Investigation of the system copper(II) carbonic anhydrase and HCO3-/CO2

Copper(II) substituted human and bovine carbonic anhydrases B in the presence of bicarbonate have been investigated in solution through water-solvent proton nuclear magnetic resonance (nmr) at variable magnetic fields. HCO3-, contrary to all the other monoanionic inhibitors, partially reduces the water proton relaxation rates. This has been accounted for on the basis of the availability within the active cavity of two coordination positions partially overlapping. 13C-nmr measurements on both CO2 and HCO3- confirm that HCO3- binds the metal, whereas CO2 interacts with the paramagnetic center at nonbonding distance. The upper limit for the CO2 in equilibrium HCO3- interconversion has been estimated to be 10 sec-1.     

Kinetic parameters of human carbonic anhydrase B as determined from NMR linewidths of 13 C in CO 2 and HCO 3 -

The linewidths of the ¹³C NMR signals of CO₂ and $\text{HCO}{}_3{}^{\mathrm{?}}$, in equilibrium aqueous solutions containing small amounts of carbonic anhydrase, are determined mainly by the rate of enzyme-induced interconversion of CO₂ and $\text{HCO}{}_3{}^{\mathrm{?}}$. We have measured these linewidths in unbuffered solutions of human carbonic anhydrase B for several values of [CO₂], at 25°C as a function of pH. From a least-squares analysis of the data, using the equations relating the linewidths to the enzyme kinetics, we have obtained values for the kinetic (Michaelis-Menten) parameters that characterize this interconversion. These preliminary results are in approximate agreement with published values for highly buffered solutions. Additionally, the results confirm that the product of the hydration reaction, and the substrate for the dehydration, is the neutral molecule H₂CO₃.     

Effect of basolateral CO2/HCO3- on intracellular pH regulation in the rabbit S3 proximal tubule

We used the absorbance spectrum of the pH-sensitive dye dimethylcarboxyfluorescein to monitor intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule, and examined the effect on pHi of switching from a HEPES to a CO2/HCO3- buffer in the lumen and/or the bath (i.e., basolateral solution). Solutions were titrated to pH 7.40 at 37 degrees C. With 10 mM acetate present bilaterally (lumen and bath), this causing steady-state pHi to be rather high (approximately 7.45), bilaterally switching the buffer from 32 mM HEPES to 5% CO2/25 mM HCO3- caused a sustained fall in pHi of approximately 0.26. However, with acetate absent bilaterally, this causing steady-state pHi to be substantially lower (approximately 6.9), bilaterally switching to CO2/HCO3- caused a transient pHi fall (due to the influx of CO2), followed by a sustained rise to a level approximately 0.18 higher than the initial one. The remainder of the experiments was devoted to examining this alkalinization in the absence of acetate. Switching to CO2/HCO3- only in the lumen caused a sustained pHi fall of approximately 0.15, whereas switching to CO2/HCO3- only in the bath caused a transient fall followed by a sustained pHi increase to approximately 0.26 above the initial value. This basolateral CO2/HCO3(-)-induced alkalinization was not inhibited by 50 microM DIDS applied shortly after CO2/HCO3- washout, but was slowed approximately 73% by DIDS applied more than 30 min after CO2/HCO3- washout. The rate was unaffected by 100 microM bilateral acetazolamide, although this drug greatly reduced CO2-induced pHi transients. The alkalinization was not blocked by bilateral removal of Na+ per se, but was abolished at pHi values below approximately 6.5. The alkalinization was also unaffected by short-term bilateral removal of Cl- or SO4=. Basolateral CO2/HCO3- elicited the usual pHi increase even when all solutes were replaced, short or long-term (> 45 min), by N-methyl-D- glucammonium/glucuronate (NMDG+/Glr-). Luminal CO2/HCO3- did not elicit a pHi increase in NMDG+/Glr-. Although the sustained pHi increase elicited by basolateral CO2/HCO3- could be due to a basolateral HCO3- uptake mechanism, net reabsorption of HCO3- by the S3 segment, as well as our ACZ data, suggest instead that basolateral CO2/HCO3- elicits the sustained pHi increase either by inhibiting an acid-loading process or stimulating acid extrusion across the luminal membrane (e.g., via an H+ pump).     

