The effects of endothelin-1 on the cardiorespiratory physiology of the freshwater trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias).

The aim of the present study was to evaluate the effects of endothelin-l-elicited cardiovascular events on respiratory gas transfer in the freshwater rainbow trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias). In both species, endothelin-1 (666 pmol kg(-1)) caused a rapid (within 4 min) reduction (ca. 30-50 mmHg) in arterial blood partial pressure of O2. The effects of endothelin-1 on arterial blood partial pressure of CO2 were not synchronised with the changes in O2 partial pressure and the responses were markedly different in trout and dogfish. In trout, arterial CO2 partial pressure was increased transiently by approximately 1.0 mmHg but the onset of the response was delayed and occurred 12 min after endothelin-1 injection. In contrast, CO2 partial pressure remained more-or-less constant in dogfish after injection of endothelin-1 and was increased only slightly (approximately 0.1 mmHg) after 60 min. Pre-treatment of trout with bovine carbonic anhydrase (5 mg ml(-1)) eliminated the increase in CO2 partial pressure that was normally observed after endothelin-1 injection. In both species, endothelin-1 injection caused a decrease in arterial blood pH that mirrored the changes in CO2 partial pressure. Endothelin-1 injection was associated with transient (trout) or persistent (dogfish) hyperventilation as indicated by pronounced increases in breathing frequency and amplitude. In trout, arterial blood pressure remained constant or was decreased slightly and was accompanied by a transient increase in systemic resistance, and a temporary reduction in cardiac output. The decrease in cardiac output was caused solely by a reduction in cardiac frequency; cardiac stroke volume was unaffected. In dogfish, arterial blood pressure was lowered by approximately 10 mmHg at 6-10 min after endothelin-1 injection but then was rapidly restored to pre-injection levels. The decrease in arterial blood pressure reflected an increase in branchial vascular resistance (as determined using in situ perfused gill preparations) that was accompanied by simultaneous decreases in systemic resistance and cardiac output. Cardiac frequency and stroke volume were reduced by endothelin-1 injection and thus both variables contributed to the changes in cardiac output. We conclude that the net consequences of endothelin-1 on arterial blood gases result from the opposing effects of reduced gill functional surface area (caused by vasoconstriction) and an increase in blood residence time within the gill (caused by decreased cardiac output.     

In situ characterization of carbonic anhydrase activity in isolated rat lungs

Lung carbonic anhydrase (CA) participates directly in plasma CO2-HCO3(-)-H+ reactions. To characterize pulmonary CA activity in situ, CO2 excretion and capillary pH equilibration were examined in isolated saline-perfused rat lungs. Isolated lungs were perfused at 25, 30, and 37 degrees C with solutions containing various concentrations of HCO3- and a CA inhibitor, acetazolamide (ACTZ). Total CO2 excretion was partitioned into those fractions attributable to dissolved CO2, uncatalyzed HCO3- dehydration, and catalyzed HCO3- dehydration. Approximately 60% of the total CO2 excretion at each temperature was attributable to CA-catalyzed HCO3- dehydration. Inhibition of pulmonary CA diminished CO2 excretion and produced significant postcapillary perfusate pH disequilibria, the magnitude and time course of which were dependent on temperature and the extent of CA inhibition. The half time for pH equilibration increased from approximately 5 s at 37 degrees C to 14 s at 25 degrees C. For the HCO3- dehydration reaction, pulmonary CA in situ displayed an apparent inhibition constant for ACTZ of 0.9-2.2 microM, a Michaelis-Menten constant of 90 mM, a maximal reaction velocity of 9 mM/s, and an apparent activation energy of 3.0 kcal/mol.     

Effects of perfusate buffer capacity on capillary CO2-HCO3(-)-H+ reactions: theory.

