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1.
A 13C nuclear magnetic resonance study of CO2/HCO-3 exchange catalyzed by human carbonic anhydrase I
Rates of CO2/HCO-3 exchange, catalyzed by human carbonic anhydrase I (or B) at chemical equilibrium, were estimated from the nuclear magnetic resonance linewidths of 13C-labeled substrates. The results show that the maximal exchange rate constant is independent of pH in the range 5.7-8.0, whereas the apparent substrate dissociation constant depends on pH. Exchange proceeds rapidly in the absence of added buffers, and the addition of buffers has negligible effects on exchange rates. Exchange is equally rapid with 1H2O or 2H2O as solvents. Chloride ions inhibit CO2/HCO-3 exchange competitively. The maximal exchange rates obtained with human carbonic anhydrase I are 50 times slower than those obtained with human isoenzyme II (or C). From a comparison of the exchange kinetics with the steady-state kinetics of CO2 hydration and HCO-3 dehydration it is tentatively concluded that the transfer of H+ between active site and medium proceeds with rates of similar magnitudes in the two isoenzymes, whereas the central catalytic step, the interconversion of enzyme-bound CO2 and HCO-3, is much slower in isoenzyme I than in isoenzyme II. 相似文献
2.
Entry and exit pathways of CO2 in rat liver mitochondria respiring in a bicarbonate buffer system 总被引:3,自引:0,他引:3
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. 相似文献
3.
We have measured the pH dependence of the kinetics of CO2 hydration catalyzed by carbonic anhydrase III from the skeletal muscle of the cat. Two methods were used: an initial velocity study in which the change in absorbance of a pH indicator was measured in a stopped flow spectrophotometer, and an equilibrium study in which the rate of exchange of 18O between CO2 and H2O was measured with a mass spectrometer. We have found that the steady state constants kCO2 cat and KCO2 m are independent of pH within experimental error in the range of pH 5.0 to 8.5; the rate of release from the enzyme of the oxygen abstracted from substrate HCO-3 in the dehydration is also independent of pH in this range. This behavior is very different from that observed for carbonic anhydrase II for which kCO2 cat and the rate of release of substrate oxygen are very pH-dependent. The rate of interconversion of CO2 and HCO-3 at equilibrium catalyzed by carbonic anhydrase III is not altered when the solvent is changed from H2O to 98% D2O and 2% H2O. Thus, the interconversion probably proceeds without proton transfer in its rate-limiting steps, similar to isozymes I and II. 相似文献
4.
W N Scott I Skipski 《Comparative biochemistry and physiology. B, Comparative biochemistry》1979,63(3):429-435
1. Two forms of carbonic anhydrase, having isoelectric points of 6.1 and 5.8, were purified from erythrocytes of the toad, Bufo marinus, and the presence of a third form, pI = 5.4, was demonstrated. 2. Each of the two purified isozymes catalyzed the hydration of CO2 and the hydrolysis of nitrophenyl acetate esters at rates characteristic of Type C (or high-activity) forms of carbonic anhydrase. 3. Both forms of the erythrocyte enzyme have similar molecular weights (approx 29,000), amino acid composition, sensitivity to acetazolamide, and kinetic properties. 4. The epithelium of the toad's urinary bladder also was found to contain significant amounts of carbonic anhydrase, which appears by isoelectric focusing to be indistinguishable from the enzyme isolated from the erythrocyte. 相似文献
5.
G L?nnerholm 《Journal of applied physiology (Bethesda, Md. : 1985)》1982,52(2):352-356
The distribution of carbonic anhydrase in the human, monkey, and rat lung was studied by the histochemical method of Hansson. High activity of this enzyme was demonstrated in the endothelium of pulmonary capillaries. In the human and the monkey lung enzyme activity was exhibited in the whole circumference of the capillaries, but in the rat enzyme activity is confined to capillary segments having close contact with alveolar epithelium forming the blood-air barrier. Staining was inhibited by 10 microM acetazolamide, but was not affected by 10 microM Cl 13,850, an inactive acetazolamide analogue. The location of carbonic anhydrase in the lung supports the idea that pulmonary carbonic anhydrase promotes CO2 elimination from the blood into the alveolar space. Its possible functions may be to act upon plasma to accelerate the conversion of HCO-3 to CO2 and to facilitate CO2 transport through the lung tissue. 相似文献
6.
