N-carboxymethanofuran (carbamate) formation from methanofuran and CO2 in methanogenic archaea. Thermodynamics and kinetics of the spontaneous reaction.

N-carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 to methane in methanogenic archaea. The reaction proceeds spontaneously. We address here the question whether the rate of spontaneous carbamate formation is high enough to account for the observed rate of methanogenesis from CO2. The rates of carbamate formation (v1) and cleavage (v2) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k1* of carbamate formation from 'MFR'(MFR + MFRH+) and 'CO2' (CO2 + H2CO3 + HCO3-+ CO32-) was found to be 7 M-1.s-1 (v1 = k1* ['MFR'] ['CO2']) while the pseudo first order rate constant k2* of carbamate cleavage was 12 s-1 (v2 = k2* [carbamate]). The equilibrium constant K* = k1*/k2* = [carbamate]/['MFR']['CO2'] was 0.6 M-1 at pH 7.0 corresponding to a free energy change DeltaG degrees ' of + 1.3 kJ.mol-1. The pH and temperature dependence of k1*, of k2* and of K* were determined. From the second order rate constant k1* it was calculated that under physiological conditions the rate of spontaneous carbamate formation is of the same order as the maximal rate of methane formation and as the rate of spontaneous CO2 formation from HCO3- in methanogenic archaea, the latter being important as CO2 is mainly present as HCO3- which has to be converted to CO2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO2. Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosarcina barkeri and Methanobacterium thermoautotrophicum or by purified formylmethanofuran dehydrogenase which catalyzes the reduction of N-carboxymethanofuran to N-formylmethanofuran. From the concentrations of 'CO2' and of 'MFR' determined by 1D-NMR spectroscopy and the pKa of H2CO3 and of MFRH+ the concentrations of CO2 and of MFR were obtained, allowing to calculate k1 (v1 = k1 [MFR] [CO2]). The second order rate constant k1 was found to be approximately 1000 M-1 x s-1 at 300 K and pH values between 7.0 and 8. 0 which is in the order of k1 values determined for other carbamate forming reactions by stopped flow.     

Hepatic urea synthesis and pH regulation. Role of CO2, HCO3-, pH and the activity of carbonic anhydrase

In isolated perfused rat liver, urea synthesis from ammonium ions was dependent on extracellular HCO3- and CO2 concentrations when the HCO3-/CO2 ratio in the influent perfusate was constant (pH 7.4). Urea synthesis was half-maximal at HCO3- = 4 mM, CO2 = 0.19 mM and was maximal at HCO3- and CO2 concentrations above 20 mM and 0.96 mM, respectively. At physiological HCO3- (25 mM) and CO2 (1.2 mM) concentrations in the influent perfusate, acetazolamide, the inhibitor of carbonic anhydrase, inhibited urea synthesis from ammonium ions (1 mM) by 50-60% and led to a 70% decrease in citrulline tissue levels. Acetazolamide concentrations required for maximal inhibition of urea synthesis were 0.01-0.1 mM. At subphysiological HCO3- and CO2 concentrations, inhibition of urea synthesis by acetazolamide was increased up to 90%. Inhibition of urea synthesis by acetazolamide was fully overcome in the presence of unphysiologically high HCO3- and CO2 concentrations, indicating that the inhibitory effect of acetazolamide is due to an inhibition of carbonic-anhydrase-catalyzed HCO3- supply for carbamoyl-phosphate synthetase, which can be bypassed when the uncatalyzed intramitochondrial HCO3- formation from portal CO2 is stimulated in the presence of high portal CO2 concentrations. With respect to HCO3- supply of mitochondrial carbamoyl-phosphate synthetase, urea synthesis can be separated into a carbonic-anhydrase-dependent (sensitive to acetazolamide at 0.5 mM) and a carbonic-anhydrase-independent (insensitive to acetazolamide) portion. Carbonic-anhydrase-independent urea synthesis linearly increased with the portal 'total CO2 addition' (which was experimentally determined to be CO2 addition plus 0.036 HCO3- addition) and was independent of the perfusate pH. At a constant 'total CO2 addition', carbonic-anhydrase-dependent urea synthesis was strongly affected by perfusate pH and increased about threefold when the perfusate pH was raised from 6.9 to 7.8. It is concluded that the pH dependent regulation of urea synthesis is predominantly due to mitochondrial carbonic anhydrase-catalyzed HCO3- supply for carbamoyl phosphate synthesis, whereas there is no control of urea synthesis by pH at the level of the five enzymes of the urea cycle. Because HCO3- provision for carbamoyl phosphate synthetase increases with increasing portal CO2 concentrations even in the absence of carbonic anhydrase activity, susceptibility of ureogenesis to pH decreases with increasing portal CO2 concentrations. This may explain the different response of urea synthesis to chronic metabolic and chronic respiratory acidosis in vivo.     

