Biochemical and Population Genetics of the Rabbit, Oryctolagus cuniculus, Carbonic Anhydrases I and II, from the Iberian Peninsula and France

Available studies on the biochemical and electrophoretic characterization of European rabbit (Oryctolagus cuniculus) carbonic anhydrases I and II (CAI, CAII) show contradictory results about their relative electrophoretic mobility and substrate specificity. After positive identification of carbonic anhydrase activity by CO2 hydration, the differential esterase activity of CAI and CAII toward beta-napththyl acetate and flourescein diacetate,respectively, were used to identify the banding patterns corresponding to each locus. Electrophoretic and hybrid isoelectric focusing analyses of the CAI and CAII loci in 1 domestic and 19 wild rabbit populations led to the recognition of genetic polymorphism at the CAI locus and of extensive variability at the CAII locus. Four and nine alleles at the CAI and CAII loci, respectively, are described. The geographic distribution of genetic variability is consistent with the existence of two evolutionary groups within O. cuniculus.     

Quantitative structure-activity relationship study on sulfanilamide schiff's bases: carbonic anhydrase (CA) inhibitors

The paper deals with quantitative structure–activity studies on a group of sulfanilamide Schiff's base inhibitors of carbonic anhydrase (CA) using distance-based topological indices. The regression analysis of the data has shown that the activities of the compounds used in inhibiting Carbonic AnhydraseII (CAII) activity can be modeled excellently in multi-parametric model in that some indicator parameters are also involved. The results are discussed critically.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

Carbonic anhydrase II binds to and enhances activity of the Na+/H+ exchanger

We examined the ability of carbonic anhydrase II to bind to and affect the transport efficiency of the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. The C-terminal region of NHE1 was expressed in Escherichia coli fused with an N-terminal glutathionine S-transferase or with a C-terminal polyhistidine tag. Using a microtiter plate binding assay we showed that the C-terminal region of NHE1 binds carbonic anhydrase II (CAII) and binding was stimulated by low pH and blocked by antibodies against the C-terminal of NHE1. The binding to NHE1 was confirmed by demonstrating protein-protein interaction using affinity blotting with CAII and immobilized NHE1 fusion proteins. CAII co-immunoprecipitated with NHE1 from CHO cells suggesting the proteins form a complex in vivo. In cells expressing CAII and NHE1, the H(+) transport rate was almost 2-fold greater than in cells expressing NHE1 alone. The CAII inhibitor acetazolamide significantly decreased the H(+) transport rate of NHE1 and transfection with a dominant negative CAII inhibited NHE1 activity. Phosphorylation of the C-terminal of NHE1 greatly increased the binding of CAII. Our study suggests that NHE1 transport efficiency is influenced by CAII, likely through a direct interaction at the C-terminal region. Regulation of NHE1 activity by phosphorylation could involve modulation of CAII binding.     

Impact of Zn,Cu, and Fe on the Activity of Carbonic Anhydrase of Erythrocytes in Ducks

The impact of zinc, copper, and iron on the duck erythrocyte carbonic anhydrase (CA) activity and the hemoglobin content in vitro culture were studied. The increase of zinc or iron addition at a low level induced the rise of CA activity, and the CA activity was inhibited by zinc or iron at a high addition level. The duck erythrocyte CA was strongly inhibited by cupric ion. The inhibition constant of duck erythrocyte CA to cupric ion is about 3.5 μM. Carbonic anhydrase compared to hemoglobin is more sensitive to zinc and copper in the environment. These findings suggest that some characteristics of duck erythrocyte CA are different from both CAI and CAII of mammals. The increase of Fe addition below 8 μM in the minimal essential medium brought about the rise of CA activity and resulted in the maximum of CA activity exceeding that induced by Zn. It provided a new evidence for the role of ferrous ion in CA.     

Carbonic anhydrase II-based metal ion sensing: Advances and new perspectives

Carbonic anhydrases are archetypical zinc metalloenzymes and as such, they have been developed as the recognition element of a family of fluorescent indicators (sensors) to detect metal ions, particularly Zn²⁺ and Cu²⁺. Subtle modification of the structure of human carbonic anhydrase II isozyme (CAII) alters the selectivity, sensitivity, and response time for these sensors. Sensors using CAII variants coupled with zinc-dependent fluorescent ligands demonstrate picomolar sensitivity, unmatched selectivity, ratiometric fluorescence signal, and near diffusion-controlled response times. Recently, these sensors have been applied to measuring the readily exchangeable concentrations of zinc in the cytosol and nucleus of mammalian tissue culture cells and concentrations of free Cu²⁺ in seawater.     

