Effects of various ion transport inhibitors on the water response in the superior laryngeal nerve in rats

The effects of inhibitors [acetazolamide, an inhibitor of carbonic anhydrase; amiloride, an inhibitor of the Na channel; furosemide, an inhibitor of the Na/K/2Cl transporter; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of the Cl channel] on the water response in the superior laryngeal nerve (SLN) were investigated using whole nerve recordings from the SLN of anesthetized and paralyzed rats. Changes in spontaneous activity in the SLN after i.v. injection of a hypo- or hypertonic solution were also investigated. The water response to higher concentration amiloride solutions (0.1, 1, 5 and 10 mM) were significantly smaller in comparison with the control, i.e. the water response to deionized water (88-59% of the control, Fisher's PLSD, P < 0.05). DIDS suppressed the water response significantly at concentrations of 0.5 and 2 mM by 18 and 33%, respectively (P < 0.05). Likewise, acetazolamide (2 mM) and furosemide (5 mM) significantly suppressed the water response by 9 and 40%, respectively (P < 0.05). An i.v. bolus injection of a hypertonic solution (1 ml of 1.5 M NaCl or 1.0 M mannitol) depressed spontaneous activity of the SLN. In contrast, an i.v. injection of a hypotonic solution (0.015 M NaCl) increased spontaneous activity. These results suggest that several ion transporters and ion channels, as well as carbonic anhydrase, that may exist in the dorsal surface in the epiglottis may regulate the water response in the SLN and that osmotic changes in the dorsal surface of the epiglottis and in the interstitial space can affect nerve activity in the SLN.     

Entry and exit pathways of CO2 in rat liver mitochondria respiring in a bicarbonate buffer system

The dynamics and pathways of CO2 movements across the membranes of mitochondria respiring in vitro in a CO2/HCO-3 buffer at concentrations close to that in intact rat tissues were continuously monitored with a gas-permeable CO2-sensitive electrode. O2 uptake and pH changes were monitored simultaneously. Factors affecting CO2 entry were examined under conditions in which CO2 uptake was coupled to electrophoretic influx of K+ (in the presence of valinomycin) or Ca2+. The role of mitochondrial carbonic anhydrase (EC 4.2.1.1) in CO2 entry was evaluated by comparison of CO2 uptake by rat liver mitochondria, which possess carbonic anhydrase, versus rat heart mitochondria, which lack carbonic anhydrase. Such studies showed that matrix carbonic anhydrase activity is essential for rapid net uptake of CO2 with K+ or Ca2+. Studies with acetazolamide (Diamox), a potent inhibitor of carbonic anhydrase, confirmed the requirement of matrix carbonic anhydrase for net CO2 uptake. It was shown that at pH 7.2 the major species leaving respiring mitochondria is dissolved CO2, rather than HCO-3 or H2CO3 suggested by earlier reports. Efflux of endogenous CO2/HCO-3 is significantly inhibited by inhibitors of the dicarboxylate and tricarboxylate transport systems of the rat liver inner membrane. The possibility that these anion carriers mediate outward transport of HCO-3 is discussed.     

Effects of 4-acetamido-4'-isothiocyano-2,2-disulfonic stilbene on ion transport in turtle bladders.

The disulfonic stilbene (4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO-3,Cl-) moiety of the short-circuiting current is eliminated by 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene, but only after its addition to the serosal bathing fluid. Whereas 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene has no effect on Na+ transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na+ + K+)-ATPase of both turtle bladder and eel electric organ.     