The importance of perfusate nonbicarbonate buffer capacity (beta nonHCO3) to intracapillary CO2-HCO3(-)-H+ reactions was assessed by theoretical analysis of CO2 exchange in saline-perfused pulmonary capillaries. Time courses for perfusate PCO2, [HCO3-], and [H+] were computed for capillaries containing different activities of luminal vascular carbonic anhydrase and different amounts of perfusate nonbicarbonate buffers. Mobilization of perfusate HCO3- toward CO2 during capillary transit is determined by the availability of HCO3- and H+. A supply of protons from the nonbicarbonate buffer pool is necessary to maintain a high rate of HCO3- dehydration. The analyses indicate that beta nonHCO3 has marked nonlinear effects on transcapillary CO2 exchange and intravascular pH equilibration. These nonlinear effects differ from those previously computed for CO2 reactions in an open system because the present model system consists of a sequential combination of open (within capillary proper) and closed (within postcapillary vasculature) systems. The role of luminal vascular carbonic anhydrase in capillary CO2 reactions is strongly dependent on beta nonHCO3. Perfusate nonbicarbonate buffer capacity must be considered when the results of experimental studies of transcapillary CO2 exchange and/or intravascular pH equilibration are interpreted.     

Carbon isotope effect on dehydration of bicarbonate ion catalyzed by carbonic anhydrase

The carbon-13 kinetic isotope effect on the dehydration of HCO3- by bovine carbonic anhydrase has been measured. To accomplish this, bicarbonate was added to a buffer solution at pH 8 containing carbonic anhydrase under conditions where purging of the product CO2 from the solution is rapid. Measurement of the isotopic composition of the purged CO2 as a function of the concentration of carbonic anhydrase permits calculation of the isotope effect on the enzymic reaction. The isotope effect on the dehydration is k12/k13 = 1.0101 +/- 0.0004. This effect is most consistent with a ping-pong mechanism for carbonic anhydrase action, in which proton transfer to or from the enzyme occurs in a step separate from the dehydration step. Substrate and product dissociation steps are at least 2-3-fold faster than the hydration/dehydration step.     

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

The effects of endothelin-1 on the cardiorespiratory physiology of the freshwater trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias)

The aim of the present study was to evaluate the effects of endothelin-1-elicited cardiovascular events on respiratory gas transfer in the freshwater rainbow trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias). In both species, endothelin-1 (666 pmol kg^-1) caused a rapid (within 4 min) reduction (ca. 30-50 mmHg) in arterial blood partial pressure of O₂. The effects of endothelin-1 on arterial blood partial pressure of CO₂ were not synchronised with the changes in O₂ partial pressure and the responses were markedly different in trout and dogfish. In trout, arterial CO₂ partial pressure was increased transiently by ~1.0 mmHg but the onset of the response was delayed and occurred 12 min after endothelin-1 injection. In contrast, CO₂ partial pressure remained more-or-less constant in dogfish after injection of endothelin-1 and was increased only slightly (~0.1 mmHg) after 60 min. Pre-treatment of trout with bovine carbonic anhydrase (5 mg ml^-1) eliminated the increase in CO₂ partial pressure that was normally observed after endothelin-1 injection. In both species, endothelin-1 injection caused a decrease in arterial blood pH that mirrored the changes in CO₂ partial pressure. Endothelin-1 injection was associated with transient (trout) or persistent (dogfish) hyperventilation as indicated by pronounced increases in breathing frequency and amplitude. In trout, arterial blood pressure remained constant or was decreased slightly and was accompanied by a transient increase in systemic resistance, and a temporary reduction in cardiac output. The decrease in cardiac output was caused solely by a reduction in cardiac frequency; cardiac stroke volume was unaffected. In dogfish, arterial blood pressure was lowered by ~10 mmHg at 6-10 min after endothelin-1 injection but then was rapidly restored to pre-injection levels. The decrease in arterial blood pressure reflected an increase in branchial vascular resistance (as determined using in situ perfused gill preparations) that was accompanied by simultaneous decreases in systemic resistance and cardiac output. Cardiac frequency and stroke volume were reduced by endothelin-1 injection and thus both variables contributed to the changes in cardiac output. We conclude that the net consequences of endothelin-1 on arterial blood gases result from the opposing effects of reduced gill functional surface area (caused by vasoconstriction) and an increase in blood residence time within the gill (caused by decreased cardiac output.     