Prokaryotic carbonic anhydrases 总被引:2,自引:0,他引:2
Carbonic anhydrases catalyze the reversible hydration of CO(2) [CO(2)+H(2)Oright harpoon over left harpoon HCO(3)(-)+H(+)]. Since the discovery of this zinc (Zn) metalloenzyme in erythrocytes over 65 years ago, carbonic anhydrase has not only been found in virtually all mammalian tissues but is also abundant in plants and green unicellular algae. The enzyme is important to many eukaryotic physiological processes such as respiration, CO(2) transport and photosynthesis. Although ubiquitous in highly evolved organisms from the Eukarya domain, the enzyme has received scant attention in prokaryotes from the Bacteria and Archaea domains and has been purified from only five species since it was first identified in Neisseria sicca in 1963. Recent work has shown that carbonic anhydrase is widespread in metabolically diverse species from both the Archaea and Bacteria domains indicating that the enzyme has a more extensive and fundamental role in prokaryotic biology than previously recognized. A remarkable feature of carbonic anhydrase is the existence of three distinct classes (designated alpha, beta and gamma) that have no significant sequence identity and were invented independently. Thus, the carbonic anhydrase classes are excellent examples of convergent evolution of catalytic function. Genes encoding enzymes from all three classes have been identified in the prokaryotes with the beta and gamma classes predominating. All of the mammalian isozymes (including the 10 human isozymes) belong to the alpha class; however, only nine alpha class carbonic anhydrase genes have thus far been found in the Bacteria domain and none in the Archaea domain. The beta class is comprised of enzymes from the chloroplasts of both monocotyledonous and dicotyledonous plants as well as enzymes from phylogenetically diverse species from the Archaea and Bacteria domains. The only gamma class carbonic anhydrase that has thus far been isolated and characterized is from the methanoarchaeon Methanosarcina thermophila. Interestingly, many prokaryotes contain carbonic anhydrase genes from more than one class; some even contain genes from all three known classes. In addition, some prokaryotes contain multiple genes encoding carbonic anhydrases from the same class. The presence of multiple carbonic anhydrase genes within a species underscores the importance of this enzyme in prokaryotic physiology; however, the role(s) of this enzyme is still largely unknown. Even though most of the information known about the function(s) of carbonic anhydrase primarily relates to its role in cyanobacterial CO(2) fixation, the prokaryotic enzyme has also been shown to function in cyanate degradation and the survival of intracellular pathogens within their host. Investigations into prokaryotic carbonic anhydrase have already led to the identification of a new class (gamma) and future research will undoubtedly reveal novel functions for carbonic anhydrase in prokaryotes. 相似文献
7.
8.
R S Rowlett N J Gargiulo F A Santoli J M Jackson A H Corbett 《The Journal of biological chemistry》1991,266(2):933-941
We have found that many dianionic species, at millimolar concentrations, significantly activate or inhibit the bovine carbonic anhydrase III-catalyzed hydration of CO2. Dianionic species such as HPO2-4 and SO2-3, with pKb values near 7, are activators, whereas weakly basis species such as SO2-4 act as inhibitors. Both activation and inhibition are partial hyperbolic in nature and do not appear to compete with monoanionic linear inhibitors like N-3. Our kinetic data are consistent with a formal mechanism of action for carbonic anhydrase III that is directly analogous to that of carbonic anhydrase II, in which Lys-64 of carbonic anhydrase III can act as an intramolecular H+ transfer group during CO2 hydration. Our data suggest that dianionic inhibitors depress the rate of H+ transfer during turnover by stabilizing the protonated form of Lys-64. We postulate that dianionic activators enhance the rate of a rate-limiting H+ transfer step in the mechanism, probably by acting directly as H+ acceptors. 相似文献
9.
10.
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. 相似文献
11.
M D Hatch 《Analytical biochemistry》1991,192(1):85-89
Apparent carbonic anhydrase activity in leaf extracts, measured as the rate of H+ production associated with the CO2 hydration reaction, varied by as much as 25-fold when the assay buffer was varied. Highest activities were usually recorded in barbitone buffer, with lower activities in imidazole, Tricine, Hepes, Tris, and phosphate buffers. The greatest differences were observed with the enzyme isolated from leaves of the monocotyledonous plants Zea mays (maize) and Triticum aestivum (wheat). Smaller differences were observed with carbonic anhydrase from dicotyledonous species and there was no effect on the erythrocyte enzyme. Leaf carbonic anhydrase activity measured by the mass spectrometric procedure was unaffected by varying the assay buffer. The low activity in certain buffers observed with the former assay system was found to be due to inhibition of the enzyme-catalyzed reaction by higher concentrations of CO2. Carbonic anhydrase from some sources was also strongly inhibited by certain inorganic and organic anions. 相似文献
12.
