Formation of Short-Chain Fatty Acids from H(2) and CO(2) by a Mixed Culture of Bacteria

The biological utilization of CO(2) and H(2) for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37 degrees C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Active transport of CO(2) and bicarbonate is induced in response to external CO(2) concentration in the green alga Chlorella kessleri

The time-course of induction of CO(2) and HCO(3)- transport has been investigated during the acclimation of high CO(2)-grown Chlorella kessleri cells to dissolved inorganic carbon (DIC)-limited conditions. The rate of photosynthesis of the cells in excess of the uncatalysed supply rate of CO(2) from HCO(3)- was taken as an indicator of HCO(3)- transport, while a stimulation of photosynthesis on the addition of bovine carbonic anhydrase was used as an indicator of CO(2) transport. The maximum rate of photosynthesis (Pmax) was similar for high CO(2)-grown and low CO(2)-grown cells, but the apparent whole cell affinity for DIC and CO(2) of high CO(2)-grown cells was found to be about 30-fold greater than in air-grown cells, which indicates a lower affinity for DIC and CO(2). It was found that HCO(3)- and CO(2) transport were induced in 5.5 h in cells acclimating to air in the light and in the presence and absence of 21% O(2), which indicates that a change in the CO(2)/O(2) ratio in the acclimating medium does not trigger induction of DIC transport. No active DIC transport was detected in high CO(2)-grown cells maintained on high CO(2) for 5.5 h in the presence of 5 mM aminooxyacetate, an aminotransferase inhibitor. These results indicate no involvement of photorespiration in triggering induction. Active DIC transport induction was inhibited in cells treated with 5 microgram ml(-1) cycloheximide, but was unaffected by chloramphenicol treatment, indicating that the induction process requires de novo cytoplasmic protein synthesis. The total DIC concentration eliciting the induction and repression of CO(2) and HCO(3)- transport was higher at pH 7.5 than at pH 6.6. The concentrations of external CO(2) required for the induction and repression of DIC transport were 0 and 120 microM, respectively, and was independent of the pH of the acclimation medium. Prolonged exposure to a critical external CO(2) concentration elicits the induction of DIC transport in C. kessleri.     

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.     

Mechanism of augmented duodenal HCO(3)(-) secretion after elevation of luminal CO(2)

The proximal duodenum is exposed to extreme elevations of P(CO(2)) because of the continuous mixture of secreted HCO(3)(-) with gastric acid. These elevations (up to 80 kPa) are likely to place the mucosal cells under severe acid stress. Furthermore, we hypothesized that, unlike most other cells, the principal source of CO(2) for duodenal epithelial cells is from the lumen. We hence examined the effect of elevated luminal P(CO(2)) on duodenal HCO(3)(-) secretion (DBS) in the rat. DBS was measured by the pH-stat method. For CO(2) challenge, the duodenum was superfused with a high Pco(2) solution. Intracellular pH (pH(i)) of duodenal epithelial cells was measured by ratio microfluorometry. CO(2) challenge, but not isohydric solutions, strongly increased DBS to approximately two times basal for up to 1 h. Preperfusion of the membrane-permeant carbonic anhydrase inhibitor methazolamide, or continuous exposure with indomethacin, fully inhibited CO(2)-augmented DBS. Dimethyl amiloride (0.1 mM), an inhibitor of the basolateral sodium-hydrogen exchanger 1, also inhibited CO(2)-augumented DBS, although S-3226, a specific inhibitor of apical sodium-hydrogen exchanger 3, did not. DIDS, an inhibitor of basolateral sodium-HCO(3)(-) cotransporter, also inhibited CO(2)-augemented DBS, as did the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. CO(2) decreased epithelial cell pH(i), followed by an overshoot after removal of the CO(2) solution. We conclude that luminal CO(2) diffused in the duodenal epithelial cells and was converted to H(+) and HCO(3)(-) by carbonic anhydrase. H(+) initially exited the cell, followed by secretion of HCO(3)(-). Secretion was dependent on a functioning basolateral sodium/proton exchanger, a functioning basolateral HCO(3)(-) uptake mechanism, and submucosal prostaglandin generation and facilitated hydration of CO(2) into HCO(3)(-) and H(+).     

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.     