Chemical kinetic and diffusional limitations on bicarbonate reabsorption by the proximal tubule.

It is accepted that bicarbonate reabsorption in the proximal tubule is mediated by H+ secretion, but several aspects of this process have remained controversial. To examine some of these issues, we have developed a model that allows for spatial variations in the concentrations of CO2, HCO3-, and H2CO3 within the tubule lumen and cell cytoplasm, passive transport of these substances across cell membranes, carbonic anhydrase-catalyzed interconversion of HCO3- and CO2 within the cell and at the luminal membrane surface, and the corresponding uncatalyzed reactions in lumen and cell. Most of the required kinetic and transport parameters were estimated from physicochemical data in the literature, whereas intracellular pH and HCO3- permeability at the basal cell membrane, found to be the most significant parameters under normal conditions, were adjusted to yield reabsorption rates of "total CO2" (tCO2, the sum of CO2, HCO3- and H2CO3) comparable to measured values in the rat. Our results suggest that for normal carbonic anhydrase activity, almost all tCO2 leaves the lumen as CO2, yet the transepithelial differences in CO2 partial pressure does not exceed approximately 2 mm Hg. Electrochemical potential gradients favor substantial passive backleak of HCO3- from cell to lumen. Gradients in CO2 partial pressure remain small during simulated inhibition of carbonic anhydrase, with approximately 70% of tCO2 leaving the lumen as H2CO3 in this case, and the remainder as CO2. Predicted tCO2 reabsorption rates for carbonic anhydrase inhibition are approximately of normal, in good agreement with recent measurements in the rat, indicating that the concept of "carbonic acid recycling" is viable.     

Adaptation to respiratory acidosis in the turtle bladder

The effect of in vivo respiratory acidosis for 4 and 48 hr was examined in the turtle bladder by placing turtles in hypercapnic chambers. Blood pH was significantly lowered and pCO2 was significantly elevated over control values both 4 and 48 hr, while blood bicarbonate was only increased after 48 hr. In vitro rates for H+ secretion determined by the reverse short-circuit current were significantly greater in bladders from 48 hr of respiratory acidosis than those of controls (27.3 +/- 2.7 vs 20.6 +/- 1.7 microA, P less than 0.05). In vitro rates for HCO3- secretion determined by pH stat were not altered. Fluorescence microscopy was used to study cell morphology. The number of carbonic anhydrase cells (corrected for the total number of cells) as determined by four different fluorescence stains (6-carboxyfluorescein, rhodamine 123, acridine orange, and 3,3'-diethyloxacarbocyaninine iodide) was increased both after 4 and 48 hr of respiratory acidosis. However, the number of HCO3(-)-secreting (beta subtype) carbonic anhydrase cells, determined by a probe for the anion exchanger, NBD-taurine, was not increased. In vitro 1% CO2 for 4 hr also resulted in an increase in H+ secretion and in the number of 6-carboxyfluorescein-positive cells, both of which could be blocked with SITS pretreatment. We conclude that CO2 changes the mucosal cells more toward the carbonic anhydrase phenotype, and that if NBD-taurine accurately identifies the beta cells, that the adaptation produces or recruits more alpha-carbonic anhydrase cells.     