C K Tu H G Thomas G C Wynns D N Silverman 《The Journal of biological chemistry》1986,261(22):10100-10103
We report three experiments which show that the hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle occurs at a site on the enzyme different than the active site for CO2 hydration. This is in contrast with isozymes I and II of carbonic anhydrase for which the sites of 4-nitrophenyl acetate hydrolysis and CO2 hydration are the same. The pH profile of kcat/Km for hydrolysis of 4-nitrophenyl acetate was roughly described by the ionization of a group with pKa 6.5, whereas kcat/Km for CO2 hydration catalyzed by isozyme III was independent of pH in the range of pH 6.0-8.5. The apoenzyme of carbonic anhydrase III, which is inactive in the catalytic hydration of CO2, was found to be as active in the hydrolysis of 4-nitrophenyl acetate as native isozyme III. Concentrations of N-3 and OCN- and the sulfonamides methazolamide and chlorzolamide which inhibited CO2 hydration did not affect catalytic hydrolysis of 4-nitrophenyl acetate by carbonic anhydrase III. 相似文献
13.
T H Maren 《The Journal of experimental zoology》1977,199(3):317-324
By means of the appropriate isotopes injected into the spiny dogfish, Squalus acanthias, the transfer of all major ions into cerebrospinal fluid (CSF), aqueous humor (A) and endolymph (E) was studied. In addition, the effect of raising pCO2 in sea-water upon HCO3- concentration of these fluids was measured. In the several types of experiments, acetazolamide or methazolamide was used to inhibit completely carbonic anhydrase. The rates of fluid formation and ion transfer in CSF and A were fairly close, but those for E were far slower. The general pattern of ion transport in the three fluids were the same, Na+ (or Na+ + K+ in E) entry greater than Cl - entry, and the difference was HCO3-. The greater rate constants for HCO3-, increase in its entry rate by elevation of pCO2, and inhibition of its appearance by the sulfonamides, show that this is a special case of transport; the ion is formed in secretory cells from gaseous CO2 + OH-. Secretory cells at sites of formation of all the fluids contain both carbonic anhydrase and Na+-K+-ATP-ase, which subserve HCO3- formation and Na+ (or K+) transport. Comparison of these results with studies in mammals show that the vertebrate pattern for secretion of these three fluids is well established in the elasmobranch. 相似文献
14.
C K Tu S R Paranawithana D A Jewell S M Tanhauser P V LoGrasso G C Wynns P J Laipis D N Silverman 《Biochemistry》1990,29(27):6400-6405
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. 相似文献
15.
Because of the very high activity and abundance of human red cell carbonic anhydrase C (carbamate hydrolase, EC 4.2.1.1), it seemed likely that the second isozyme, B, might not be essential for CO2 metabolism. It was then found that physiological concentrations of Cl- inhibited catalysis of CO2 hydration by the B enzyme (but not by type C), suggesting further that type B does not function in vivo as a carbonic anhydrase. The versatility of the catalytic activity of carbonic anhydrase for a number of 'artificial' substrates suggested that enzyme B may be utilized in reactions of intermediary metabolism. A number of hydration, dehydration, decarboxylation, kinase, and phosphatase systems were tested to determine a possible physiological function for the enzyme. Results with eighteen possible substrates were negative and the possibility is discussed that mammalian carbonic anhydrase B is an evolutionary accident. 相似文献
16.
Chemical kinetic and diffusional limitations on bicarbonate reabsorption by the proximal tubule.
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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. 相似文献
17.
A comparison of the mechanisms of CO2 hydration by native and Co2+-substituted carbonic anhydrase II
We have measured the pH dependence of kcat and kcat/Km for CO2 hydration catalyzed by both native Zn2+-and metallo-substituted Co2+-bovine carbonic anhydrase II in the absence of inhibitory ions. For the Zn2+-enzyme, the pKa values controlling kcat and kcat/Km profiles are similar, but for the Co2+-enzyme the values are about 0.6 pH units apart. Computer simulations of a metal-hydroxide mechanism of carbonic anhydrase suggest that the data for both native and Co2+-carbonic anhydrase can be accounted for by the same mechanism of action, if we postulate that the substitution of Co2+ for Zn2+ in the active site causes a separation of about 0.6 pH units in the pKa values of His-64 and the metal-bound water molecule. We have also measured the activation parameters for kcat and kcat/Km for Co2+-substituted carbonic anhydrase II-catalyzed CO2 hydration and have compared these values to those obtained previously for the native Zn2+-enzyme. For kcat and kcat/Km we obtain an enthalpy of activation of 4.4 +/- 0.6 and approximately 0 kcal mol-1, respectively. The corresponding entropies of activation are -18 +/- 2 and -27 +/- 2 cal mol-1 K-1. 相似文献
18.
