Intracellular Carbonic Anhydrase Activity Research Articles

Evidencing the role of carbonic anhydrase in the formation of carbonate minerals by bacterial strains isolated from extreme environments in Qatar

Calcium carbonate, one of the most abundant minerals in the geological records is considered as primary source of the carbon reservoir. The role of microorganisms in the biotic precipitation of calcium carbonate has been extensively investigated, especially at extreme life conditions. In Qatar, Sabkhas which are microbial ecosystems housing biomineralizing bacteria, have been carefully studied as unique sites of microbial dolomite formation. Dolomite (CaMg(CO3)2 is an important carbonate mineral forming oil reservoir rocks; however, dolomite is rarely formed in modern environments. The enzyme carbonic anhydrase is present in many living organisms, performs interconversion between CO2 and the bicarbonate ion. Thus, carbonic anhydrase is expected to accelerate both carbonate rock dissolution and CO2 uptake at the same time, serving as carbonite source to carbonites-forming bacteria. This study gathered cross-linked data on the potential role of the carbonic anhydrase excreted by mineral-forming bacteria, isolated from two different extreme environments in Qatar. Dohat Faishakh Sabkha, is a hypersaline coastal Sabkha, from where various strains of the bacterium Virgibacillus were isolated. Virgibacillus can -not only-mediate carbonate mineral formation, but also contributes to magnesium incorporation into the carbonate minerals, leading to the formation of high magnesium calcite. The latter is considered as precursor for dolomite formation. In addition, bacterial strains isolated from marine sediments, surrounding coral reef in Qatar sea, would provide additional knowledge on the role of carbonic anhydrase in mineral formation. Here, the quantification of the two mostly described activities of carbonic anhydrase; esterase and hydration reactions were performed. Mineral-forming strains were shown to exhibit high activities as opposed to the non-forming minerals, which confirms the relation between the presence of active carbonic anhydrase combined with elevated metabolic activity and the biomineralizing potential of the bacterial strains. The highest specific intracellular carbonic anhydrase activity; as both esterase and hydration (i.e., 66 ± 3 and 583000 ± 39000 WAU/108 cells respectively), was evidenced in mineral-forming strains as opposed to non-mineral forming strains (i.e., 6 ±. 0.5 and 1223 ± 61 WAU/108cells) respectively. These findings would contribute to the understanding of the mechanism of microbially mediated carbonate precipitation. This role may be both in capturing CO2 as source of carbonate, and partial solubilization of the formed minerals allowing incorporation of Mg instead of calcium, before catalyzing again the formation of more deposition of carbonates.

Induction of vitamin B12 to purify biogas slurry and upgrade biogas using co-culture of microalgae and fungi.

Different gradient concentrations of vitamin B12 (0, 10, 100, 1,000ngL-1 ) were used in the symbiosis system (Chlorella vulgaris-Ganoderma lucidum or Chlorella vulgaris-Pleurotus ostreatus) to assess their effect on simultaneous purification of biogas and removal of nutrients in biogas slurry using co-culture of microalgae and fungi. When B12 was added to the symbiosis system, biomass growth, intracellular carbonic anhydrase activity (CA), chlorophyll a content (CHL-a), photosynthetic characteristics of the two cultivation system, and removal efficiency of nutrients in biogas slurry and CO2 in biogas were significantly higher than those in the control group. The optimal concentration of B12 was determined to be 100ngL-1 considering the removal efficiency of nutrients and CO2 . Maximum mean chemical oxygen demand (COD), total nitrogen (TN), total phosphorus (TP), and CO2 removal efficiencies were 75.98±6.26%, 78.46±6.21%, 80.21±6.83% and 61.08±5.21% in Chlorella vulgaris-Ganoderma lucidum, respectively. This study showed the potential of microalgae and fungi symbiosis system with B12 addition for nutrient removal and biogas upgrading. PRACTITIONER POINTS: Vitamin B12 had positive effects on algal-fungal pellets growth. The optimal vitamin B12 concentration was 100ngL-1 . The highest CO2 remove rate was 61.08% by G.lucidum/C.vulgaris pellets. Vitamin B12 significantly improved photosynthetic performance of pellets.