Role of carbonic anhydrase in photosynthetic CO2 fixation in Chlorella

Chlorella vulgaris 11h cells grown in air enriched with 4% CO₂(high-CO² cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO₂, low-CO₂ cells). The CO₂ concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO₂. When high-CO₂ cells were transferred to low CO₂ conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic¹⁴CO₂ fixation under ¹⁴CO₂-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic ¹⁴CO₂fixation at low ¹⁴CO₂ concentrations decreased when low-CO₂cells were transferred to high CO₂ conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO₂ cells at high CO₂ concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO₂ concentrations, and the lowerthe CO₂ concentration, the greater was the inhibition. Consequently,the CO₂ concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO₂ concentration was higher than 1%, the photosyntheticrate in low-CO₂ cells decreased, while that in high-CO₂ cellsincreased. Fractionation of the low-CO₂ cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO₂-limiting conditions, but inhibits it at CO₂ concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed. ¹This work was reported at the International Symposium on PhotosyntheticCO₂-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; )     

Kinetics of CO2 excretion and intravascular pH disequilibria during carbonic anhydrase inhibition

Cardenas, Victor, Jr., Thomas A. Heming, and Akhil Bidani.Kinetics of CO₂ excretion andintravascular pH disequilibria during carbonic anhydrase inhibition.J. Appl. Physiol. 84(2): 683-694, 1998.Inhibition of carbonic anhydrase (CA) activity (activity in redblood cells and activity available on capillary endothelium) results indecrements in CO₂ excretion(CO₂) and plasma-erythrocyteCO₂--H⁺disequilibrium as blood travels around the circulation. To investigate the kinetics of changes in blood PCO₂and pH during progressive CA inhibition, we used our previouslydetailed mathematical model of capillary gas exchange to analyzeexperimental data of CO₂ and blood-gas/pH parameters obtained from anesthetized, paralyzed, andmechanically ventilated dogs after treatment with acetazolamide (Actz,0-100 mg/kg iv). Arterial and mixed venous blood samples werecollected via indwelling femoral and pulmonary arterial catheters, respectively. Cardiac output was measured by thermodilution. End-tidal PCO₂, as a measure of alveolarPCO₂, was obtained from continuousrecords of airway PCO₂ above thecarina. Experimental results were analyzed with the aid of amathematical model of lung and tissue-gas exchange. Progressive CAinhibition was associated with stepwise increments in the equilibratedmixed venous-alveolar PCO₂ gradient(9, 19, and 26 Torr at 5, 20, and 100 mg/kg Actz, respectively). Themaximum decrements in CO₂were 10, 24, and 26% with 5, 20, and 100 mg/kg Actz, respectively,without full recovery ofCO₂ at 1 h postinfusion. Equilibrated arterial PCO₂overestimated alveolar PCO₂, andtissue PCO₂ was underestimated by themeasured equilibrated mixed venous bloodPCO₂. Mathematical model computations predicted hysteresis loops of the instantaneousCO₂--H⁺relationship and in vivo bloodPCO₂-pH relationship due to thefinite reaction times forCO₂--H⁺reactions. The shape of the hysteresis loops was affected by the extentof Actz inhibition of CA in red blood cells and plasma.