A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria

Carboxysomes are proteinaceous biochemical compartments that constitute the enzymatic "back end" of the cyanobacterial CO2-concentrating mechanism. These protein-bound organelles catalyze HCO3- dehydration and photosynthetic CO2 fixation. In Synechocystis sp. strain PCC6803 these reactions involve the beta-class carbonic anhydrase (CA), CcaA, and Form 1B ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The surrounding shell is thought to be composed of proteins encoded by the ccmKLMN operon, although little is known about how structural and catalytic proteins integrate to form a functional carboxysome. Using biochemical activity assays and molecular approaches we have identified a catalytic, multiprotein HCO3- dehydration complex (BDC) associated with the protein shell of Synechocystis carboxysomes. The complex was minimally composed of a CcmM73 trimer, CcaA dimer, and CcmN. Larger native complexes also contained RbcL, RbcS, and two or three immunologically identified smaller forms of CcmM (62, 52, and 36 kDa). Yeast two-hybrid analyses indicated that the BDC was associated with the carboxysome shell through CcmM73-specific protein interactions with CcmK and CcmL. Protein interactions between CcmM73 and CcaA, CcmM73 and CcmN, or CcmM73 and itself required the N-terminal gamma-CA-like domain of CcmM73. The specificity of the CcmM73-CcaA interaction provided both a mechanism to integrate CcaA into the fabric of the carboxysome shell and a means to recruit this enzyme to the BDC during carboxysome biogenesis. Functionally, CcaA was the catalytic core of the BDC. CcmM73 bound H14CO3- but was unable to catalyze HCO3- dehydration, suggesting that it may potentially regulate BDC activity.     

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.     

Basolateral membrane Na/base cotransport is dependent on CO2/HCO3 in the proximal convoluted tubule

The mechanism of basolateral membrane base transport was examined in the in vitro microperfused rabbit proximal convoluted tubule (PCT) in the absence and presence of ambient CO2/HCO3- by means of the microfluorometric measurement of cell pH. The buffer capacity of the cells measured using rapid NH3 washout was 42.8 +/- 5.6 mmol.liter-1.pH unit-1 in the absence and 84.6 +/- 7.3 mmol.liter-1.pH unit-1 in the presence of CO2/HCO3-. In the presence of CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.30 pH units and lowering peritubular Na from 147 to 0 mM acidified the cell by 0.25 pH units. Both effects were inhibited by peritubular 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). In the absence of exogenous CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.25 pH units and lowering peritubular Na from 147 to 0 mM decreased cell pH by 0.20 pH units. Lowering bath pH from 7.4 to 6.8 induced a proton flux of 643 +/- 51 pmol.mm-1.min-1 in the presence of exogenous CO2/HCO3- and 223 +/- 27 pmol.mm-1.min-1 in its absence. Lowering bath Na from 147 to 0 mM induced proton fluxes of 596 +/- 77 pmol.mm-1.min-1 in its absence. The cell acidification induced by lowering bath pH or bath Na in the absence of CO2/HCO3- was inhibited by peritubular SITS or by acetazolamide, whereas peritubular amiloride had no effect. In the absence of exogenous CO2/HCO3-, cyanide blocked the cell acidification induced by bath Na removal, but was without effect in the presence of exogenous CO2/HCO3-. We reached the following conclusions. (a) The basolateral Na/base n greater than 1 cotransporter in the rabbit PCT has an absolute requirement for CO2/HCO3-. (b) In spite of this CO2 dependence, in the absence of exogenous CO2/HCO3-, metabolically produced CO2/HCO3- is sufficient to keep the transporter running at 30% of its control rate in the presence of ambient CO2/HCO3-. (c) There is no apparent amiloride-sensitive Na/H antiporter on the basolateral membrane of the rabbit PCT.     