Bone-targeted carbonic anhydrase inhibitors: effect of a proinhibitor on bone resorption in vitro

Many investigations have indicated a functional role for carbonic anhydrase in the mediation of hormone-stimulated bone resorption. These studies depend heavily on the use of heterocyclic sulfonamide inhibitors of carbonic anhydrase. These drugs have effects on many tissues other than bone, and some of these effects confound the interpretation of studies of the role of carbonic acid in bone metabolism. A novel, "bone-targeted" sulfonamide has been produced to obviate these extraosseous effects. This compound (designated WP-1) is the combination of tetracycline and acetazolamide, such that the acetazolamide is not an active inhibitor. Hydrolysis of WP-1 yields an active carbonic anhydrase inhibitor. WP-1 has a marked affinity for bone mineral, allowing deposition of the drug in bone. At a concentration of 10(-5) M, WP-1 attenuates parathyroid hormone stimulated net release of calcium from neonatal rat calvaria in culture. WP-1 is the first member of a class of drugs which may prove useful as pharmacological probes in the study of bone metabolism.     

Gastric acid secretion in the lizard. Ionic requirements and effects of inhibitors.

1. The effects of ion substitution and various inhibitors on the transmucosal potential, short circuit current, mucosal resistance and acid secretion of the lizard gastric mucosa, incubated in an Ussing chamber, have been determined. 2. Ion substitution experiments indicate that the serosal potential step consists of a combined C1- and K+ diffusion potential, and that the mucosal potential step is Na+ dependent and behaves primarily as a Na+ diffusion potential. 3. Experiments with ouabain indicate that the major (Na+, K+)-ATPase activity responsible for maintenance of cation gradients is located on the serosal side of the mucosal cells, and that this pump activity is non-electrogenic. 4. Experiments with amiloride indicate that a passive sodium influx on the mucosal side is essential for the maintenance of the transmucosal potential and short circuit current. 5. Acid secretion requires the presence of sodium and chloride on the serosal side and the maintenance of a high intracellular potassium level through the (Na+, K+)-ATPase system. 6. The effects of acetazolamide and thiocyanate are compatible with an involvement of carbonic anhydrase and anion-dependent ATPase in acid secretion. 7. Upon initiation of acid secretion the serosal membrane permeability for chloride increases and that for potassium decreases.     

Carbonic anhydrase inhibitors. Inhibition studies with anions and sulfonamides of a new cytosolic enzyme from the scleractinian coral Stylophora pistillata

The catalytic activity and the inhibition of a new coral carbonic anhydrase (CA, EC 4.2.1.1), from the scleractinian coral Stylophora pistillata, STPCA-2, has been investigated. STPCA-2 has high catalytic activity for the physiological reaction being less sensitive to anion and sulfonamide inhibitors compared to STPCA, a coral enzyme previously described. The best STPCA-2 anion inhibitors were sulfamide, sulfamic acid, phenylboronic acid, and phenylarsonic acid (K_Is of 5.7-67.2 μM) whereas the best sulfonamide inhibitors were acetazolamide and dichlorophenamide (K_Is of 74-79 nM). Because this discriminatory effect between these two coral CAs, sulfonamides may be useful to better understand the physiological role of STPCA and STPCA-2 in corals and biomineralization processes.     

The effects of carbonic anhydrase inhibitors formate,bicarbonate, acetazolamide,and imidazole on photosystem II in maize chloroplasts

Inhibitors of carbonic anhydrase were tested for their effects on Photosystem II (PS II) activity in chloroplasts. We find that formate inhibition of PS II turnover rates increases as the pH of the reaction medium is lowered. Bicarbonate ions can inhibit PS II turnover rates. The relative potency of the anionic inhibitors N₃^?, I^?, OA_c^?, and Cl^? is the same for both carbonic anhydrase and PS II. The inhibitory effect of acetazolamide on PS II increases as light intensity decreases, indicating a lowering of quantum yields in the presence of the inhibitor. Imidazole inhibition of PS II increases with pH in a manner suggesting that the unprotonated form of the compound is inhibitory. Formate, bicarbonate, acetazolamide, and imidazole all inhibit DCMU-insensitive, silicomolybdate-supported oxygen evolution, indicating that the site(s) of action of the inhibitors is at, or before, the primary stable PS II electron acceptor Q. This inhibitory effect of low levels of HCO₃^? along with the known enhancement by HCO₃^? of quinone-mediated electron flow suggests an antagonistic control effect on PS II photochemistry. We conclude that the responses of PS II to anions (formate, bicarbonate), acetazolamide, and imidazole are analogous to the responses shown by carbonic anhydrase. These findings suggest that the enzyme carbonic anhydrase may provide a model system to gain insight into the “bicarbonate-effect” associated with PS II in chloroplasts.     