不同植物的碳酸酐酶活力差异研究

碳酸酐酶是催化二氧化碳的可逆水合反应的一种含锌金属酶。测定不同植物、同一植物不同部位、同一植物同一部位不同时间的碳酸酐酶的活力,研究诸葛菜和油菜碳酸酐酶及其胞外酶活力的差异,初步探讨碳酸酐酶活力与植物抗干旱能力之间的关系。研究结果为诸葛菜的喀斯特适生性的研究提供依据。     

Carbonic anhydrase II increases the activity of the human electrogenic Na+/HCO3- cotransporter

Several acid/base-coupled membrane transporters, such as the electrogenic sodium-bicarbonate cotransporter (NBCe1), have been shown to bind to different carbonic anhydrase isoforms to create a "transport metabolon." We have expressed NBCe1 derived from human kidney in oocytes of Xenopus leavis and determined its transport activity by recording the membrane current in voltage clamp, and the cytosolic H(+) and Na(+) concentrations using ion-selective microelectrodes. When carbonic anhydrase isoform II (CAII) had been injected into oocytes, the membrane current and the rate of cytosolic Na(+) rise, indicative for NBCe1 activity, increased significantly with the amount of injected CAII (2-200 ng). The CAII inhibitor ethoxyzolamide reversed the effects of CAII on the NBCe1 activity. Co-expressing wild-type CAII or NH(2)-terminal mutant CAII together with NBCe1 provided similar results, whereas co-expressing the catalytically inactive CAII mutant V143Y had no effect on NBCe1 activity. Mass spectrometric analysis and the rate of cytosolic H(+) change following addition of CO(2)/HCO(3)(-) confirmed the catalytic activity of injected and expressed CAII in oocytes. Our results show that the transport capacity of NBCe1 is enhanced by the catalytic activity of CAII, in line with the notion that CAII forms a transport metabolon with NBCe1.     

Cytosolic carbonic anhydrase activity in chronic myeloid disorders with different clinical phenotype

Carbonic anhydrase family (CAs) plays an important role in the extracellular acidification and several studies suggest a possible involvement of such enzymes in the increased tumor progression due to the acidic extracellular pH. We measured the activities of carbonic anhydrase I and II isoforms in a group of patients affected by four specific chronic haematological diseases, sharing a common origin but characterized by a different neoplastic evolution: agnogenic myeloid metaplasia (AMM), essential thrombocythemia (ET), chronic myeloid leukemia (CML) and polycythemia vera (PV) in order to understand the correlation between CAs activities and neoplastic outcome. In comparison to controls, our data demonstrate an increase of CAI and CAII activities in all our patients with a specific increase of the CAI activity in the group of the diseases with major malignancy (CML and AMM). These results suggest a possible role of such isozymes in the progression of the myeloid disorders and CAs specific inhibitors should be useful in slowing the progression of the disease.     

Selective tissue distribution of carbonic anhydrase isozymes in the ox.

By affinity chromatography the isozymic distribution of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been studied in extract from various bovine tissues. Carbonic anhydrase II forms isolated from erythrocyte, kidney and brain are indistinguishable by specific activity, amino acid composition, fingerprint, electrophoretic and immunological behaviour. By these criteria they differ from carbonic anhydrase I isolated from rumen epithelium.     

Carbonic anhydrase II isoenzyme in rat liver is under hormonal control.

A specific and sensitive radioimmunoassay for rat carbonic anhydrase II (CAII) was developed. Rat livers were perfused with Ringer's solution and the hepatic CAII was measured. Concentrations of CAII in female liver were significantly higher than those in male liver. Castration increased the concentration in male liver, though not to that in females, and diethylstilboestrol treatment of castrated males gave values higher than those in females.     

Radioimmunoassay of carbonic anhydrase I and II in rats. Application to the central nervous system during ontogeny]

A specific radioimmunoassay method for rat erythrocyte carbonic anhydrases I and II was developed using a double antibody system. Its sensitivity was in the nanogram range for each of the two isozymes. The method has been applied to the assay of cerebral carbonic anhydrase. Only CAII has been found in brain extracts of perfused rats. Accordingly, the assay of CAI in cerebral tissue can be used to quantify erythrocyte contamination on condition that the ratio CAII/CAI in blood had been worked out. The developmental change in the soluble and the Triton X-100 solubilized brain CAII from birth to adult is reported.     