CO2 induced acute respiratory acidosis and brain tissue intracellular pH: a 31P NMR study in swine

High concentration carbon dioxide (CO(2)) is used to promote pre-slaughter anaesthesia in swine and poultry, as well as short-lasting surgical anaesthesia and euthanasia in laboratory animals. Questions related to animal welfare have been raised, as CO(2) anaesthesia does not set in momentarily. Carbon dioxide promotes anaesthesia by lowering the intracellular pH in the brain cells, but the dynamics of the changes in response to a high concentration of CO(2) is not known. Based on (31)P NMR spectroscopy, we describe CO(2)-induced changes in intracellular pH in the brains of five pigs inhaling 90% CO(2) in ambient air for a period of 60 s, and compare the results to changes in arterial blood pH, P(CO2), O(2) saturation and HCO(3)(-) concentration. The intracellular pH paralleled the arterial pH and P(CO2) during inhalation of CO(2); and it is suggested that the acute reaction to CO(2) inhalation mainly reflects respiratory acidosis, and not metabolic regulation as for example transmembrane fluxes of H(+)/HCO(3)(-). The intracellular pH decreased to approximately 6.7 within the 60 s inhalation period, and the situation was metabolically reversible after the end of CO(2) inhalation. The fast decrease in intracellular pH supports the conclusion that high concentration CO(2) leads to anaesthesia soon after the start of inhalation.     

Aerobically generated CO(2) stored during early exercise.

Previous studies have shown that a metabolic alkalosis develops in the muscle during early exercise. This has been linked to phosphocreatine hydrolysis. Over a similar time frame, the femoral vein blood pH and plasma K(+) and HCO(-)(3) concentrations increase without an increase in PCO(2). Thus CO(2) from aerobic metabolism is converted to HCO(-)(3) rather than being eliminated by the lungs. The purpose of this study was to quantify the increase in early CO(2) stores and the component due to the exercise-induced metabolic alkalosis (E-I Alk). To avoid masking the increase in CO(2) stores by CO(2) released as HCO(-)(3) buffers lactic acid, the transient increase in CO(2) stores was measured only for work rates (WRs) below the lactic acidosis threshold (LAT). The increase in CO(2) stores was evident at the airway starting at approximately 15 s; the increase reached a peak at approximately 60 s and was complete by approximately 3 min of exercise. The increase in CO(2) stores was greater, but the kinetics were unaffected at the higher WR. Three components of the change in aerobically generated CO(2) stores were considered relevant: the carbamate component of the Haldane effect, the increase in CO(2) stores due to increase in tissue PCO(2), and the E-I Alk. The Haldane effect was calculated to be approximately 5%. Physically dissolved CO(2) in the tissues was approximately 30% of the store increase. The remaining E-I Alk CO(2) stores averaged 61 and 68% for 60 and 80% LAT WRs, respectively. The kinetics of O(2) uptake correlated with the time course of the increase in CO(2) stores; the size of the O(2) deficit correlated with the size of the E-I Alk component of the CO(2) stores. We conclude that a major component of the aerobically generated increase in CO(2) stores is the new HCO(-)(3) generated as phosphocreatine is converted to creatine.     

CO2 uptake and fixation by endosymbiotic chemoautotrophs from the bivalve Solemya velum

Chemoautotrophic symbioses, in which endosymbiotic bacteria are the major source of organic carbon for the host, are found in marine habitats where sulfide and oxygen coexist. The purpose of this study was to determine the influence of pH, alternate sulfur sources, and electron acceptors on carbon fixation and to investigate which form(s) of inorganic carbon is taken up and fixed by the gamma-proteobacterial endosymbionts of the protobranch bivalve Solemya velum. Symbiont-enriched suspensions were generated by homogenization of S. velum gills, followed by velocity centrifugation to pellet the symbiont cells. Carbon fixation was measured by incubating the cells with (14)C-labeled dissolved inorganic carbon. When oxygen was present, both sulfide and thiosulfate stimulated carbon fixation; however, elevated levels of either sulfide (>0.5 mM) or oxygen (1 mM) were inhibitory. In the absence of oxygen, nitrate did not enhance carbon fixation rates when sulfide was present. Symbionts fixed carbon most rapidly between pH 7.5 and 8.5. Under optimal pH, sulfide, and oxygen conditions, symbiont carbon fixation rates correlated with the concentrations of extracellular CO(2) and not with HCO(3)(-) concentrations. The half-saturation constant for carbon fixation with respect to extracellular dissolved CO(2) was 28 +/- 3 microM, and the average maximal velocity was 50.8 +/- 7.1 micromol min(-1) g of protein(-1). The reliance of S. velum symbionts on extracellular CO(2) is consistent with their intracellular lifestyle, since HCO(3)(-) utilization would require protein-mediated transport across the bacteriocyte membrane, perisymbiont vacuole membrane, and symbiont outer and inner membranes. The use of CO(2) may be a general trait shared with many symbioses with an intracellular chemoautotrophic partner.     