     

Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA

Human NBC3 is an electroneutral Na⁺/HCO₃^– cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO₃^– metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO₂ + H₂O HCO₃^– + H⁺ in many tissues. We investigated whether NBC3, like some Cl^–/HCO₃^– exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with K_d = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 ± 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 µM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH_i) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pH_i recovery rate by 69 ± 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [-³²P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO₃^– transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct. pH regulation; bicarbonate transport; metabolon     

Hydration of carbon dioxide by carbonic anhydrase: internal proton transfer of Zn2+-bound HCO3-

Proton transfer within HCO3- has been examined under various conditions through molecular orbital methods: partial retention of diatomic differential overlap and 4-31G self-consistent field programs. These conditions include the absence or presence of Zn2+, Zn2+(NH3)3, or a water ligand on Zn2+. In addition, 4-31G+ and some MP2/4-31G results are obtained. The use of Be2+ to simulate Zn2+ reproduces reaction pathways and energy barriers, except for marginal cases. The barrier of 35.6 kcal/mol for direct internal proton transfer is reduced to 3.5 kcal/mol when one water molecule, not bound to Zn2+, is included for proton relay and to 1.4 kcal/mol when two such water molecules are included. In the enzyme, either Thr-199 or solvent molecules could perform this relay function. Our results favor this facilitated proton transfer over a mechanism in which Zn2+-bound OH- attacks CO2, a bidentate intermediate forms, and the OH moiety of the resulting HCO3- dissociates from Zn2+, thus leaving one of the oxygens of the original CO2 as a ligand to Zn2+.     

Dual role of CO2/HCO3(-) buffer in the regulation of intracellular pH of three-dimensional tumor growths

Intracellular pH (pH(i)), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H(+) ions (e.g. produced by respiration) are extruded by transporters such as Na(+)/H(+) exchange, or neutralized by HCO(3)(-) taken up by carriers such as Na(+)-HCO(3)(-) cotransport. Using fluorescence pH(i) imaging, we show that cancer-derived cell lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, and cervical HeLa) extrude acid by H(+) efflux and HCO(3)(-) influx, largely sensitive to dimethylamiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), respectively. The magnitude of HCO(3)(-) influx was comparable among the cell lines and may represent a constitutive element of tumor pH(i) regulation. In contrast, H(+) efflux varied considerably (MDA-MB-468 > HCT116 > HT29 > MiaPaca2 > HeLa). When HCO(3)(-) flux was pharmacologically inhibited, acid extrusion in multicellular HT29 and HCT116 spheroids (～10,000 cells) was highly non-uniform and produced low pH(i) at the core. With depth, acid extrusion became relatively more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H(+) flux more than HCO(3)(-) flux. HCO(3)(-) flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO(2)/HCO(3)(-), acid extrusion by H(+) flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core. This is because H(+) transporters require extracellular mobile pH buffers, such as CO(2)/HCO(3)(-), to overcome low H(+) ion mobility and chaperone H(+) ions away from cells. CO(2)/HCO(3)(-) exerts a dual effect: as substrate for membrane-bound HCO(3)(-) transporters and as a mobile buffer for facilitating extracellular diffusion of H(+) ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO(2)/HCO(3)(-) is important for maintaining uniformly alkaline pH(i) in small, non-vascularized tumor growths and may be important for cancer disease progression.     

Branchial membrane-associated carbonic anhydrase activity maintains CO2 excretion in severely anemic dogfish

Plasma CO(2) reactions in Pacific spiny dogfish (Squalus acanthias) have access to plasma and gill membrane-associated carbonic anhydrase (CA). Acute severe experimental anemia and selective CA inhibitors were used to investigate the role of extracellular CA in CO(2) excretion. Anemia was induced by blood withdrawal coupled to volume replacement with saline. Lowering hematocrit from 14.2 +/- 0.4% (mean +/- SE; N = 31) to 5.2 +/- 0.1% (N = 31) had no significant impact on arterial or venous CO(2) tensions (Pa(CO(2)) and Pv(CO(2)), respectively) over the subsequent 2 h. PCO(2) was maintained despite the reduction in red cell number and a significant 32% increase in cardiac output (V(b)), both of which have been found to cause Pa(CO(2)) increases in teleost fish. By contrast, treatment of anemic dogfish with the CA inhibitors benzolamide (1.3 mg/kg) or F3500 (50 mg/kg), to selectively inhibit extracellular CA, elicited rapid and significant increases in Pa(CO(2)) of 0.68 +/- 0.17 Torr (N = 6) and 0.53 +/- 0.11 Torr (N = 7), respectively, by 30 min after treatment. These findings provide a functional context in which extracellular CA in dogfish contributes substantially to CO(2) excretion. Additionally, the apparent lack of effect of V(b) changes on PCO(2) suggests that, in contrast to teleost fish, CO(2) excretion in dogfish does not behave as a diffusion-limited system.     