Characterization of proton-transporting membranes from resting pig gastric mucosa

Membrane vesicles were purified from resting corpus mucosa of pig stomachs by velocity-sedimentation on a sucrose-Ficoll step gradient. Two vesicular fractions containing the (H+ + K+)-ATPase were obtained. One fraction was tight towards KCl, the other was leaky. At 21 degrees C maximal (H+ + K+)-ATPase activities of 0.8 and 0.4 mumol X mg-1 X min-1, respectively, were observed in lyophilized vesicles. The vesicles contained a membrane-associated carbonic anhydrase, the activity of which was in 100-fold excess of the maximal ATPase activity. Both vesicular fractions were rich in phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol. The characteristics of ion permeability and transport in the tight vesicles were in agreement with corresponding data for vesicles of a tubulovesicular origin in the parietal cell. Measurement of the rate of K+ uptake into the vesicles was based on the ability of K+ to promote H+ transport. The uptake was slow and dependent on the type of anion present. The effectiveness in promoting uptake of K+ by anions was SCN- greater than NO3- greater than Cl- much greater than HCO3- greater than SO4(2-). Uptake of K+ was much more rapid at alkaline pH than at neutral or at acidic pH. Addition of CO2 at alkaline pH strongly stimulated the rate of H+ accumulation in the vesicles. The initial part of this stimulation was sensitive to acetazolamide, an inhibitor of carbonic anhydrase. A model how the (H+ + K+)-ATPase and the carbonic anhydrase may co-operate is presented. It is concluded that membrane vesicles of a tubulovesicular origin can produce acid.     

Pulmonary vasodilation by acetazolamide during hypoxia is unrelated to carbonic anhydrase inhibition

Acute hypoxic pulmonary vasoconstriction can be inhibited by high doses of the carbonic anhydrase inhibitor acetazolamide. This study aimed to determine whether acetazolamide is effective at dosing relevant to human use at high altitude and to investigate whether its efficacy against hypoxic pulmonary vasoconstriction is dependent on carbonic anhydrase inhibition by testing other potent heterocyclic sulfonamide carbonic anhydrase inhibitors. Six conscious dogs were studied in five protocols: 1) controls, 2) low-dose intravenous acetazolamide (2 mg.kg(-1).h(-1)), 3) oral acetazolamide (5 mg/kg), 4) benzolamide, a membrane-impermeant inhibitor, and 5) ethoxzolamide, a membrane-permeant inhibitor. In all protocols, unanesthetized dogs breathed spontaneously during the first hour (normoxia) and then breathed 9-10% O(2) for the next 2 h. Arterial oxygen tension ranged between 35 and 39 mmHg during hypoxia in all protocols. In controls, mean pulmonary artery pressure increased by 8 mmHg and pulmonary vascular resistance by 200 dyn.s.cm(-5) (P <0.05). With intravenous acetazolamide, mean pulmonary artery pressure and pulmonary vascular resistance remained unchanged during hypoxia. With oral acetazolamide, mean pulmonary artery pressure increased by 5 mmHg (P < 0.05), but pulmonary vascular resistance did not change during hypoxia. With benzolamide and ethoxzolamide, mean pulmonary artery pressure increased by 6-7 mmHg and pulmonary vascular resistance by 150-200 dyn.s.cm(-5) during hypoxia (P < 0.05). Low-dose acetazolamide is effective against acute hypoxic pulmonary vasoconstriction in vivo. The lack of effect with two other potent carbonic anhydrase inhibitors suggests that carbonic anhydrase is not involved in the mediation of hypoxic pulmonary vasoconstriction and that acetazolamide acts on a different receptor or channel.     