Evidence for K+-dependent HCO3- utilization in the marine diatom Phaeodactylum tricornutum

Photosynthetic utilization of inorganic carbon in the marine diatom Phaeodactylum tricornutum was investigated by the pH drift experiment, measurement of K(1/2) values of dissolved inorganic carbon (DIC) with pH change, and comparison of the rate of photosynthesis with the rate of the theoretical CO(2) formation from uncatalyzed HCO(3)(-) conversion in the medium. The higher pH compensation point (10.3) and insensitivity of the photosynthetic rate to acetazolamide indicate that the alga has good capacity for direct HCO(3)(-) utilization. The photosynthetic rate reached 150 times the theoretical CO(2) supply rate at 100 micromol L(-1) DIC (pH 9.0) in the presence of 10 mmol L(-1) K(+) and 46 times that in the absence of K(+), indicating that for pH 9.4-grown P. tricornutum, HCO(3)(-) in the medium is taken up through K(+)-dependent and -independent HCO(3)(-) transporters. The K(1/2) (CO(2)) values at pH 8.2 were about 4 times higher than those at pH 9.0, whereas the K(1/2) (HCO(3)(-)) values at pH 8.2 were slightly lower than those at pH 9.0 whether without or with K(+), providing further evidence for the presence of the two HCO(3)(-) transport patterns in this alga. Photosynthetic rate and affinity for HCO(3)(-) in the presence of K(+), respectively, were about 2- and 7-fold higher than those in the absence of K(+), indicating that K(+)-dependent HCO(3)(-) transport is a predominant pattern of HCO(3)(-) cellular uptake in low DIC concentration. However, as P. tricornutum was cultured at pH 7.2 or 8.0, photosynthetic affinities to HCO(3)(-) were not affected by K(+), implying that K(+)-dependent HCO(3)(-) transport is induced when P. tricornutum is cultured at high alkaline pH.     

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.     

On-line monitoring of CO2 production in Lactococcus lactis during physiological pH decrease using membrane inlet mass spectrometry with dynamic pH calibration

Monitoring CO2 production in systems, where pH is changing with time is hampered by the chemical behavior and pH-dependent volatility of this compound. In this article, we present the first method where the concentration and production rate of dissolved CO2 can be monitored directly, continuously, and quantitatively under conditions where pH changes rapidly ( approximately 2 units in 15 min). The method corrects membrane inlet mass spectrometry (MIMS) measurements of CO2 for pH dependency using on-line pH analysis and an experimentally established calibration model. It is valid within the pH range of 3.5 to 7, despite pH-dependent calibration constants that vary in a non-linear fashion with more than a factor of 3 in this interval. The method made it possible to determine the carbon dioxide production during Lactococcus lactis fermentations, where pH drops up to 3 units during the fermentation. The accuracy was approximately 5%. We used the method to investigate the effect of initial extracellular pH on carbon dioxide production during anarobic glucose fermentation by non-growing Lactocoocus lactis and demonstrated that the carbon dioxide production rate increases considerably, when the initial pH was increased from 6 to 6.8.     

Uptake of CO(2) and bicarbonate by intact cells and chloroplasts of Tetraedron minimum and Chlamydomonas noctigama

Using a mass-spectrometric disequilibrium technique, net uptake of HCO(3)(-) and CO(2) during steady-state photosynthesis was studied in whole cells and chloroplasts from the green algae Tetraedron minimum and Chlamydomonas noctigama, grown in air enriched with 5% (v/v) CO(2) (high-CO(2) cells) or in air [0.035% (v/v) CO(2); low-CO(2) cells]. High- and low-CO(2) cells of both species were able to take up CO(2) and HCO(3)(-), with maximum rates being largely unaffected by the growth conditions. High- and low-CO(2) cells of T. minimum showed a pronounced preference for HCO(3)(-) while the rates of net HCO(3)(-) and CO(2) uptake were similar in C. noctigama. The most significant differences between high- and low-CO(2) cells of the two species were the 5- to 6-fold increase in the apparent affinities of net HCO(3)(-) uptake and CO(2) uptake after acclimation to air. The high-affinity uptake systems for inorganic carbon were almost completely induced within 4 h in both algae. Photosynthetically active chloroplasts isolated from both species were also able to take up CO(2) and HCO(3)(-). As in whole cells, HCO(3)(-) was the dominant carbon species taken up by chloroplasts from T. minimum while CO(2) and HCO(3)(-) were taken up at similar rates in plastids from C. noctigama. In addition, high-affinity uptake systems for CO(2) and HCO(3)(-) were detected in chloroplasts preparations after acclimation of the parent cells to air. Isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase revealed K(m) values of 13 and 42 micro M CO(2) for the enzymes from T. minimum and C. noctigama, respectively. These results are consistent with the presence of inducible and energy-dependent high-affinity HCO(3)(-) and CO(2) uptake systems associated with chloroplasts, indicating that these organelles play an important role in the CO(2)-concentrating mechanism.     