Urea synthesis and CO2/HCO3- compartmentation in isolated perfused rat liver

With physiological portal HCO3- and CO2 concentrations of 25mM and 1.2mM in the perfusate, respectively, acetazolamide inhibited urea synthesis from NH4Cl in isolated perfused rat liver by 50-60%, whereas urea synthesis from glutamine was inhibited by only 10-15%. A decreased sensitivity of urea synthesis from glutamine to acetazolamide inhibition was also observed when the extracellular HCO3- and CO2 concentrations were varied from 0-50mM and 0-2.4mM, respectively. Stimulation of intramitochondrial CO2 formation at pyruvate dehydrogenase with high pyruvate concentrations (7mM) was without effect on the acetazolamide sensitivity of urea synthesis from NH4Cl. Urea synthesis was studied under conditions of a limiting HCO3- supply for carbamoyl-phosphate synthesis. In the absence of externally added HCO3- or CO2, when 14CO2 was provided intracellularly by [U-14C]glutamine or [1-14C]-glutamine oxidation, acetazolamide had almost no effect on label incorporation into urea, whereas label incorporation from an added tracer H14CO3- dose was inhibited by about 70%. 14CO2 production from [U-14C]glutamine was about twice as high as from [1-14C]glutamine, indicating that about 50% of the CO2 produced from glutamine is formed at 2-oxoglutarate dehydrogenase. The fractional incorporation of 14CO2 into urea was about 13% with [1-14C]-as well as with [U-14C]glutamine. Addition of small concentrations of HCO3- (1.2mM) to the perfusate increased urea synthesis from glutamine by about 70%. This stimulation of urea synthesis was fully abolished by acetazolamide. The carbonate-dehydratase inhibitor prevented the incorporation of added HCO3- into urea, whereas incorporation of CO2 derived from glutamine degradation was unaffected. Without HCO3- and CO2 in the perfusion medium, when 14CO2 was provided by [1-14C]-pyruvate oxidation, acetazolamide inhibited urea synthesis from NH4Cl as well as 14C incorporation into urea by about 50%. Therefore carbonate-dehydratase activity is required for the utilization of extracellular CO2 or pyruvate-dehydrogenase-derived CO2 for urea synthesis, but not for CO2 derived from glutamine oxidation. This is further evidence for a special role of glutamine as substrate for urea synthesis.     

Sensing inorganic carbon: CO2 and HCO3-

Enzymes and transporters that catalyse reactions involving inorganic carbon are well characterized with respect to the species of inorganic carbon (CO2 or HCO3-) with which they interact. There is less information on the species recognized by proteins that sense inorganic carbon. In this issue of the Biochemical Journal, Hammer and colleagues show conclusively that cyanobacterial adenylyl cyclases are activated by CO2 and not HCO3-, as was believed previously. While in some circumstances a similar in vivo regulatory outcome is achieved from sensing HCO3- as from sensing CO2, there are cases in which the outcomes are significantly different. The most striking example is where a compartment lacks carbonic anhydrase yet supports large metabolic fluxes of inorganic carbon species so that CO2 and HCO3- are not at equilibrium. Other examples involve changes in pH, or temperature, of a compartment containing an equilibrium mixture of CO2 and HCO3-.     