Effects of acetazolamide on cerebral acid-base balance

Acetazolamide (AZ) inhibition of brain and blood carbonic anhydrase increases cerebral blood flow by acidifying cerebral extracellular fluid (ECF). This ECF acidosis was studied to determine whether it results from high PCO2, carbonic acidosis (accumulation of H2CO3), or lactic acidosis. Twenty rabbits were anesthetized with pentobarbital sodium, paralyzed, and mechanically ventilated with 100% O2. The cerebral cortex was exposed and fitted with thermostatted flat-surfaced pH and PCO2 electrodes. Control values (n = 14) for cortex ECF were pH 7.10 +/- 0.11 (SD), PCO2 42.2 +/- 4.1 Torr, PO2 107 +/- 17 Torr, HCO3- 13.8 +/- 3.0 mM. Control values (n = 14) for arterial blood were arterial pH (pHa) 7.46 +/- 0.03 (SD), arterial PCO2 (PaCO2) 32.0 +/- 4.1 Torr, arterial PO2 (PaO2) 425 +/- 6 Torr, HCO3- 21.0 +/- 2.0 mM. After intravenous infusion of AZ (25 mg/kg), end-tidal PCO2 and brain ECF pH immediately fell and cortex PCO2 rose. Ventilation was increased in nine rabbits to bring ECF PCO2 back to control. The changes in ECF PCO2 then were as follows: pHa + 0.04 +/- 0.09, PaCO2 -8.0 +/- 5.9 Torr, HCO3(-)-2.7 +/- 2.3 mM, PaO2 +49 +/- 62 Torr, and changes in cortex ECF were as follows: pH -0.08 +/- 0.04, PCO2 -0.2 +/- 1.6 Torr, HCO3(-)-1.7 +/- 1.3 mM, PO2 +9 +/- 4 Torr. Thus excess acidity remained in ECF after ECF PCO2 was returned to control values. The response of intracellular pH, high-energy phosphate compounds, and lactic acid to AZ administration was followed in vivo in five other rabbits with 31P and 1H nuclear magnetic resonance spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of proton availability on intracapillary CO2-HCO3(-)-H+ reactions in isolated rat lungs.

Transcapillary CO2 exchange entails a transient perfusate CO2-HCO3(-)-H+ disequilibrium, leading to net loading or unloading of blood HCO3-. Perfusate reequilibration may or may not reach completion during the time of capillary transit, depending on the rate of intracapillary CO2-HCO3(-)-H+ reactions. Failure to reestablish equilibrium within the "open" capillary system leads to continued reequilibration in the "closed" postcapillary vasculature with resultant shifts in postcapillary perfusate PCO2, pH, and [HCO3-]. In the present study, we determined the effects of perfusate nonbicarbonate buffer capacity (beta) on intracapillary CO2-HCO3(-)-H+ reactions in isolated saline-perfused rat lungs. Effects of beta on the rate of transcapillary CO2 excretion (VCO2) and the magnitude of the postcapillary perfusate pH disequilibrium were measured as a function of luminal vascular carbonic anhydrase (CA) activity. The data indicate that beta markedly influenced the kinetics and dynamics of intravascular CO2-HCO3(-)-H+ reactions. beta affected VCO2 and the relative enhancement of VCO2 by luminal vascular CA. The data emphasize the inadequacies of using traditional "equilibrium" models of the CO2-HCO3(-)-H+ system to investigate capillary CO2 transport and exchange, even in organs (e.g., lungs) that contain significant luminal vascular CA activity.     

Differential modification of specificity in carbonic anhydrase catalysis

Incubation of carbonic anhydrase II with acrolein results in a rapid, time-dependent loss of all but approximately 3-6% of the original catalytic activity toward CO2 hydration and HCO3- dehydration, with the inactivation rate being first-order in both acrolein and the enzyme. The pH dependence of the inactivation rate constant can be adequately described with a function incorporating a pK alpha of 7.15 and a maximal value for kinact [corrected] of 26.2 M-1 min-1, indicating that at least one of the catalytically essential residues that ionizes at this pH is involved in the modification scheme. The amount of residual CO2 hydratase activity is proportional to the molar excess of acrolein over carbonic anhydrase II with 5 histidyl and 3 lysyl residues being subject to alkylation under conditions where [acrolein] to [carbonic anhydrase II] ratio is greater than 100. Because all lysyl residues were shown previously to be amidinated without detectable loss of activity, it was assumed that the modification of one (or more) of the histidines was primarily responsible for the observed inactivation. The number of modified histidyl residues could be related to residual activity by using the statistical analysis of Tsou (Tsou, C.-L. (1962) Sci. Sin. (Engl. Ed.) 11, 1535-1558) which indicates that one essential histidine reacts approximately four times faster than the other (histidyl) residues. In sharp contrast with the phenomenon observed in connection with CO2 hydration and HCO3- dehydration, acrolein improves the catalytic efficiency of the enzyme toward p-nitrophenyl acetate hydrolysis and acetaldehyde hydration, with the relative activity increasing by approximately 12 and 34%, respectively. The widely differing effects imparted by the same reagent represent the first step toward differential control of the specificity of carbonic anhydrase II.     

