Rat lung carbonic anhydrase: activity, localization, and isozymes

sCarbonic 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.

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A micromethod for measuring carbonic anhydrase activity using 18O exchange between CO2 and H2O

Journal of Applied Physiology ◽

10.1152/jappl.1988.65.4.1902 ◽

1988 ◽

Vol 65 (4) ◽

pp. 1902-1906 ◽

Cited By ~ 3

Author(s):

N. Bitterman ◽

L. Lin ◽

R. E. Forster

Keyword(s):

Enzyme Activity ◽

Carbonic Anhydrase ◽

High Activity ◽

Intact Cell ◽

Porous Membrane ◽

Carbonic Anhydrase Activity ◽

Published Data ◽

Hydration Reaction ◽

Activity Type ◽

Reaction Velocity

We have developed a method of measuring the activity and characteristics of carbonic anhydrase (CA) using the disappearance of 18O from CO2 in 1 ml of gas contained in a glass chamber as it exchanges with H2O in 0.01 ml 0.25 M NaHCO3 solution in a thin (25 micron) porous membrane. Serial gas samples (approximately 0.02 ml) are analyzed in a mass spectrometer to obtain the rate of disappearance of the label. The enzyme activity can be measured inside intact cell or particle membranes. As little as 10(-15) mol of high-activity type CA can be detected at 25 degrees C, and the activity of 200 times this amount can be measured. The uncatalyzed hydration reaction velocity constant was 0.056 +/- 0.004 s-1, in agreement with published data.

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Carbonic anhydrase distribution in rodent embryos and its relationship to acetazolamide teratogenesis.

Journal of Histochemistry & Cytochemistry ◽

10.1177/29.10.6795259 ◽

1981 ◽

Vol 29 (10) ◽

pp. 1213-1218 ◽

Cited By ~ 10

Author(s):

C M Schreiner ◽

K S Hirsch ◽

W J Scott

Keyword(s):

Enzyme Activity ◽

Carbonic Anhydrase ◽

Staining Pattern ◽

Carbonic Anhydrase Activity ◽

Primary Site ◽

Mouse Embryos ◽

Histochemical Method ◽

Site Of Action ◽

Activity Form ◽

Low Activity

The carbonic anhydrase inhibitor, acetazolamide, leads to a unique distal postaxial right forelimb deformity in rats and CBA/J mice, but SWV mice are completely resistant. Using Hansson's histochemical method, the distribution of carbonic anhydrase and its inhibition by acetazolamide in rat, CBA/J mouse, and SWV mouse embryos were compared. Carbonic anhydrase activity was demonstrable in many tissues of sensitive rat and CBA/J mouse embryos and in resistant SWV mouse embryos. The forelimb buds of resistant and sensitive embryos possess carbonic anhydrase activity in the area between the ectoderm and adjacent mesenchyma with no localization of enzyme activity corresponding to the malformation seen in acetazolamide teratogenesis. This suggests that carbonic anhydrase in the forelimbs is not the primary site of action for acetazolamide. A distinctive staining pattern of nucleated erythrocytes in resistant embryos indicated the presence of a low activity form of carbonic anhydrase in nearly half of the erythrocytes. A five-to tenfold greater amount of acetazolamide was needed to completely inhibit carbonic anhydrase activity in nucleated erythrocytes from resistant embryos than in those from sensitive embryos. The existence of a low activity form of carbonic anhydrase in SWV embryo erythrocytes may be the basis of resistance to acetazolamide teratogenesis.