A nuclear-magnetic-resonance study of the binding of novel N-hydroxybenzenesulphonamide carbonic anhydrase inhibitors to native and cadmium-111-substituted carbonic anhydrase

Various ring- and nitrogen-substituted benzenesulphonamides have been prepared and tested as potential inhibitors of carbonic anhydrase. N-Methoxysulphonamides showed no inhibitory activity, as predicted by the classic work of Krebs on N-substituted inhibitors. By contrast, N-hydroxysulphonamides proved to be very effective inhibitors of carbonic anhydrase. Using 111Cd-NMR it has been possible to analyse the molecular interaction of 4-fluoro-N-hydroxybenzenesulphon[15N]amide, with 111Cd-substituted bovine carbonic anhydrase. A large cadmium-111:nitrogen-15 spin-coupling shows that this inhibitor is directly bound to the metal via its nitrogen rather than through an oxygen atom. The mode of this binding is similar to that for the unsubstituted sulphonamide inhibitor, 4-fluorobenzenesulphon[15N]amide. The 111Cd-chemical shift of the signal for the inhibited enzyme shows that the N-hydroxysulphonamide is bound as its anion. From the relative intensities of free and complexed enzyme signals it can be deduced that the cadmium enzyme complex with the N-hydroxysulphonamide has a longer life-time than that formed with the unsubstituted sulphonamide. By contrast, native zinc-containing bovine carbonic anhydrase shows similar I50 values with both of these sulphonamides. Attempts to monitor the binding using 15N-NMR were unsuccessful, possibly due to a very long relaxation time for the nitrogen nucleus in the N-hydroxysulphonamide when bound to the enzyme leading to loss of the 15N signal.     

Carbonic anhydrase supports electrolyte transport in Drosophila Malpighian tubules. Evidence by X-ray microanalysis of cryosections

Electron probe X-ray microanalytical studies on the role of carbonic anhydrase in electrolyte transport in the cells of Drosophila Malpighian tubules indicate that carbonic anhydrase delivers protons and bicarbonate ions to ion transport systems in the cell membrane. After injection and after feeding acetazolamide or hydrochlorothiazide, known inhibitors of carbonic anhydrase, the contents of potassium, magnesium and chloride in the apical cytoplasm and in the cytoplasm close to the basal plasma membrane decreased. We explain our measurements by the hypothesis of a basal Mg-H-antiport system in parallel with Cl-HCO(3)-antiport, inhibitable by DIDS. Zinc is supposed to enters cells and intracellular Zn storage vacuoles by a negatively charged Zn-anion-complex in exchange for HCO(3)(-) ions. This antiport is inhibitable by SITS. The content of the Zn storage vacuoles is acid, as shown by red fluorescence after incubation of Malpighian tubules with acridine orange. Red fluorescence is absent after preincubation in a medium containing an inhibitor of carbonic anhydrase. Carbonic anhydrase was demonstrated cytochemically in the Golgi-ER complex, Golgi vesicles and intercellular space. We suppose that carbonic anhydrase is synthesized and stored in the Golgi-ER-complex from where it is released into the tubule lumen.     