Comparative properties of mammalian and insect carbonic anhydrases: effects of potassium and chloride on the rate of carbon dioxide hydration.

1. Carbonic anhydrase (E.C.4.2.1.1) catalysed CO2 hydration was studied with enzymes from mammalian and insect sources at CO2 concentrations of 7.6-30.8 mM. 2. At 0.01-0.15 M, potassium chloride (KCl) or choline chloride (ChCl) markedly inhibited all 8 mammalian enzymes studied. 3. Inhibition by KCl is always greater than that associated with ChCl. 4. KCl non-competitively inhibits and choline chloride competitively inhibits bovine carbonic anhydrase. 5. Carbonic anhydrase obtained from fat body, integumentary epithelium and midgut tissues of larval tobacco hornworms, Manduca sexta, is greatly stimulated by KCl and slightly inhibited by ChCl. 6. We propose that the effect of K+ on mammalian and insect carbonic anhydrases if fundamentally different.     

Affinity chromatography of carbonic anhydrase

An insoluble support for affinity chromatography of carbonic anhydrase has been prepared by coupling Sulfamylon (p-aminomethylbenzene sulfonamide) to Sepharose 4B. Carbonic anhydrase binds to Sulfamylon-Sepharose very strongly and can be eluted under mild conditions by the addition of enzyme inhibitors. The gel was used to purify carbonic anhydrase from human erythrocytes and to separate isozymes B and C. It was also employed to separate native enzyme from modified carbonic anhydrases. The apoenzyme and the carboxymethyl enzyme of human carbonic anhydrase B were both isolated by this method.     

Differential expression of the L- and S-isoforms of myelin associated glycoprotein (MAG) in oligodendrocyte unit phenotypes in the adult rat anterior medullary velum

Brain Cell Biology - We have previously demonstrated differences in the expression of carbonic anhydrase II (CAII) in oligodendrocyte units myelinating small and large diameter fibres in the...     

Increased carbonic anhydrase activity in Friend erythroleukemia cells during DMSO-stimulated erythroid differentiation and its inhibition by BrdU.

Carbonic anhydrase activity is increased in Friend erythroleukemia (FL) cells during the enhancement of erythroid differentiation in the presence of dimethylsulfoxide (DMSO) or butyric acid. Untreated FL cells show an increase in enzyme activity associated with logarithmic growth. The increase in the specific activity of carbonic anhydrase in the differentiating treated cells, however, appears to be due to at least two additional general mechanisms: (1) an induction of carbonic anhydrase paralleling the stimulation of hemoglobin synthesis and (2) the stability and/or retention of active carbonic anhydrase as compared to most of the other cell proteins. The stimulation of carbonic anhydrase activity in the treated cells is inhibited by 5-bromo-2'-deoxyuridine (BrdU). This is the first demonstration of BrdU inhibition of a DMSO induced product not directly related to hemoglobin.     

Biochemical and Immunocytochemical Evidence for a Deficiency of Normal Interfascicular Oligodendroglia in the CNS of the Dysmyelinating Mutant (md) Rat

Carbonic anhydrase was assayed and carbonic anhydrase and 5'-nucleotidase were localized in the CNS of myelin-deficient mutant rats and normal littermates. The carbonic anhydrase specific activities were reduced by 61% and 29% in the mutants' forebrains and cerebella, respectively, and the total carbonic anhydrase activity in the spinal cords was reduced by 35%. Immunostained cells were found in gray matter from both normal and mutant rats, but, in the mutants, there was a marked deficiency of interfascicular oligodendrocytes in the regions that are normally occupied by white matter. It is suggested that a developmental study could indicate the step(s) at which normal differentiation of interfascicular oligodendroglia is blocked in this mutant.     

A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers

The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50-60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 +/- 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.     

Carbonic anhydrase III is not required in the mouse for normal growth, development, and life span

Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.     

Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone,cartilage and giant cell tumor

Summary Carbonic anhydrase isoenzymes I and II have been localized in human bone and cartilage. Osteoclasts are strongly positive for carbonic anhydrase II but very little if any reaction is observed for carbonic anhydrase I. In tendon giant cell tumor osteoclastlike-giant cells contained high amounts of carbonic anhydrase II suggesting the close relation of these cells to normal osteoclasts. In growth plate cartilage strong staining was obtained in late proliferative and hypertrophic chondroxytes as well as in extracellular matrix of hypertrophic zone also only with anti-human carbonic anhydrase II.