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CARBONIC ANHYDRASE ACTIVITY IN RAT NEUROHYPOPHYSIS FOLLOWING OSMOTIC STRESS

Journal of Histochemistry & Cytochemistry ◽

10.1177/20.8.621 ◽

1972 ◽

Vol 20 (8) ◽

pp. 621-626 ◽

Cited By ~ 1

Author(s):

G. J. BOER

Keyword(s):

Enzyme Activity ◽

Carbonic Anhydrase ◽

Osmotic Stress ◽

Surface Coating ◽

Incubation Medium ◽

Water Deprivation ◽

Carbonic Anhydrase Activity ◽

Neural Lobe ◽

Modified Method ◽

Considerable Loss

Using the colorimetric micromethod of Mattenheimer and deBruin for the assay of carbonic anhydrase activity, a considerable loss of substrate (CO2 gas) from the incubation medium was measured during spontaneous hydration. Adding gaseous CO2 and coating the surface of the incubation medium with oil were employed in order to prevent this loss, which otherwise led to serious errors in the measurement of the enzyme activity. A mixture of 80% paraffin and 20% hexadecane as surface coating was satisfactory for reducing the CO2 exchange to negligible proportions. With this modified method, the carbonic anhydrase activity of frozen-dried samples of rat neurohypophysis was studied as a possible "marker" for pituicyte activity following osmotic stress. The enzyme activity increased about 130% after 6 days of water deprivation. Experiments with perfused tissue, however, indicated that this increase in enzyme activity was caused by an increase in the blood content of the neural lobe.

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Pulmonary carbonic anhydrase in the human, monkey, and rat

Journal of Applied Physiology ◽

10.1152/jappl.1982.52.2.352 ◽

1982 ◽

Vol 52 (2) ◽

pp. 352-356 ◽

Cited By ~ 21

Author(s):

G. Lonnerholm

Keyword(s):

Enzyme Activity ◽

Carbonic Anhydrase ◽

High Activity ◽

Lung Tissue ◽

Close Contact ◽

Alveolar Epithelium ◽

Rat Lung ◽

Alveolar Space ◽

Co2 Transport ◽

Pulmonary Capillaries

The distribution of carbonic anhydrase in the human, monkey, and rat lung was studied by the histochemical method of Hansson. High activity of this enzyme was demonstrated in the endothelium of pulmonary capillaries. In the human and the monkey lung enzyme activity was exhibited in the whole circumference of the capillaries, but in the rat enzyme activity is confined to capillary segments having close contact with alveolar epithelium forming the blood-air barrier. Staining was inhibited by 10 microM acetazolamide, but was not affected by 10 microM Cl 13,850, an inactive acetazolamide analogue. The location of carbonic anhydrase in the lung supports the idea that pulmonary carbonic anhydrase promotes CO2 elimination from the blood into the alveolar space. Its possible functions may be to act upon plasma to accelerate the conversion of HCO-3 to CO2 and to facilitate CO2 transport through the lung tissue.

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Carbonic anhydrase in mouse salivary glands and saliva: a histochemical, immunohistochemical, and enzyme activity study.

Journal of Histochemistry & Cytochemistry ◽

10.1177/32.6.6427328 ◽

1984 ◽

Vol 32 (6) ◽

pp. 625-635 ◽

Cited By ~ 21

Author(s):

T Ikejima ◽

S Ito

Keyword(s):

Enzyme Activity ◽

Carbonic Anhydrase ◽

Salivary Glands ◽

Acinar Cell ◽

Secretory Granules ◽

Salivary Secretion ◽

Carbonic Anhydrase Activity ◽

Duct Systems ◽

Activity Measurements ◽

Granule Membrane

Mouse parotid gland and saliva were studied by histochemical, immunohistochemical, and activity measurements for carbonic anhydrase. Hansson 's histochemical reaction for carbonic anhydrase revealed positive enzyme activity in the parotid acinar cell cytoplasm and little or no reaction in the secretory granules. The luminal contents in all of the glandular duct systems also reacted positively, but the duct cells themselves were only weakly positive. Ultrastructural observations confirmed the light microscope histochemical localization and, in addition, revealed luminal content activity in intercellular ducts. Purified carbonic anhydrase isolated from mouse salivary glands was used to raise antibodies in rabbits. Localization of carbonic anhydrase by direct immunolabeling with fluorescein-coupled antibody and indirect immunoperoxidase labeling revealed enzyme localization on or in the acinar cell secretory granule membrane and not in the surrounding cytoplasm. The luminal contents of the intercalated and striated ducts were also strongly positive. Stimulation of salivary secretion with phenylephrine or pilocarpine increased the amount of carbonic anhydrase in saliva. Acetatazolamide and potassium cyanate inhibited carbonic anhydrase activity. Reasons underlying the discrepancy between the histochemical and immunolabeling localization of carbonic anhydrase are discussed. It is concluded that the parotid acinar cells of mice appear to be a significant source of carbonic anhydrase in saliva but its role is enigmatic.