Influence of CO(2)-HCO(3) Levels and pH on Growth, Succinate Production, and Enzyme Activities of Anaerobiospirillum succiniciproducens

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO(2)-HCO(3) growth conditions and suggest that there exists a threshold level of CO(2) for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO(2)-HCO(3) growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K') was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO(2)-HCO(3) as a consequence of regulating electron sink metabolism.     

The mechanisms of reductive carboxylation reactions. Carbon dioxide or bicarbonate as substrate of nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase and `malic'' enzyme

1. A simple kinetic method was devised to show whether dissolved CO(2) or HCO(3)- ion is the substrate in enzyme-catalysed carboxylation reactions. 2. The time-course of the reductive carboxylation of 2-oxoglutarate by NADPH, catalysed by isocitrate dehydrogenase, was studied by a sensitive fluorimetric method at pH7.3 and pH6.4, with large concentrations of substrate and coenzyme and small carbon dioxide concentrations. 3. Reaction was initiated by the addition of carbon dioxide in one of three forms: (i) as the dissolved gas in equilibrium with bicarbonate; (ii) as unbuffered bicarbonate solution; (iii) as the gas or as an unbuffered solution of the gas in water. Different progress curves were obtained in the three cases. 4. The results show that dissolved CO(2) is the primary substrate of the enzyme, and that HCO(3)- ion is at best a very poor substrate. The progress curves are in quantitative agreement with this conclusion and with the known rates of the reversible hydration of CO(2) under the conditions of the experiments. The effects of carbonic anhydrase confirm the conclusions. 5. Similar experiments on the reductive carboxylation of pyruvate catalysed by the ;malic' enzyme show that dissolved CO(2) is the primary substrate of this enzyme also. 6. The results are discussed in relation to the mechanisms of these enzymes, and the effects of pH on the reactions. 7. The advantages of the method and its possible applications to other enzymes involved in carbon dioxide metabolism are discussed.     

The role of CO2 in cobalt-catalyzed peroxidations

Augmentation, by CO(2)/HCO(3)(-), of Co(II)-catalyzed peroxidations was explored to clarify whether the rate enhancement was due to CO(2) or to HCO(3)(-). The rate of oxidation of NADH by Co(II) plus H(2)O(2), in Tris or phosphate, was markedly enhanced by CO(2)/HCO(3)(-). Phosphate was seen to inhibit the Co(II)-catalyzed peroxidation, probably due to its sequestration of the Co(II). When CO(2) was used, there was an initial burst of NADH oxidation followed by a slower linear rate. The presence of carbonic anhydrase eliminated this initial burst; establishing that CO(2) rather than HCO(3)(-) was the species responsible for the observed rate enhancements. Both kinetic and spectral data indicated that Co(II) was converted by H(2)O(2) into a less active form from which Co(II) could be regenerated. This less active form absorbed in both the UV and visible regions, and is assumed to be a peroxy bridged binuclear complex. The rate of formation of this absorbing form was increased by HCO(3)(-)/CO(2). A minimal mechanism consistent with these observations is proposed.     

Examination of the role of Gln-158 in the mechanism of CO(2) hydration catalyzed by beta-carbonic anhydrase from Arabidopsis thaliana

We have cloned and overexpressed a variant of Arabidopsis thaliana beta-carbonic anhydrase (Q158A) that deletes the functional equivalent of the backbone amide NH of Thr-199 in human alpha-carbonic anhydrase II. The latter residue is hypothesized to be important in catalyzing the rate of CO(2)(-) HCO (3)(-) interconversion in alpha-carbonic anhydrase but this hypothesis is not directly testable in that enzyme. Kinetic studies of a variant of the functionally equivalent residue in A. thaliana beta-carbonic anhydrase provide direct evidence for the role of this residue in beta-carbonic anhydrase. Namely, the mutation of Gln-158 to Ala results in a significant decrease in the maximal k(cat) (33% of wild type) at steady state and the maximal rate of CO(2)(-) HCO(2)(-) exchange at chemical equilibrium as measured by R(1)/[E] (7% of wild type), while leaving the maximal rate of H(+) transfer, as measured by k(cat) at steady state, or R(H(2)O)) at chemical equilibrium, largely unaffected.     