Effect of GR24 concentrations on biogas upgrade and nutrient removal by microalgae-based technology

Freshwater microalgae Chlorella vulgaris was cultured and induced with strigolactone (GR24) to simultaneously eliminate nutrients in biogas slurry and purify biogas. Treatment with 10−7 M GR24 yielded maximum growth rate and mean daily productivity for algae at 0.187 ± 0.06 d−1 and 0.097 ± 0.008 g L−1 d−1, respectively. Results from chlorophyll fluorescence transients method demonstrated that moderate concentration of GR24 could enhance the photosynthetic performance of microalgae. In addition, GR24 affected intracellular carbonic anhydrase activity and chlorophyll-a content. Maximum chemical oxygen demand, total nitrogen, and CO2 removal efficiencies were 78.62 ± 2.36%, 76.47 ± 1.53% and 64.05 ± 1.15% with 10−7 M GR24 induction, respectively. Further, highest total phosphorus removal efficiency (80.27 ± 1.93%) was observed at 10−9 M. The optimal GR24 concentration range was determined to be between 10−9 and 10−7 M in consideration with nutrient and CO2 removal efficiencies.

Overexpression of Chlamydomonas reinhardtii LCIA (CrLCIA) gene increases growth of Nannochloropsis salina CCMP1776

Most aquatic photosynthetic organisms have developed inorganic carbon-concentrating mechanisms (CCMs) to compensate for the kinetic constraints of CO2 concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which functions in the first steps of carbon fixation. CCMs, which are widespread in various types of algae, evolved independently among algal lineages and accordingly play a fundamental role in algal photosynthesis, metabolism, growth, and biomass production. Nannochloropsis, a marine eustigmatophycean microalga, is a candidate organism for biofuel production due to its high lipid content; however, inorganic carbon (Ci) availability in Nannochloropsis cells is not sufficient for complete carbon fixation due to inherently weak CCM machinery, including a CO2-leaking HCO3− pump. CrLCIA, a member of the formate-nitrite transporter family, functions as a HCO3− transporter in Chlamydomonas reinhardti. Thus, in this study, we overexpressed the CrLCIA gene heterologously in N. salina CCMP1776 in an attempt to reinforce its bicarbonate transport activity. CrLCIA expression in N. salina increased intracellular Ci and carbonic anhydrase (CA) activity, resulting in increased growth (30%) and biomass (2-fold). These results indicate that constitutively expressed CrLCIA leads to increased Ci uptake and CA activity, contributing to high availability of CO2 for photosynthesis under low CO2 conditions. The total fatty acid (FA) per cell mass in the transgenic lines was similar to that of wild-type Nannochloropsis cells, indicating that total FA productivity in the transgenic lines is increased approximately 2-fold. These findings will be useful to improve the CCM function for high biomass and lipid production using genetic modification tools in Nannochloropsis species, contributing to improved biodiesel production.

CO2 permeability and carbonic anhydrase activity of rat cardiomyocytes.

To determine the CO2 permeability (PCO2 ) of plasma membranes of cardiomyocytes. These cells were chosen because heart possesses the highest rate of O2 consumption/CO2 production in the body. Cardiomyocytes were isolated from rat hearts using the Langendorff technique. Cardiomyocyte suspensions exhibited a vitality of 2-14% and were studied by the previously described mass spectrometric 18 O-exchange technique deriving PCO2 . We showed by mass spectrometry and by carbonic anhydrase (CA) staining that non-vital cardiomyocytes are free of CA and thus do not contribute to the mass spectrometric signal, which is determined exclusively by the fully functional vital cardiomyocytes. Lysed cardiomyocytes yielded an intracellular CA activity for vital cells of 5070; that is, the rate of CO2 hydration inside the cell is accelerated 5071-fold. Using this number, analyses of the mass spectrometric recordings from cardiomyocyte suspensions yield a PCO2 of 0.10 cm s-1 (SD ± 0.06, n = 15) at 37 °C. In comparison with the PCO2 of other cells, this value is quite high and about identical to that of the human red cell membrane. As no major protein CO2 channels such as aquaporins 1 and 4 are present in rat cardiac sarcolemma, the high PCO2 of this membrane is likely due to its low cholesterol content of about 0.2 (mol cholesterol)·(mol total membrane lipids)-1 . Previous work predicted a PCO2 of ≥0.1 cm s-1 from this level of cholesterol. We conclude that the low cholesterol establishes a PCO2 high enough to render the membrane resistance to CO2 diffusion almost negligible, even under conditions of maximal O2 consumption of the heart.