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.     

Acid-base disequilibrium in the venous blood of rainbow trout (Oncorhynchus mykiss)

Acid-base equilibria/disequilibria were evaluated in vivo in post-branchial arterial blood and pre-branchial venous blood of freshwater rainbow trout (Oncorhynchus mykiss). This was accomplished using arterial and venous extracorporeal circuits in conjunction with a stopped-flow apparatus. After the abrupt stoppage of circulating post-branchial blood within the stopped-flow apparatus, pH increased slowly ([Delta]pH = +0.032 ± 0.004 pH units; n = 15), thus confirming the existence of an acid-base disequilibrium state in the arterial blood of rainbow trout. The slow downstream pH changes were unaffected by prior treatment of fish with the carbonic anhydrase inhibitor benzolamide (1.2 mg kg^-1; [Delta]pH = +0.032 ± 0.01 pH units; n = 5) but were eliminated after intra-vascular injection of 10 mg kg^-1 bovine carbonic anhydrase ([Delta]pH = -0.011 ± 0.003 pH units; n = 8). These results demonstrate that the acid-base disequilibrium in the arterial blood reflects a total absence of extracellular carbonic anhydrase activity. Similar stopped-flow experiments revealed the existence of a reduced, yet significant, acid-base disequilibrium in the venous blood circulating within the caudal vein ([Delta]pH = +0.004 ± 0.003 pH units; n = 15). Selective inhibition of extracellular carbonic anhydrase using benzolamide did not significantly influence the magnitude of the venous pH disequilibrium ([Delta]pH = +0.007 ± 0.007 pH units; n = 8) whereas intra-vascular injection of carbonic anhydrase eliminated the pH disequilibrium. These results demonstrate that extracellular carbonic anhydrase, although reported to be present within the skeletal muscle of rainbow trout, does not accelerate post-capillary pH changes in the venous circulation.     

Effect of plasma carbonic anhydrase on ventilation in exercising dogs

Recent studies suggest pH sampled by arterial chemoreceptors may not equal that sampled by external pH electrodes, because the uncatalyzed hydration of CO2 in plasma is a slow reaction (t 1/2 approximately 9 S). The importance of this reaction rate to ventilatory control (particularly during exercise) is not known. We studied the effect of catalyzing the CO2-pH reaction in three awake exercising dogs with chronic tracheostomies and carotid loops; the dogs were trained to run on a treadmill. Respiration frequency, tidal volume, total ventilation, and end-tidal partial pressure of CO2 (PCO2) were continuously monitored. Periodically, carotid artery blood was drawn and analyzed for partial pressure of O2 (PO2), PCO2, pH, and plasma carbonic anhydrase (CA) activity. Measurements were made during steady-state exercise (3 mph and 10% grade), during a control period, after injection of a 5 ml bolus of saline, and after injection of 5 mg/kg of bovine CA dissolved in 5 ml of saline. This dose of CA increased the reaction rate by more than 80-fold. Neither the control nor the CA injections significantly altered the ventilatory parameters. Saline and CA date differed by less than 5% in ventilation, 1 Torr in arterial PCO2, 0.01 in pH units, and 1.5 Torr in end-tidal PCO2. Thus the of CO2 hydration in plasma is not a significant factor in ventilatory control.     