Novel role of extracellular carbon dioxide in lymphocyte proliferation in culture

CO(2)/HCO(3)(-) buffering system is indispensable to maintain the pH of culture media for long-term cell culture. Now-a-days, the zwiterionic hydrogen buffer HEPES is widely used as an additional buffer in the commonly used culture media. There are reports on the successful use of HEPES-buffered media, under CO(2)/HCO(3)(-) free conditions, for long-term cell cultures. However, still CO(2)/HCO(3)(-) buffering system is widely used. We aimed at investigating the reason for this. We found that lymphocytes proliferate in response to concanavalin A only in HCO(3)(-)-buffered medium in the presence of 5% CO(2), but not in the HEPES-buffered medium in the absence of CO(2). However, lymphocyte proliferation was observed in HEPES-buffered medium in the presence of 5% CO(2) and in the absence of HCO(3)(-). On the other hand, a low level proliferation was observed in HEPES-buffered medium supplemented with HCO(3)(-) in the absence of CO(2). Supplementation of the culture medium with TCA cycle intermediates and the precursors for the salvage pathway of nucleotide synthesis did not support the lymphocyte proliferation at all. Based on these findings and other reports, we suggest that extracellular CO(2) plays a novel role in cell proliferation.     

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (II. Evidence for Induction in Response to External CO2 Concentration)

The critical species and concentrations of dissolved inorganic carbon (DIC) required for the induction of DIC transport during adaptation to low CO2 were determined for the green alga Chlorella ellipsoidea. The concentration of dissolved CO2 needed for the induction of both CO2 and HCO3- transport was independent of pH during adaptation, whereas the total DIC concentration required increased at alkaline pH. At pH 7.5, the minimum equilibrium DIC concentration at which high CO2 characteristics were maintained, i.e. transport was repressed, was 2100 [mu]M, whereas the maximum equilibrium DIC concentration below which DIC transport was fully induced (DICIND) was 500 [mu]M. Intracellular DIC concentration during adaptation to DICIND decreased temporarily after 2 h to 60% of the maximum level but recovered after 3 h of adaptation. After 3 h of adaptation to DICIND, cells exhibited maximum O2 evolution rate at DICIND. When cells partially adapted to DICIND were returned to high CO2, there was an immediate halt to the induction of transport and a gradual decrease in transport capacity over 23 h. The capacity for the induction of transport was unaffected by the absence of light. These results indicate that changes in the internal DIC pool during adaptation to low CO2 do not trigger the induction of DIC transport and that the induction is not light dependent. Induction of DIC transport in C. ellipsoidea appears to occur in response to the continuous exposure of cells to a critical CO2 concentration in the external medium.     

Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCO(2) or pH.

Accumulation of CO(2) in animal cell cultures can be a significant problem during scale-up and production of recombinant glycoprotein biopharmaceuticals. By examining the cell-surface polysialic acid (PSA) content, we show that elevated CO(2) partial pressure (pCO(2)) can alter protein glycosylation. PSA is a high-molecular-weight polymer attached to several complex N-linked oligosaccharides on the neural cell adhesion molecule (NCAM), so that small changes in either core glycosylation or in polysialylation are amplified and easily measured. Flow-cytometric analysis revealed that PSA levels on Chinese hamster ovary (CHO) cells decrease with increasing pCO(2) in a dose-dependent manner, independent of any change in NCAM content. The results are highly pH-dependent, with a greater decrease in PSA at higher pH. By manipulating medium pH and pCO(2), we showed that decreases in PSA correlate well with bicarbonate concentration ([HCO(3)(-)]). In fact, it was possible to offset a 60% decrease in PSA content at 120 mm Hg pCO(2) by decreasing the pH from 7.3 to 6.9, such that [HCO(3)(-)] was lowered to that of control (38 mm Hg pCO(2)). When the increase in osmolality associated with elevated [HCO(3)(-)] was offset by decreasing the basal medium [NaCl], elevated [HCO(3)(-)] still caused a decrease in PSA, although less extensive than without osmolality control. By increasing [NaCl], we show that hyperosmolality alone decreases PSA content, but to a lesser extent than for the same osmolality increase due to elevated [NaHCO(3)]. In conclusion, we demonstrate the importance of pH and pCO(2) interactions, and show that [HCO(3)(-)] and osmolality can account for the observed changes in PSA content over a wide range of pH and pCO(2) values.