Effects of ocean acidification on the physiological performance and carbon production of the Antarctic sea ice diatom Nitzschia sp. ICE-H

Ocean acidification (OA) resulting from increasing atmospheric CO2 strongly influences marine ecosystems, particularly in the polar ocean due to greater CO2 solubility. Here, we grew the Antarctic sea ice diatom Nitzschia sp. ICE-H in a semicontinuous culture under low (~400ppm) and high (1000ppm) CO2 levels. Elevated CO2 resulted in a stimulated physiological response including increased growth rates, chlorophyll a contents, and nitrogen and phosphorus uptake rates. Furthermore, high CO2 enhanced cellular particulate organic carbon production rates, indicating a greater shift from inorganic to organic carbon. However, the cultures grown in high CO2 conditions exhibited a decrease in both extracellular and intracellular carbonic anhydrase activity, suggesting that the carbon concentrating mechanisms of Nitzschia sp. ICE-H may be suppressed by elevated CO2. Our results revealed that OA would be beneficial to the survival of this sea ice diatom strain, with broad implications for global carbon cycles in the future ocean.

Carbonic anhydrase inhibitors modify intracellular pH transients and contractions of rat middle cerebral arteries during CO2/HCO3– fluctuations

The CO2/HCO3– buffer minimizes pH changes in response to acid–base loads, HCO3– provides substrate for Na+,HCO3–-cotransporters and Cl–/HCO3–-exchangers, and H+ and HCO3– modify vasomotor responses during acid–base disturbances. We show here that rat middle cerebral arteries express cytosolic, mitochondrial, extracellular, and secreted carbonic anhydrase isoforms that catalyze equilibration of the CO2/HCO3– buffer. Switching from CO2/HCO3–-free to CO2/HCO3–-containing extracellular solution results in initial intracellular acidification due to hydration of CO2 followed by gradual alkalinization due to cellular HCO3– uptake. Carbonic anhydrase inhibition decelerates the initial acidification and attenuates the associated transient vasoconstriction without affecting intracellular pH or artery tone at steady-state. Na+,HCO3–-cotransport and Na+/H+-exchange activity after NH4+-prepulse-induced intracellular acidification are unaffected by carbonic anhydrase inhibition. Extracellular surface pH transients induced by transmembrane NH3 flux are evident under CO2/HCO3–-free conditions but absent when the buffer capacity and apparent H+ mobility increase in the presence of CO2/HCO3– even after the inhibition of carbonic anhydrases. We conclude that (a) intracellular carbonic anhydrase activity accentuates pH transients and vasoconstriction in response to acute elevations of pCO2, (b) CO2/HCO3– minimizes extracellular surface pH transients without requiring carbonic anhydrase activity, and (c) carbonic anhydrases are not rate limiting for acid–base transport across cell membranes during recovery from intracellular acidification.