Ouabain-sensitive bicarbonate secretion and acid absorption by the marine teleost fish intestine play a role in osmoregulation

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.     

CO2 enhanced peroxidase activity of SOD1: the effects of pH

At pH 7.4, CO2, rather than HCO3-, markedly enhances the oxidation of diverse substrates by SOD1 plus H2O2. Since the concentration of CO2 would fall with rising pH in HCO3- buffers, it was of interest to explore the effects of pH on the peroxidase activity of SOD1 in the presence and in the absence of HCO3-. The rate of NADPH peroxidation in the HCO3- buffer was minimally affected by pH in the range of 8-10.5; in a pyrophosphate buffer, the rate increased markedly, such that at pH 10.5 the rates in the two buffers were nearly identical. Similar results were obtained when urate was used as the peroxidizeable substrate. These results are explicable on the basis of an increase in the rate with pH due to the ionization of H2O2 to the effective HO2- coupled with a decrease in [CO2] due to the ionizations of H2CO3, which displaces the hydration equilibrium to the right. These two opposing effects counteract in the HCO3(-)-buffered reaction mixtures; in the pyrophosphate buffer, only the effect of increasing [H02-] was seen.     

Dynamic 13C NMR investigations of substrate interaction and catalysis by cobalt(II) human carbonic anhydrase I

Using 13C NMR spectroscopy, we have further investigated the binding of HCO3- in the active site of an artificial form of human carbonic anhydrase I in which the native zinc is replaced by Co(II). The Co(II) enzyme, unlike all other metal-substituted derivatives, has functional properties closely similar to those of the native zinc enzyme. By measuring the spin-lattice relaxation rate and the line width for both the CO2 and HCO3- at two field strengths, we have determined both the paramagnetic effects that reflect substrate binding and the exchange effects due to catalysis at chemical equilibrium. The following are the results at 14 degrees C and pH 6.3 (1) HCO3- is bound in the active site of the catalytically competent enzyme with the 13C of the HCO3- located 3.22 +/- 0.02 A from the Co(II); (2) the apparent equilibrium dissociation constant for the bound HCO3- is 7.6 +/- 1.5 mM, determined by using the paramagnetic effects on the line widths, and 10 +/- 2 mM, determined by using the exchange effects; (3) the lifetime of HCO3- bound to the metal is (4.4 +/- 0.4) X 10(-5) s; (4) the overall catalyzed CO2 in equilibrium HCO3- exchange rate constant of the Co(II) enzyme is (9.6 +/- 0.4) X 10(3) s-1; (5) the electron spin relaxation time of the Co(II), determined by using paramagnetic effects on the bound HCO3-, is (1.1 +/- 0.1) X 10(-11) s. The data did not provide any direct information on the binding of CO2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Considerations for osmolality measurement under elevated pCO(2): comparison of vapor pressure and freezing point osmometry

Osmolality increases with pCO(2) in bioreactors with pH control, and it has been shown that osmolality compensation by decreasing the basal NaCl concentration partially mitigates the adverse effects of elevated pCO(2) on animal cell growth, protein production, and glycosylation. Thus, measurement of osmolality is important for a complete characterization of the culture environment under elevated pCO(2). However, osmolality measurement may be compromised by CO(2) evolution. Freezing point depression and vapor pressure depression osmometry were directly compared for the measurement of osmolality in samples at elevated pCO(2) (up to 250 mmHg) and at a variety of pH values (6.7-7.5). More extensive degassing may be expected with the vapor pressure osmometer due to the smaller sample volume and larger surface area employed. However, both types of osmometer yielded similar results for all pCO(2) and pH values studied. Moreover, the measured values agreed with osmolality values calculated using a semi-empirical model. Further analysis showed that, while sample degassing may result in a large decrease in pCO(2), there is little associated decrease in osmolality. The great majority of total CO(2) in solution is present as bicarbonate (HCO(3)(-)). Although a small amount of HCO(3)(-) is converted to CO(2) to compensate for CO(2) evolution, further depletion of HCO(3)(-) is inhibited by the associated increase in medium pH and by the need for HCO(3)(-) to maintain charge neutrality in solution. This explanation is consistent with the observed similarity in osmolality values for the two types of osmometer. It was also observed that osmolality did not change in samples that were frozen at -20 degrees C for up to 1 year.     