pH shifts evoked by neuronal stimulation in slices of rat hippocampus

Extracellular pH was measured with ion-selective microelectrodes in 500-microns thick slices of the CA1 region of the rat hippocampus. The center of the slice was 0.24 pH units more acidic than the surface, thus creating a decreasing pH gradient from the surface to the center, most likely owing to increased anaerobic metabolism. Stimulation at various frequencies created a transient alkaline shift of 0.03 pH unit, followed by a sustained acidification (0.05 pH unit above baseline). The same pattern was seen in both cell body and dendritic layers. The presence of the alkaline shift in slices in vitro is especially significant, implying that it is not due to alterations in blood flow. The HCO3- -Cl- transport inhibitor SITS and the carbonic anhydrase inhibitor acetazolamide increased the alkaline shift to 0.06 and 0.23, respectively. The acid shift was influenced by the Na+-H+ transport inhibitor amiloride, with a reduction of about 50%. Possible mechanisms for these stimulus-evoked changes as discussed. The most likely cause for the alkaline shift is bicarbonate accumulation in the extracellular space. Hydrogen ion and lactic acid release are seen as the major factors contributing to the sustained acid shift.     

Carbonic anhydrase inhibitors: the very weak inhibitors dithiothreitol, beta-mercaptoethanol, tris(carboxyethyl)phosphine and threitol interfere with the binding of sulfonamides to isozymes II and IX

The inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) with dithiothreitol, 2-mercaptoethanol, tris(carboxyethyl)phosphine (reducing agent frequently added to enzyme assay buffers) and threitol has been investigated. The agents were very weak inhibitors of isozymes CA II and CA IX, but unexpectedly, strongly influenced the binding of the low nanomolar sulfonamide inhibitor acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide). Acetazolamide affinity for all investigated CAs diminished orders of magnitude with increasing concentrations of these agents in the assay system. DTT and similar derivatives should not be added to the assay buffers used in monitoring CA activity/inhibition, as they lead to under-estimation of the binding constants, by a mechanism probably involving the formation of ternary complexes.     

Histomorphometric identification of carbonic anhydrase in fetal rat bone embedded in glycolmethacrylate

Carbonic anhydrase was identified in bone-resorbing cells present in sections of fetal rat femur embedded in glycolmethacrylate. Using a slight modification of the Hansson's histochemical method, we demonstrated that most chondroclasts (91.8-95.4%) and osteoclasts (95.1-96.3%) display a positive histochemical reaction for carbonic anhydrase. This staining was consistently inhibited in the presence of very low concentrations (10(-6), 10(-7) M) of the specific inhibitor acetazolamide. The number of chondroclasts reacting for carbonic anhydrase was identical to the number of acid phosphatase-stained chondroclasts determined on adjacent sections. A large majority of osteoclasts (96.3%) stained for carbonic anhydrase and for acid phosphatase (97.2%), with more osteoclasts reacting for the latter enzyme than the former (76.8 +/- 8.5 (SD) vs 85.3 +/- 9.2 cells/mm2 of endosteal bone; p less than 0.01). The observation that acetazolamide at a concentration as low as 10(-7) M inhibited Hansson's reaction, together with our histomorphometric results, validates the use of histochemical staining for carbonic anhydrase to evaluate activity of bone-resorbing cells identified in plastic-embedded fetal bone tissue.     

Prevention of non-steroidal anti-inflammatory agents induced acute gastric mucosal lesions by carbonic anhydrase inhibitors. An endoscopic study

The present paper studies the effect of acetazolamide, an inhibitor of carbonic anhydrase, on acute gastric mucosal damage induced by non-steroidal anti-inflammatory drugs. The study was performed on healthy male subjects. The drugs tested were aspirin (1.5 g/day), indomethacin (75 mg/day), phenylbutazone (600 mg/day) and ibuprofen (600 mg/day) given for 7 days in 3 divided doses. Each drug was given to 5 cases in two separate periods, during which they were given acetazolamide 20 mg/kg/day or placebo in random order. Dyspeptic symptoms were evaluated. Endoscopy was performed before, and 3 and 7 days after NOSAC administration. Gastric mucosal lesions were evaluated according to the scale proposed by Lanza (J. Clin. Pharmacol., 24: 1984, 89) and the severity of the lesions was calculated. All drugs tested produced dyspeptic symptoms and acute mucosal damage of the gastric mucosa. Inhibition of gastric mucosa carbonic anhydrase by acetazolamide cessated promptly dyspeptic symptoms and reduced significantly the number and severity of drug-associated mucosal lesions.     

