in glucose synthesis from pyruvate.

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.

Carbonic anhydrase activity of intact erythrocytes from seven mammals.

Carbonic anhydrase activity of intact erythrocytes from seven mammalian species was determined at 25 degrees C, pH 7.4, by mass spectrometry using the 18O-exchange technique. The seven species were Cavia porcellus, Mustela putorius furo, Felis domesticus, Canis familiaris, Homo sapiens, Equus caballus, and Bos taurus. Carbonic anhydrase activities determined as a function of hemoglobin concentration (std kcat) for intact erythrocytes at pH 7.4 were not significantly different from those determined for lysed erythrocytes at pH 7.20 for each species. The carbonic anhydrase activity of intact erythrocytes was not changed by a concentration of acetazolamide that inhibited it 85% in lysate (10(-7) M) in the 5-10 min needed for the assay. However, ethoxzolamide, another carbonic anhydrase inhibitor, produced the same fractional inhibition of enzyme activity in erythrocyte suspensions as in lysate in 1-2 min. Thus the inhibition constant, Ki, was approximately the same in both intact and lysed cells from each species, and it was possible to measure the apparent molar enzyme concentration inside the erythrocytes from the concentration of bound inhibitor. Intracellular enzyme concentrations were greater in those species with larger cells, but the specific activity of the carbonic anhydrase per molecule was less so that the overall enzyme activity, std kcat, was not related to mean cell volume. The effective permeability of the cells to the self-exchange of bicarbonate ion, P(HCO3-), averaged 2 X 10(-4) cm x s-1 and did not vary among the species.

The reaction kinetics of four sheep haemoglobins with identical alpha-chains.

Sheep haemoglobins A, B, C and F are known to have identical alpha-chains but differing beta-chains. Rate constants were determined for the combination of CO with these four deoxy haemoglobins (l') and for the dissociation of O2 from the fully saturated tetramers (k4) from 15 to 38 degrees C at physiological pH in the presence of CO2, and at pH 9.1 in the absence of CO2. The constant for the replacement of O2 from the tetramer by CO (m' infinity) and the ratio of the combination constants of CO and O2 with the three parts saturated tetramer (l'4/k'4), were also determined over this range of temperature at physiological pH in the presence of CO2. In no respect did Hb A differ from Hb C. Apart from this the values for l' differed significantly between each pair of haemoglobins. The constant k4 showed significant differences only when Hb B was compared with Hb A or Hb C. Values of m' infinity were similar for the four haemoglobins; and the values of the ratio l'4/k'4 were similar for Hbs A, C and F, and about half as great for Hb B. The results do not support the hypothesis of predominance of the alpha-chains in determining the rate of ligand combination with the desaturated tetramer. It is suggested that faster rate of release of O2 from Hb B may be due to its having lysine at position HCl in the beta-chain whereas the other haemoglobins have arginine.

Comparison of carbonic anhydrase activity and other haematological values of two Eutherian and three Australian marsupial mammals.

1. Carbonic anhydrase activity and 2,3-diphosphoglycerate (2,3-DPG) concentration were determined in whole blood from humans (Homo sapiens), rabbits (Oryctolagus cuniculus), eastern grey kangaroos (Macropus giganteus), pademelons (Thylogale billardierii) and brush-tailed possums (Trichosurus vulpecula). 2. Marsupial blood carbonic anhydrase activity increased as species body size decreased. 3. T. billardierii haemoglobin was found to have a polymorphism which may be the same (beta 2 = histidine or glutamine) as that of M. giganteus. 4. The concentration of 2,3-DPG int e red cells of T. billardierii was approximately equal to that of the haemoglobin tetramer. Levels of 2,3-DPG in the other species were similar to those previously reported.

Rat renal proximal tubular gluconeogenesis: possible involvement of nonmitochondrial carbonic anhydrase isozymes.

The carbonic anhydrase (CA) inhibitor ethoxzolamide decreases the rate of glucose synthesis from 10 mM pyruvate by tubules incubating in 25 mM HCO3- but not in 50 mM HCO3-: this is evidence that rat renal cortical mitochondrial CA (CA V) provides HCO3- for pyruvate carboxylation in renal tubular gluconeogenesis at physiological total CO2 (CO2 + HCO3-). In renal proximal tubules prepared from 48-h-starved rats and incubating in 10 mM pyruvate in 25 mM HCO3- buffered saline (Krebs-Henseleit buffer) the CA inhibitors acetazolamide (AZ) and benzolamide (BZ) decreased the rate of glucose synthesis. Maximal inhibition was reached with 125 microM AZ or with 450 microM BZ. The rate of glucose synthesis increased with increasing pyruvate concentration from 3.33 to 20 mM; including 600 microM BZ or 188 microM AZ results in glucose synthesis becoming independent of increasing pyruvate concentration. Doubling the physiological concentration of bicarbonate restored the dependence of glucose synthesis on pyruvate concentration and partly, but not completely, alleviated the inhibitory effect of AZ and BZ, leading to the conclusion that AZ and BZ influence gluconeogenesis by affecting enzymes in addition to CA V. Tubules were incubated with substrates which do not require pyruvate carboxylation for synthesis of oxaloacetate. When tubules were incubated in 10 mM malate the rate of glucose synthesis was unaffected by less than 100 microM AZ or 400 microM BZ and was decreased maximally by 40 and 20%, respectively, by 125 microM AZ, 450 microM BZ, and higher concentrations of these drugs. Increasing the malate concentration from 3.33 to 20 mM increased the rate of glucose synthesis; 600 microM BZ inhibited the rate of glucose synthesis only when the malate concentration was greater than 10 mM but 188 microM AZ decreased the rate of glucose synthesis at each concentration of malate. Results were similar when tubules were incubated in glutamine with CA inhibitors. The rate of glucose synthesis differed with the substrate metabolized and the substrate concentration except when 600 microM BZ was included.(ABSTRACT TRUNCATED AT 400 WORDS)