Grosell M Genz J 《American journal of physiology. Regulatory, integrative and comparative physiology》2006,291(4):R1145-R1156
The gulf toadfish (Opsanus beta) intestine secretes base mainly in the form of HCO3- via apical anion exchange to serve Cl- and water absorption for osmoregulatory purposes. Luminal HCO3- secretion rates measured by pH-stat techniques in Ussing chambers rely on oxidative energy metabolism and are highly temperature sensitive. At 25 degrees C under in vivo-like conditions, secretion rates averaged 0.45 micromol x cm(-2) x h(-1), of which 0.25 micromol x cm(-2) x h(-1) can be accounted for by hydration of endogenous CO2 partly catalyzed by carbonic anhydrase. Complete polarity of secretion of HCO3- and H+ arising from the CO2 hydration reaction is evident from equal rates of luminal HCO3- secretion via anion exchange and basolateral H+ extrusion. When basolateral H+ extrusion is partly inhibited by reduction of serosal pH, luminal HCO3- secretion is reduced. Basolateral H+ secretion occurs in exchange for Na+ via an ethylisopropylamiloride-insensitive mechanism and is ultimately fueled by the activity of the basolateral Na+-K+-ATPase. Fluid absorption by the toadfish intestine to oppose diffusive water loss to the concentrated marine environment is accompanied by a substantial basolateral H+ extrusion, intimately linking osmoregulation and acid-base balance. 相似文献
19.
Transport of cations and anions across forestomach epithelia: conclusions from in vitro studies 总被引:1,自引:0,他引:1
Leonhard-Marek S Stumpff F Martens H 《Animal : an international journal of animal bioscience》2010,4(7):1037-1056
Secretion of saliva as well as absorptive and secretory processes across forestomach epithelia ensures an optimal environment for microbial digestion in the forestomachs. Daily salivary secretion of sodium (Na+) exceeds the amount found in plasma by a factor of 2 to 3, while the secretion of bicarbonate (HCO3-) is 6 to 8 times higher than the amount of HCO3- in the total extracellular space. This implies a need for efficient absorptive mechanisms across forestomach epithelia to allow for an early recycling. While Na+ is absorbed from all forestomachs via Na+/H+ exchange and a non-selective cation channel that shows increased conductance at low concentrations of Mg2+, Ca2+ or H+ in the luminal microclima and at low intracellular Mg2+, HCO3- is secreted by the rumen for the buffering of ingesta but absorbed by the omasum to prevent liberation of CO2 in the abomasum. Fermentation provides short chain fatty acids and ammonia (NH3) that have to be absorbed both to meet nutrient requirements and maintain ruminal homeostasis of pH and osmolarity. The rumen is an important location for the absorption of essential minerals such as Mg2+ from the diet. Other ions can be absorbed, if delivered in sufficient amounts (Ca2+, Pi, K+, Cl- and NH4+). Although the presence of transport mechanisms for these electrolytes has been described earlier, our knowledge about their nature, regulation and crosstalk has increased greatly in the last years. New transport pathways have recently been added to our picture of epithelial transport across rumen and omasum, including an apical non-selective cation conductance, a basolateral anion conductance, an apical H+-ATPase, differently expressed anion exchangers and monocarboxylate transporters. 相似文献
20.
Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa 总被引:3,自引:0,他引:3
The effects of K+, Na+ and ATP on the gastric (H+ + K+)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min-1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min-1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min-1. Additions of 5 mM K+ or Na+, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K+, but not on Na+. Alkaline pH increased the rate of dephosphorylation. K+ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K+ was inhibitory. Below pH 7.0 Na+ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na+ inhibited both activities. The effect of extravesicular pH on transport of H+ was investigated. At pH 6.5 the apparent Km for ATP was 2.7 microM and increased little when K+ was added extravesicularly. At pH 7.5, millimolar concentrations of K+ increased the apparent Km for ATP. Extravesicular K+ and Na+ inhibited the transport of H+. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H+-producing reactions as possible candidates as physiological regulators of (H+ + K+)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO2 and the subsequent formation of H+ and HCO3-. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H+/ATP ratio was about 1 at pH 8.0. When CO2 was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO2. The results indicate a possible co-operation in the production of acid between the H+ + K+-ATPase and a carbonic anhydrase associated with the vesicular membrane. 相似文献