Kinetics of carbonic anhydrase catalysis in solvents of increased viscosity: a partially diffusion-controlled reaction

Steady-state kinetic studies of the bovine carbonic anhydrase B-catalyzed hydration of CO2, dehydration of HCO3-, and hydrolysis of p-nitrophenylacetate were made in glycerol/water solvents of increased viscosity in order that the effect of diffusion-control on the substrate association reactions could be determined. The minimum association rate constants (kmin = V/(Km[E0])) were obtained at low substrate concentrations. The esterase activity did not depend upon the solvent viscosity. However, both the CO2 hydration and HCO3- dehydration reactions depended upon the solvent viscosity consistent with partial diffusion control. Thus both chemical activation and diffusion control processes contribute to the observed kmin. In low-viscosity aqueous solutions both hydration and dehydration are largely controlled by chemical activation. However, at higher viscosities, equal to that found in the interior of the erythrocyte, both reactions are largely diffusion controlled. This result can be interpreted to mean that carbonic anhydrase is a highly evolved enzyme that has approached its maximum efficiency. The extent of diffusion control observed rules out H2CO3 as a significant reactant with the enzyme. Several models that yield minimum steric requirements for access of substrate to the active site are examined. Minimum steric constraints are less for the smaller CO2. The slower esterase reaction is not influenced by diffusion.     

Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the beta- and gamma-class

Catalysis of the dehydration of HCO(3)(-) by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the alpha, beta, and gamma classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the alpha class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (beta class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (gamma class). A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO(2) hydration at steady state and as proton donors of the exchange of (18)O between CO(2) and water at chemical equilibrium. Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pK(a) of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers. Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the beta and gamma class, similar to results obtained for mutants of carbonic anhydrase in the alpha class. The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.     

Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure

We found that a carbonic anhydrase, YadF, is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli. However, mutant strains lacking both of them grew at high CO2 concentrations (5%), where non-enzymatic mechanisms generate HCO3-. This suggests that these carbonic anhydrases are essential because they maintain HCO3- levels at ambient CO2 concentrations.     

Effect of acetazolamide on gas exchange and acid-base control after maximal exercise.

To investigate the interactions between the systems that contribute to acid-base homeostasis after severe exercise, we studied the effects of carbonic anhydrase inhibition on exchange of strong ions and CO2 in six subjects after 30 s of maximal isokinetic cycling exercise. Each subject exercised on two randomly assigned occasions, a control (CON) condition and 30 min after intravenous injection of 1,000 mg acetazolamide (ACZ) to inhibit blood carbonic anhydrase activity. Leg muscle power output was similar in the two conditions; peak O2 uptake (VO2) after exercise was lower in ACZ (2,119 +/- 274 ml/min) than in CON (2,687 +/- 113, P less than 0.05); peak CO2 production (VCO2) was also lower (2,197 +/- 241 in ACZ vs. 3,237 +/- 87 in CON, P less than 0.05) and was accompanied by an increase in the recovery half-time from 1.7 min in CON to 2.3 min in ACZ. Whereas end-tidal PCO2 was lower in ACZ than in CON, arterial PCO2 (PaCO2) was higher, and a large negative end-tidal-to-arterial difference (less than or equal to 20 Torr) was present in ACZ on recovery. In ACZ, postexercise increases in arterial plasma [Na+] and [K+] were greater but [La-] was lower. Arteriovenous differences across the forearm showed a greater uptake of La- and Cl- in CON than in ACZ. Carbonic anhydrase inhibition with ACZ, in addition to impairing equilibration of the CO2 system to the acid-base challenge of exercise, was accompanied by changes in equilibration of strong inorganic ions. A lowered plasma [La-] was not accompanied by greater uptake of La- by inactive muscle.     

Control of flatfish sperm motility by CO2 and carbonic anhydrase

Sperm motility in flatfishes shows unique characteristics. The flagellar movement either in vivo or in permeabilized models is arrested by the presence of 25-100 mM HCO3-, or by gentle perfusion with CO2 gas. To understand the molecular basis of this property, sperm Triton-soluble proteins and flagellar proteins from several species were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An abundant 29-kDa protein was observed only in flatfish species. Partial amino acid sequences identified this protein as a carbonic anhydrase, an enzyme involved in the interconversion of CO2 and HCO3-. 6-ethoxyzolamide, a specific inhibitor of carbonic anhydrase inhibits sperm motility, especially at low pH. In the case of HCO3(-)-arrested sperm, the motility is restored by addition of 6-ethoxyzolamide. Taken together, these results suggest that a novel pH/HCO3(-)-dependent regulatory mechanism mediated by carbonic anhydrase is involved in the motility control in flatfish sperm.