Carbonic anhydrase activity of intact erythrocytes from seven mammals

1983 ◽  
Vol 55 (4) ◽  
pp. 1292-1298 ◽  
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.

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

1981 ◽  
Vol 29 (10) ◽  
pp. 1213-1218 ◽  
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.

Localization of carbonic anhydrase in the cytoplasmic membrane of Neisseria sicca (strain 19)

1981 ◽  
Vol 27 (1) ◽  
pp. 87-92 ◽  
Author(s):  
M. N. MacLeod ◽  
I. W. DeVoe
Keyword(s):  
Carbonic Anhydrase ◽  
Density Gradient ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Carbonic Anhydrase Activity ◽  
Protein Patterns ◽  
Inner Membrane ◽  
Outer Membranes ◽  
Neisseria Sicca

The carbonic anhydrase activity and the growth of Neisseria sicca 19 were inhibited by the sulfonamide acetazolamide (10−5 M). Such inhibition was completely overcome by the addition of exogenous bicarbonate. Some carbonic anhydrase activity associated with the membranous envelope fraction of the cell was released when cells were broken by sonic treatment but not during cell breakage by high-pressure extrusion. After the selective solubilization (4 °C) of the inner membrane of envelopes by treatment with 1% sodium lauroyl sarcosinate, all detectable carbonic anhydrase activity was found in the soluble (inner membrane) fraction. After fractionation of the cell envelope into inner and outer membranes by treatment with ethylenediaminetetraacetate (EDTA) followed by sucrose density gradient centrifugation, the total and specific activity of carbonic anhydrase paralleled that of succinate dehydrogenase, an inner membrane enzyme marker. The Coomassie blue stained protein patterns after polyacrylamide gel electrophoresis of the bands from the sucrose density gradient provided confirmation that the inner and outer membranes had indeed been separated.

Rat lung carbonic anhydrase: activity, localization, and isozymes

1986 ◽  
Vol 60 (2) ◽  
pp. 638-645 ◽  
Author(s):  
R. P. Henry ◽  
S. J. Dodgson ◽  
R. E. Forster ◽  
B. T. Storey
Keyword(s):  
Enzyme Activity ◽  
Carbonic Anhydrase ◽  
Carbonic Anhydrase Activity ◽  
Hydration Reaction ◽  
Rat Lung ◽  
Cell Debris ◽  
Blood Contamination ◽  
Total Enzyme Activity ◽  
Erythrocyte Lysis ◽  
A Cell

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.

Why do hagfish have gill "chloride cells" when they need not regulate plasma NaCl concentration?

1987 ◽  
Vol 65 (8) ◽  
pp. 1956-1965 ◽  
Author(s):  
Jon Mallatt ◽  
David M. Conley ◽  
Richard L. Ridgway
Keyword(s):  
Carbonic Anhydrase ◽  
Ion Transport ◽  
Specific Activity ◽  
Carbonic Anhydrase Activity ◽  
Chloride Cells ◽  
Gillichthys Mirabilis ◽  
The Pacific ◽  
Nacl Concentration ◽  
Tubular System ◽  
Freshwater Teleosts

Two enzymes implicated in branchial ion transport, Na+-K+-ATPase and carbonic anhydrase, were localized in gill ionocytes ("chloride cells") of the Pacific hagfish, Eptatretus stouti, by light microscopic histochemical techniques. In hagfish, ouabain-sensitive Na+-K+-ATPase activity was confined to apical halves of ionocytes, where most of the cytoplasmic tubular system is located. In marine teleosts, Na+-K+-ATPase was noted in chloride cells and erythrocytes. Acetazolamide and potassium cyanate sensitive carbonic anhydrase activity occurred throughout the cytoplasm and nucleus of hagfish ionocytes. Biochemical assay of hagfish gill homogenates for Na+-K+-ATPase yielded a specific activity of 3.1 μmol Pi∙mg protein−1∙h−1 at 37 °C. This resembles values we obtained for freshwater fish (Carassius auratus: 3.3 μmol Pi∙mg protein−1∙h−1; Tilapia shirana: 3.7 μmol Pi∙mg protein−1∙h−1), and is less than values we obtained for marine teleosts (Pomacentrus spp.: 13 μmol Pi∙mg protein−1∙h−1; Gillichthys mirabilis: 6.7 μmol Pi∙mg protein−1∙h−1). Hagfish resemble freshwater teleosts in many other gill features related to ion transport. The presence of carbonic anhydrase in gill ionocytes of hagfish supports the proposal that these cells function in acid–base regulation, i.e., that they exchange H+ for Na+ and [Formula: see text] for Cl−.