Paramagnetic carbon-13 NMR relaxation studies on the kinetics and mechanism of the HCO3-/CO2 exchange catalyzed by manganese(II) human carbonic anhydrase I

A detailed analysis of the stability and activity of Mn(II) human carbonic anhydrase I and the kinetics and mechanism of its catalysis of the HCO3-/CO2 exchange have been performed at pH 8.5. The analysis was based on the paramagnetic relaxation rates R1p and R2p of the 13C atom of HCO3- in the Mn2+/apoenzyme/HCO3-/CO2 system and the HCO3(-)----CO2 interconversion rate obtained by the magnetization-transfer technique. The R1p and R2p rates were measured as functions of the temperature, magnetic field strength, and substrate and apoenzyme concentrations and were interpreted on the basis of the Solomon-Bloembergen-Morgan theories and general equations for the ligand exchange [Led, J. J., & Grant, D. M. (1977) J. Am. Chem. Soc. 99, 5845-5858]. From the analysis of the data, a formation constant for the Mn(II) enzyme of log KMAM = 5.8 +/- 0.4 was obtained while the activity of the Mn(II) enzyme, measured as the HCO3-/CO2 interconversion rate at [HCO3-] = 0.100 M and pH 8.5, was found to be about 4% of that of the native Zn(II) enzyme. However, an effective dissociation constant KeffHCO3- less than or approximately 12 mM and a maximal exchange rate constant kcatexch approximately equal to 400 s-1, also derived by the analysis, result in an apparent second-order rate constant kcatexch/KeffHCO3- only a factor of 4 smaller than the corresponding rate constant for the native Zn(II) isoenzyme I. Most conspicuously, the resulting distance of only 2.71 +/- 0.03 A between the Mn2+ ion of the enzyme and the 13C atom of HCO3- in the enzyme-bicarbonate complex indicates that the bicarbonate is bound to the metal ion by two of its oxygen atoms in the central catalytic step, thereby supporting the modified Zn(II)-OH mechanism [Lindskog, S., Engberg, P., Forsman, C., Ibrahim, S. A., Jonsson, B.-H., Simonsson, I., & Tibell, L. (1984) Ann. N.Y. Acad. Sci. 429, 61-75 (and references cited therein)]. In contrast, this binding mode differs from the structure of the complexes suggested in the rapid-equilibrium kinetic model [Pocker, Y., & Deits, T. L. (1983) J. Am. Chem. Soc. 105, 980-986; Pocker, Y., & Deits, T. L. (1984) Ann. N.Y. Acad. Sci. 429, 76-83].     

Design,synthesis, and carbonic anhydrase inhibition activity of benzenesulfonamide-linked novel pyrazoline derivatives

Carbonic anhydrases (CA, EC 4.2.1.1) are Zinc metalloenzymes and are present throughout most living organisms. Among the catalytically active isoforms are the cytosolic CA I and II, and tumor-associated CA IX and CA XII. The carbonic anhydrase (CA) inhibitory activities of newly synthesized pyrazoline-linked benzenesulfonamides 18–33 against human CA (hCA) isoforms I, II, IX, and XII were measured and compared with that of acetazolamide (AAZ), a standard inhibitor. Potent inhibitory activity against hCA I was exerted by compounds 18–25, with inhibition constant (K_I) values of 87.8–244.1 nM, which were greater than that of AAZ (K_I, 250.0 nM). Compounds 19, 21, 22, 29, 30, and 32 were proven to have inhibitory activities against hCA IX with K_I values (5.5–37.0 nM) that were more effective than or nearly equal to that of AAZ (K_I, 25.0 nM). Compounds 20–22, and 30 exerted potent inhibitory activities (K_Is, 7.1–10.1 nM) against hCA XII, in comparison with AAZ (K_I, 5.7 nM).     

Localization of the Cl-/HCO3- anion exchanger binding site to the amino-terminal region of carbonic anhydrase II