Plasma membrane Cl-/HCO3-exchangers: Structure,mechanism and physiology

Anion exchanger proteins facilitate the exchange of bicarbonate for chloride across the plasma membrane. When bicarbonate combines with a proton it undergoes conversion into CO2, either spontaneously, or catalyzed by carbonic anhydrase enzymes. The CO2/HCO3- equilibrium is the body’s central pH buffering system. Rapid bicarbonate transport across the plasma membrane is essential to maintain cellular and whole body pH, to dispose of metabolic waste CO2, and to control fluid movement in our bodies. Cl-/HCO3- exchangers are found in two distinct gene families: SLC4A and SLC26A. Differences in the tissue distribution, electrogenicity, and regulation of the specific anion exchanger proteins allow for precise regulation of bicarbonate transport throughout the human body. This review provides a look into the structural and functional features that make this family of proteins unique, as well as the physiological significance of the different anion exchangers.     

Electrogenic Na-independent HCO3 transport in OK cells.

We have shown previously that OK cells recover from an acid load in a medium nominally CO2-free by extruding H via a Na/H exchanger and a passive H-conductive pathway. In this work, the regulation of cell pH (pHi) was studied after addition or withdrawal of CO2/HCO3 (5% CO2, 95 mM HCO3, pH = 8) using the fluoroprobe BCECF. In the presence of Na and amiloride to inhibit Na/H exchange, the recovery of pHi after CO2 entry and CO2 exit were found to depend in part on HCO3 entry and exit, respectively. Efflux of H per se also contributed to restoring pHi after CO2 addition, whereas H influx may have played a smaller role to normalize pHi after CO2 removal. DIDS, 0.5 mM, significantly inhibited both recovery phases of pHi. Removal of Na failed to inhibit the recovery of pHi after CO2 addition and removal. Cl removal also failed to inhibit pHi recovery after CO2 removal. Cell depolarization in the presence of Na moderately stimulated the pHi recovery rate after CO2 addition whereas it markedly inhibited the normalization of pHi after CO2 removal. Cell depolarization in the absence of sodium had only a slight effect to increase pHi recovery after CO2 addition but markedly prevented the pHi recovery after CO2 removal. These results indicate that OK cells lack Na or Cl-dependent HCO3 transport systems. The OK cell possesses a novel stilbene-sensitive electrogenic HCO3 transport system that is involved in the regulation of cell pH.     

pH and buffering in the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay

The HCO3/CO3(2-) buffer used in the bicinchoninic acid (BCA) protein assay has only weak buffering capacity at the recommended pH (11.25). Consequently the assay is rather sensitive to interference from effectively acid or alkaline samples, particularly in the micro method. Adjustment of pH in these alkaline solutions of high [Na+] is complicated by Na+ errors on the pH electrode. Hence it is recommended to prepare the buffers from known amounts of NaHCO3 and Na2CO3, and to reduce the pH to around 10.7; this offers much better buffering capacity with only a limited reduction in color development.     

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H(+) absorption from the lumen. We thus examined the effect of inhibition of Na(+)/H(+) exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO(2) diffusion and HCO(3)(-) secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO(2) solution or pH 2.2 low CO(2) solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR(inh)-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO(2)]). Luminal high CO(2) solution increased CO(2) absorption and HCO(3)(-) secretion, accompanied by PV acidification and PV Pco(2) increase. During CO(2) challenge, CFTR(inh)-172 induced HCO(3)(-) absorption, while inhibiting PV acidification. S3226 reversed CFTR(inh)-associated HCO(3)(-) absorption. Luminal pH 2.2 challenge increased H(+) and CO(2) absorption and acidified the PV, inhibited by CFTR(inh)-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO(3)(-) secretion to absorption and inhibited PV acidification during CO(2) challenge, suggesting that HCO(3)(-) secretion helps facilitate CO(2)/H(+) absorption. Furthermore, CFTR inhibition prevented CO(2)-induced cellular acidification reversed by S3226. Reversal of increased HCO(3)(-) loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H(+) available to neutralize luminal HCO(3)(-) with consequent CO(2) absorption. NHE3, by secreting H(+) into the luminal microclimate, facilitates net transmucosal HCO(3)(-) absorption with a mechanism similar to proximal tubular HCO(3)(-) absorption.     