Renal carbonic anhydrase

1982 ◽  
Vol 243 (4) ◽  
pp. F311-F324 ◽  
Author(s):  
D. C. Dobyan ◽  
R. E. Bulger
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Mammalian Species ◽  
Carbonic Anhydrase Activity ◽  
The Body ◽  
Carbonic Anhydrases ◽  
Duct System ◽  
Renal Carbonic Anhydrase ◽  
Membrane Bound ◽  
Editorial Review

Carbonic anhydrase is a zinc metalloenzyme widely distributed throughout the tissues of the body. This enzyme exists in a number of isozymic forms in most mammalian species. Significant advances over the past decade have been made in characterizing the nature of renal carbonic anhydrase. In the kidney, this enzyme is thought to play a pivotal role in urinary acidification and bicarbonate reabsorption. Two distinct isozymes of carbonic anhydrase have now been identified in the mammalian kidney. A soluble cytoplasmic form, similar if not identical to human erythrocyte carbonic anhydrase C, accounts for the bulk of the renal carbonic anhydrase activity. In addition, a membrane-bound form constituting only about 2--5% of the renal activity has been found in the brush border and basolateral fractions of kidney homogenates. The histochemical and immunocytochemical localization of these isozymes along the nephron and collecting duct system of various mammalian species suggests that marked heterogeneity exists. The Editorial Review examines the biochemical and morphological approaches that have been used to elucidate the nature of renal carbonic anhydrase and to assess its distribution along the urinary tubule. Possible physiological roles for the renal carbonic anhydrases are considered for the different segments of the nephron and collecting duct system.

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

1988 ◽  
Vol 65 (4) ◽  
pp. 1902-1906 ◽  
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.

Temperature-Related Changes in the Erythrocytic Carbonic Anhydrase (Acetazolamide-Sensitive Esterase) Activity of Goldfish, Carassius Auratus

1979 ◽  
Vol 78 (1) ◽  
pp. 255-264
Author(s):  
ARTHUR H. HOUSTON ◽  
KAREN M. MEAROW
Keyword(s):  
Carbonic Anhydrase ◽  
Carassius Auratus ◽  
Specific Activity ◽  
Thermal Sensitivity ◽  
Carbonic Anhydrase Activity ◽  
Red Cell ◽  
Goldfish Carassius Auratus ◽  
Species Activity ◽  
Plasma Chloride ◽  
Stable Plasma

1. Carbonic anhydrase activity in ‘membrane’ and ‘cytosol’ fractions of goldfish erythrocytes was assayed by the p-nitrophenyl acetate procedure following thermal acclimation. 2. The thermal sensitivity of ‘membrane’-associated activity was apparently unaltered by acclimation. ‘Cytosol’ activity in warm-acclimated specimens was somewhat more thermosensitive than that of animals maintained at low temperature. 3. Significant increases in specific activity, and activity per unit volume of packed cells and blood were observed at higher temperatures when assays were conducted at the temperatures at which the system actually functions in the fish. By contrast, when determinations were carried out at a standard temperature (41 °C) corresponding to the upper incipient lethal for this species, activity was either unaffected, or declined as acclimation temperatures increased. 4. Changes in carbonic anhydrase activity following acclimation are consistent with the hypothesis that this system is implicated in the maintenance of stable plasma chloride levels, and the suggestion that alterations in red cell chloride levels with temperature are, in part at least, attributable to concomitant variations in enzyme activity.

scholarly journals CARBONIC ANHYDRASE ACTIVITY IN RAT NEUROHYPOPHYSIS FOLLOWING OSMOTIC STRESS

1972 ◽  
Vol 20 (8) ◽  
pp. 621-626 ◽  
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.

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

1984 ◽  
Vol 32 (6) ◽  
pp. 625-635 ◽  
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.

scholarly journals Why is there no carbonic anhydrase activity available to fish plasma?

1995 ◽  
Vol 198 (1) ◽  
pp. 31-38 ◽  
Author(s):  
J Lessard ◽  
A Val ◽  
S Aota ◽  
D Randall
Keyword(s):  
Carbonic Anhydrase ◽  
Blood Cell ◽  
Cell Volume ◽  
Red Blood Cell ◽  
Short Circuit ◽  
Hemoglobin Concentration ◽  
Carbonic Anhydrase Activity ◽  
Control Group ◽  
Acid Loading

Carbonic anhydrase (CA) is absent from the plasma of vertebrates. In vitro, CA in fish plasma will short-circuit the effect of catecholamines, which is to increase red blood cell (RBC) pH and volume, both of which enhance the affinity of hemoglobin for O2. CA was infused into trout for a period of 6 h and injected after 48 h, during which the animal was submitted to deep hypoxia (PO2=30­35 mmHg; 4.0­4.7 kPa). O2 content, lactate content, catecholamine levels, hematocrit, hemoglobin concentration and pHi were similar to those in the saline-infused control group. In contrast, cell volume was significantly higher and pHe, total CO2 content and organic phosphate levels were significantly lower than in the control group. The concentration of CA was not high enough completely to short-circuit the increase in pHi and red blood cell volume caused by catecholamines. The lower pHe in the CA-infused animals could enhance the activity of the Na+/H+ pump, which would keep the nucleotide triphosphate levels low. pH is a balance between acid loading at the muscle and acid excretion at the gills or the kidneys; we cannot distinguish between which of these resulted in a decrease of plasma pH. In conclusion, CA in plasma did not cause the expected reduction in blood oxygen content but did have a marked effect on plasma total CO2 content.

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