Human carbonic anhydrase II (CAII) possesses a binding site for an acidic motif (D887ADD) within the carboxyl-terminal region (Ct) of the human erythrocyte chloride/bicarbonate anion exchanger, AE1. In this study, the amino acid sequence comprising this AE1 binding site was localized to the first 17 residues of CAII, which form a basic patch on the surface of the protein. Truncation of the amino terminal of CAII by five residues resulted in a 3-fold reduction in the apparent affinity of the interaction with a GST fusion protein of the Ct of AE1 (GST-Ct) measured by a sensitive microtiter plate binding assay. Further amino-terminal truncation of CAII by 17 or 24 residues caused a loss of binding. The homologous isoform CAI does not bind AE1, despite having 60% sequence identity to CAII. One major difference between the two CA isoforms, within the amino-terminal region, is a high content of histidine residues in CAII (His3, -4, -10, -15, -17) not found in CAI. Mutation of pairs of these histidines (and one lysine) in CAII to the analogous residues in CAI (H3P/H4D or K9D/H10K or H15Q/H17S), or combinations of these various double mutants, did not greatly affect binding between GST-Ct and the mutant CAII. However, when all six of the targeted CAII residues were mutated to the corresponding sequence in CAI, binding of GST-Ct was lost. These results indicate that the AE1 binding site is located within the first 17 residues of CAII, and that the interaction is mediated by electrostatic interactions involving histidine and/or lysine residues. Further specificity for the interaction of AE1 and CAII is provided by a conserved leucine residue (L886) in AE1 that, when mutated to alanine, resulted in loss of GST-Ct binding to immobilized CAII. The binding of the basic amino-terminal region of CAII to an acidic Ct in AE1 provides a structural basis for linking bicarbonate transport across the cell membrane to intracellular bicarbonate metabolism.     

Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3- transport

We have used pH-, Na-, and Cl-sensitive microelectrodes to study basolateral HCO3- transport in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum. In one series of experiments, we lowered basolateral pH (pHb) from 7.5 to 6.8 by reducing [HCO3-]b from 10 to 2 mM at a constant pCO2. This reduction of pHb and [HCO3-]b causes a large (approximately 0.35), rapid fall in pHi as well as a transient depolarization of the basolateral membrane. Returning pHb and [HCO3-]b to normal has the opposite effects. Similar reductions of luminal pH (pHl) and [HCO3-]l have only minor effects. The reduction of [HCO3-]b and pHb also produces a reversible fall in aiNa. In a second series of experiments, we reduced [Na+]b at constant [HCO3-]b and pHb, and also observed a rapid fall in pHi and a transient basolateral depolarization. These changes are reversed by returning [Na+]b to normal. The effects of altering [Na+]l in the presence of HCO3-, or of altering [Na+]b in the nominal absence of HCO3-, are substantially less. Although the effects on pHi and basolateral membrane potential of altering either [HCO3-]b or [Na+]b are largely blocked by 4-acetamido-4- isothiocyanostilbene-2,2'-disulfonate (SITS), they are not affected by removal of Cl-, nor are there accompanying changes in aiCl consistent with a tight linkage between Cl- fluxes and those of Na+ and HCO3-. The aforementioned changes are apparently mediated by a single transport system, not involving Cl-. We conclude that HCO3- transport is restricted to the basolateral membrane, and that HCO3- fluxes are linked to those of Na+. The data are compatible with an electrogenic Na/HCO3 transporter that carries Na+, HCO3-, and net negative charge in the same direction.     

Role of zinc in catalytic activity of carbonic anhydrase IX

The carbonic anhydrases (CAs) in the α class are zinc-dependent metalloenzymes. Previous studies have reported that recombinant forms of carbonic anhydrase IX (CAIX), a membrane-bound form of CA expressed in solid tumors, appear to be activated by low levels of zinc independent of its well-studied role at the catalytic site. In this study, we sought to determine if CAIX is stimulated by zinc in its native environment. MDA-MB-231 breast cancer cells express CAIX in response to hypoxia. We compared CAIX activity associated with membrane ghosts isolated from hypoxic cells with that in intact hypoxic cells. We measured CA activity directly using (18)O exchange from (13)CO(2) into water determined by membrane inlet mass spectrometry. In membrane ghosts, there was little effect of zinc at low concentrations on CAIX activity, although at high concentration zinc was inhibitory. In intact cells, zinc had no significant effect on CAIX activity. This suggests that there is an appreciable decrease in sensitivity to zinc when CAIX is in its natural membrane milieu compared to the purified forms.