The activation of ribulose-1,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications.

Ribulose-1,5-bisphosphate carboxylase was activated by incubation with CO2 and Mg2++, and inactivated upon removal of CO2 and Mg2+ by gel filtration. The activation process involved CO2 rather than HCO3-. The activity of the enzyme was dependent upon the preincubation concentrations of CO2 and Mg2+ and upon the preincubation pH, indicating that activation involved the reversible formation of an equilibrium complex of enzyme-CO2-Mg. The initial rate of activation was linearly dependent upon the CO2 concentration but independent of the Mg2+ concentration. Kinetic analyses indicated that the enzyme reacted first with CO2 in a rate-determining and reversible step, followed by a rapid reaction with Mg2+ to form an active ternary complex (see eq 1 in text). The pseudo-first order rate constant, kobsd, for the activation process at constant pH was derived: kobsd=k1[CO2] + (k2k4/k3[Mg2+]). Experimentally, kobsd was shown to be linearly dependent upon the CO2 concentration and inversely dependent upon the Mg2+ concentration. The activity of the enzyme after preincubation to equilibrium at constant concentrations of CO2 and Mg2+ increased as the preincubation pH was raised, indicating that CO2 reacted with an enzyme group whose pK was distinctly alkaline. It is proposed that the activation of ribulose-1, 5-biphosphate carboxylane involves the formation of a carbamate.     

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.     

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-.     

An investigation of the nature of Bohr,Root, and haldane effects inOctopus dofleini hemocyanin

1. The pH dependence of Octopus dofleini hemocyanin oxygenation is so great that below pH 7.0 the molecule does not become fully oxygenated, even in pure O2 at 1 atm pressure. However, the curves describing percent oxygenation as a function of PO2 appear to be gradually increasing in oxygen saturation, rather than leveling out at less than full saturation. Hill plots indicate that at pH 6.6 and below the molecule is stabilized in its low affinity conformation. Thus, the low saturation of this hemocyanin in air is due to the very large Bohr shift, and not to the disabling of one or more functionally distinct O2 binding sites on the native molecule. 2. Experiments in which pH was monitored continuously while oxygenation was manipulated in the presence of CO2 provide no evidence of O2 linked binding of CO2. While CO2 does influence O2 affinity independently of pH, its effect may be due to high levels of HCO3- and CO3-, rather than molecular CO2, and it may entail a lowering of the activities of the allosteric effectors Mg2+ and Ca2+.     

C1/HCO3 exchange in human placental brush border membrane vesicles

Membrane transport pathways for transplacental transfer of CO2/HCO3 were investigated by assessing the possible presence of a Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. Cl/HCO3 exchange was tested for in preparations of purified brush border membrane vesicles by 36Cl tracer flux measurements and determinations of acridine orange fluorescence changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake to levels approximately 2-fold greater than observed at equilibrium. Maneuvers designed to offset the development of ion gradient-induced diffusion potentials (valinomycin, Ko = Ki) significantly reduced HCO3- gradient-driven Cl- uptake but concentrative accumulation of Cl- persisted. Early time point determinations performed in the presumed absence of membrane potential suggests the reduced level of HCO3- gradient-driven Cl- uptake resulted from a more rapid dissipation of the HCO3- concentration gradient. Concentrative accumulation of Cl- was not observed in the presence of a pH gradient alone under 100% N2, suggesting a preference of HCO3- over OH- as a substrate for transport. As monitored by acridine orange fluorescence the Cl- gradient-dependent collapse of an imposed pH gradient (pHo 8.5/pHi 6) was accelerated in the presence of CO2/HCO3 when compared with its absence, indicating coupling of HCO3- influx to Cl- efflux. Increasing concentrations of the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were observed to cause a stepwise reduction in HCO3- gradient-driven Cl- uptake (I50 approximately 25 microM) further suggesting the presence of a Cl/HCO3 exchange mechanism. The results of this study provide evidence for a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. The identification of an ion-coupled HCO3- transport pathway in placental epithelia may suggest functional roles in mediating transplacental transfer of CO2 as well as maintenance of fetal acid/base balance.     

Bicarbonate is a recycling substrate for cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.