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Carbonic anhydrase levels in blue-green algae

Canadian Journal of Botany ◽

10.1139/b75-263 ◽

1975 ◽

Vol 53 (20) ◽

pp. 2385-2387 ◽

Cited By ~ 31

Author(s):

R. K. Ingle ◽

Brian Colman

Keyword(s):

Enzyme Activity ◽

Carbonic Anhydrase ◽

Green Algae ◽

Carbonic Anhydrase Activity ◽

Anacystis Nidulans ◽

Blue Green Algae ◽

Membrane Bound

Carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1) activity was detected in four species of blue-green algae, Anabaena flos-aquae, Anacystis nidulans, Coccochloris peniocystis, and Oscillatoria sp., grown with air, but no significant enzyme activity was detected in these algae when grown with 5% CO2 in air. The enzyme was found not to be membrane-bound, was inhibited by 1 mM Diamox, and had an apparent Km of 15.6 mM. Carbonic anhydrase activity in all four blue-green algae was significantly lower than that reported for unicellular green algae.

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In vivo changes in carbonic anhydrase activity and histopathology of gill and liver tissues after acute exposure to chlorpyrifos in rainbow trout

Archives of Industrial Hygiene and Toxicology ◽

10.2478/10004-1254-65-2014-2547 ◽

2014 ◽

Vol 65 (4) ◽

pp. 377-385 ◽

Cited By ~ 22

Author(s):

Ahmet Topal ◽

Muhammed Atamanalp ◽

Ertan Oruç ◽

Yeliz Demir ◽

Şükrü Beydemir ◽

...

Keyword(s):

Enzyme Activity ◽

Rainbow Trout ◽

Carbonic Anhydrase ◽

Carbonic Anhydrase Activity ◽

Organophosphate Pesticide ◽

Histopathological Changes ◽

Liver Changes ◽

Trout Gill ◽

Liver Tissues

Abstract Chlorpyrifos is an organophosphate pesticide widely used in agriculture and aquaculture. This study investigated its effects on carbonic anhydrase (CA) enzyme activity and histopathology of rainbow trout gill and liver. The fish were exposed to 2.25 (25 % of 96 h LC50), 4.5 (50 % of 96 h LC50), and 6.75 μg L-1 (75 % of 96 h LC50) of chlorpyrifos for 24, 48, 72, and 96 h. CA activity was measured in liver and gills and histopathological changes were examined by light microscopy. The most common liver changes at most of the chlorpyrifos concentrations were hyperaemia and degenerative changes. Gill tissues were characterised by lamellar hyperaemia, lamellar oedemas, clumping, cellular degeneration, hyperplasia, and lamellar atrophy. CA enzyme activity in the gills decreased at all concentrations at 48, 72, and 96 h after exposure to chlorpyrifos (p<0.05). Similarly, there was a time-dependent decrease in CA activity at all of the concentrations in liver tissues (p<0.05). The present study indicated that chlorpyrifos inhibits CA enzyme activity and causes histopathological damage in gill and liver tissues

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Carbonic anhydrase activity of intact erythrocytes from seven mammals

Journal of Applied Physiology ◽

10.1152/jappl.1983.55.4.1292 ◽

1983 ◽

Vol 55 (4) ◽

pp. 1292-1298 ◽

Cited By ~ 19

Author(s):

S. J. Dodgson ◽

R. E. Forster

Keyword(s):

Enzyme Activity ◽

Carbonic Anhydrase ◽

Bos Taurus ◽

Specific Activity ◽

Homo Sapiens ◽

Mammalian Species ◽

Hemoglobin Concentration ◽

Carbonic Anhydrase Activity ◽

Mean Cell Volume ◽

Intracellular Enzyme

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.

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