Possible role of carbonic anhydrase in rat pancreatic islets: enzymatic, secretory, metabolic, ionic, and electrical aspects

The presence of carbonic anhydrase (type V) was recently documented in rat and mouse pancreatic islet beta-cells by immunostaining and Western blotting. In the present study, the activity of carbonic anhydrase was measured in rat islet homogenates and shown to be about four times lower than in rat parotid cells. The pattern for the inhibitory action of acetazolamide on carbonic anhydrase activity also differed in islet and parotid cell homogenates, suggesting the presence of different isoenzymes. NaN3 inhibited carbonic anhydrase activity in islet homogenates and both D-[U-14C]glucose oxidation and glucose-stimulated insulin secretion. Acetazolamide (0.3-10.0 mM) also decreased glucose-induced insulin output but failed to affect adversely D-[U-14C]glucose oxidation, although it inhibited the conversion of D-[5-3H]glucose to [3H]OH and that of D-[U-14C]glucose to acidic metabolites. Hydrochlorothiazide (3.0-10.0 mM), which also caused a concentration-related inhibition of the secretory response, like acetazolamide (5.0-10.0 mM), decreased H(14)CO3- production from D-[U-14C]glucose (16.7 mM). Acetazolamide (5.0 mM) did not affect the activity of volume-sensitive anion channels in beta-cells but lowered intracellular pH and adversely affected both the bioelectrical response to d-glucose and its effect on the cytosolic concentration of Ca2+ in these cells. The lowering of cellular pH by acetazolamide, which could well be due to inhibition of carbonic anhydrase, might in turn account for inhibition of glycolysis. The perturbation of stimulus-secretion coupling in the beta-cells exposed to acetazolamide may thus involve impaired circulation in the pyruvate-malate shuttle, altered mitochondrial Ca2+ accumulation, and perturbation of Cl- fluxes, resulting in both decreased bioelectrical activity and insulin release.     

Carbonic anhydrase inhibitors: E7070, a sulfonamide anticancer agent, potently inhibits cytosolic isozymes I and II, and transmembrane, tumor-associated isozyme IX

E7070 [N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide] is an anticancer drug candidate under clinical development for the treatment of several types of cancers. We prove here that this compound also acts as a potent carbonic anhydrase (CA) inhibitor. Similarly to the clinically used drugs acetazolamide, methazolamide and topiramate, E7070 showed inhibition constants in the range of 15-31nM against isozymes I, II and IX, being slightly less effective as a CA IV inhibitor (K(i) of 65nM). The X-ray crystal structure of the adduct of hCA II with E7070 revealed unprecedented interactions between the inhibitor and the active site, with three different conformations of the chloroindole fragment of the inhibitor interacting with different amino acid residues/water molecules of the enzyme. A superimposition of these conformations with those of other sulfonamide/sulfamate CA inhibitors indicated that similar regions of the hCA II active site could be involved in the interaction with inhibitors.     

Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.     

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.     

The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from Arabidopsis leaves

Supernatant obtained after high-speed centrifugation of disrupted thylakoids that had been washed free from extrathylakoid carbonic anhydrases demonstrated carbonic anhydrase activity that was inhibited by the specific inhibitors acetazolamide and ethoxyzolamide. A distinctive feature of the effect of Triton X-100 on this activity also suggested that the source of the activity is a soluble protein. Native electrophoresis of a preparation obtained using chromatography with agarose/mafenide as an affinity sorbent revealed one protein band with carbonic anhydrase activity. The same protein was revealed in a mutant deficient in soluble stromal carbonic anhydrase β-CA1, and this indicated that the newly revealed carbonic anhydrase is not a product of the At3g01500 gene. These data imply the presence of soluble carbonic anhydrase in the thylakoid lumen of higher plants.