Velocity of CO2 exchange in muscle and liver.
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Animals↗
Biomedical subjects
S J Dodgson
Publications and source records attributed to S J Dodgson.
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)
Adult↗
Carbonic anhydrase activity of chick osteoclasts is increased by parathyroid hormone.
Embryonic chick osteoclasts were harvested, cultured for 18 h, and separated from erythrocytes, and carbonic anhydrase (CA) activity was assayed. Cultures contained 75-85% osteoclasts by acid phosphatase stain. CA activity was also determined after exposure to parathyroid hormone (PTH). Two methods of measurement were used: a delta pH method, which detects the generation of hydrogen ion by carbonic anhydrase, and a mass spectrometric method, which follows the disappearance of 18O-enriched CO2. Unstimulated osteoclasts showed CA activity that was one-third that of erythrocytes but was comparable with macrophages and chondrocytes. Osteoclasts exposed to PTH showed a twofold increase in activity, which occurred after 30 min. Ethoxzolamide (10(-8) M) inhibited enzyme activity by 90%. Treatment of osteoclasts with calcitonin did not prevent the PTH-associated increase in CA activity. The doubling of CA activity with PTH stimulation supports the hypothesis that osteoclastic CA is intimately involved in bone resorption.
Animals↗
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.
Acetazolamide↗
Inhibition of CA V decreases glucose synthesis from pyruvate.
The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 5 mM pyruvate, 5 mM lactate, 3 mM ornithine, 10 mM NH4Cl, 1 mM oleate; with these inclusions both urea and glucose synthesis start with HCO3- -requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated Ki for each approximately 0.1 mM. In other experiments hepatocytes were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 10 mM glutamine, 1 mM oleate; with these inclusions glucose synthesis no longer starts with a HCO3- -requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated Ki of 0.1 mM, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 degrees C, with 7.5 mM pyruvate, 3 mM ATP, and 10 mM NaHCO3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.
Animals↗
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.
Animals↗
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.
Animals↗
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).
Acetazolamide↗
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.
Acidosis, Renal Tubular↗
Measurement of carbonic anhydrase activity inside cells and subcellular particles.
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Animals↗
The role of carbonic anhydrase in hepatocyte metabolism.
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Acetazolamide↗
Contribution of matrix carbonic anhydrase to citrulline synthesis in isolated guinea pig liver mitochondria.
Synthesis of citrulline from ornithine, NH4+, and HCO3- by isolated pig liver mitochondria is inhibited by acetazolamide, a specific inhibitor of carbonic anhydrase, at the same concentrations which inhibit the mitochondrial matrix carbonic anhydrase. At an acetazolamide concentration sufficient to give complete inhibition of matrix carbonic anhydrase, the rate of citrulline synthesis is reduced by 71%, but no further decrease in citrulline is observed at higher concentrations of acetazolamide. Stimulation of O2 uptake by ornithine under conditions of maximal citrulline synthesis is also inhibited by acetazolamide. At pH 6.7, the ratio of citrulline synthesis is depressed relative to the rates observed over the range 7.2-7.7, and acetazolamide inhibits this rate by only 20%. These results support the hypothesis that the physiological role of carbonic anhydrase in liver mitochondria is to supply HCO3- as the substrate for the enzyme carbamoyl phosphate synthetase I, which provides the intermediate carbamoylphosphate in the rate-limiting step of citrulline synthesis. Since the uncatalyzed rate of CO2 hydration is rapid enough that it should not be rate-limiting for the carbamoylphosphate synthetase reaction, carbonic anhydrase appears to regulate access of HCO3- in the synthetase and so should be considered as one of the enzymes participating in the biosynthetic pathway leading to urea formation in the hepatocyte.
Acetazolamide↗
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.
Amino Acid Sequence↗
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.
Animals↗
Determination of intramitochondrial pH by means of matrix carbonic anhydrase activity measured with 18O exchange.
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Animals↗
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.
2,3-Diphosphoglycerate↗
Mitochondrial carbonic anhydrase.
We have assayed carbonic anhydrase activity (carbonate dehydratase, carbonate hydro-lyase, EC 4.2.1.1) and bicarbonate permeability in suspensions of broken and intact guinea pig mitochondria by monitoring the disappearance of C16O18O. We found significant activity in preparations from liver and skeletal muscle, but not in preparations from heart muscle, brain, and kidney. Intact mitochondria containing carbonic anhydrase produce a two-phase acceleration of the disappearance of the labeled CO2, which indicates that the enzyme is located in a region more accessible to CO2 than to HCO3-. Acetazolamide inhibits the enzyme activity instantly in broken mitochondria but only after a delay in intact mitochondria, indicating that the enzyme is in a region not immediately accessible to the inhibitor. Sonication of mitochondria containing carbonic anhydrase activity releases the enzyme, which remains in the supernatant after sedimentation of the submitochondrial particles. This shows that mitochondrial carbonic anhydrase is in the matrix compartment and not in, or bound to, the inner membrane. The activity of the enzyme increases markedly with increasing pH. The enzyme activity of intact mitochondria is greater than that of the broken mitochondria at the same pH of the suspending fluid, corresponding to an intramitochondrial pH that is 0.2-0.5 unit more alkaline.
Animals↗
Erythrocyte phosphoglycerate kinase deficiency: enzymatic and oxygen binding studies.
Enzyme and oxygen transport studies have been made on seven members of a kindred with red cell deficiency of phosphoglycerate kinase (PGK), an enzyme known to be linked to the X-chromosome. The two affected males were children, had little detectable PGK activity and had severe neurological damage. Both had moderate haemolytic anaemia complicated by the occurrence of haemolytic crises. Red cell ATP levels were substantially decreased in both subjects. However, they had more than double the normal level of 2,3-diphosphoglycerate (2,3-DPG) in their red cells, associated with a rightward shift in their oxygen-haemoglobin equilibrium curves. This shift in the curve was sufficient to permit oxygen delivery to most body tissues, including the brain, at better tensions than normal, except during a haemolytic crisis. Two of the three known heterozygotes were aged over 70. They had moderate haemolytic anaemia, considerable reduction of red cell PGK activity (19% and 35% of normal, respectively), and considerable increase in red cell 2,3-DPG with rightward shifting of the oxygen-haemoglobin equilibrium curve. The other known heterozygote was aged 22 and showed little abnormality in her blood oxygen transport.
Adenosine Triphosphate↗