Photosynthetic responses of the marine diatom Thalassiosira pseudonana to CO2-induced seawater acidification

Ocean acidification due to atmospheric CO2 rise is expected to influence marine phytoplankton. Diatoms are responsible for about 40% of the total primary production in the ocean. In order to investigate the physiological response of marine diatom Thalassiosira pseudonana to ocean acidification, we grew the cells under ambient CO2 level (380 µatm) versus the elevated CO2 level (800 µatm) at a light level of 180 µmol m−2 s−1 for 30 generations. Our results showed that the elevated CO2 concentration caused a decrease of the effective photochemical efficiency of PSII $$\left( {{{F_{{\text{v}}}^{\prime } } \mathord{\left/ {\vphantom {{F_{{\text{v}}}^{\prime } } {F_{{\text{m}}}^{\prime } }}} \right. \kern-\nulldelimiterspace} {F_{{\text{m}}}^{\prime } }}} \right)$$ and increase of the dark respiration in T. pseudonana. The intracellular carbonic anhydrase activity was suppressed and the photosynthetic affinity for CO2 was lowered in the high CO2-grown cells, reflecting a downregulation of the CO2 concentrating mechanism (CCM). PSI activity was enhanced to support an increase in ATP synthesis by cyclic electron transfer as required for transport of inorganic carbon and regulation of intracellular pH. The energetic benefit from the downregulation of CCM to growth as reported in other diatom species was not observed here in T. pseudonana.

Phosphate Chemical Probes Designed for Location Specific Inhibition of Intracellular Carbonic Anhydrases.

Chemical probes are small molecules designed to bind to a specific protein and disrupt the proteins function. Although many inhibitors are reported for human carbonic anhydrase (CA) enzymes, few may be considered useful as chemical probes as they exhibit broad action against the 12 catalytically active CA isozymes. In addition, most do not possess an appropriate physicochemical profile to discriminate intracellular CA activity from either global or extracellular CA activity. We report herein the synthesis of three monophosphate CA proinhibitors (compounds 2, 3, and 5) that are derived from cyclosaligenyl (cycloSal) phosphate and S-acyl-2-thioethyl (SATE) phosphate as protecting groups. The proinhibitors are designed as neutral, membrane-permeable compounds that once inside the cell may be hydrolyzed by pH-driven or enzymatic-driven mechanisms to release a negatively charged monophosphate. The resulting monophosphate compound is trapped intracellularly and available for locality specific inhibition of intracellular CAs.

Intracellular Carbonic Anhydrase Activity Sensitizes Cancer Cell pH Signaling to Dynamic Changes in CO2 Partial Pressure

Carbonic anhydrase (CA) enzymes catalyze the chemical equilibration among CO2, HCO3(-) and H(+). Intracellular CA (CAi) isoforms are present in certain types of cancer, and growing evidence suggests that low levels correlate with disease severity. However, their physiological role remains unclear. Cancer cell CAi activity, measured as cytoplasmic CO2 hydration rate (kf), ranged from high in colorectal HCT116 (∼2 s(-1)), bladder RT112 and colorectal HT29, moderate in fibrosarcoma HT1080 to negligible (i.e. spontaneous kf = 0.18 s(-1)) in cervical HeLa and breast MDA-MB-468 cells. CAi activity in cells correlated with CAII immunoreactivity and enzymatic activity in membrane-free lysates, suggesting that soluble CAII is an important intracellular isoform. CAi catalysis was not obligatory for supporting acid extrusion by H(+) efflux or HCO3(-) influx, nor for maintaining intracellular pH (pHi) uniformity. However, in the absence of CAi activity, acid loading from a highly alkaline pHi was rate-limited by HCO3(-) supply from spontaneous CO2 hydration. In solid tumors, time-dependence of blood flow can result in fluctuations of CO2 partial pressure (pCO2) that disturb cytoplasmic CO2-HCO3(-)-H(+) equilibrium. In cancer cells with high CAi activity, extracellular pCO2 fluctuations evoked faster and larger pHi oscillations. Functionally, these resulted in larger pH-dependent intracellular [Ca(2+)] oscillations and stronger inhibition of the mTORC1 pathway reported by S6 kinase phosphorylation. In contrast, the pHi of cells with low CAi activity was less responsive to pCO2 fluctuations. Such low pass filtering would "buffer" cancer cell pHi from non-steady-state extracellular pCO2. Thus, CAi activity determines the coupling between pCO2 (a function of tumor perfusion) and pHi (a potent modulator of cancer cell physiology).

Abstract 2056: Evidence against hypoxic-dependent activation of carbonic anhydrase IX in MDA-MB-231 breast cancer cells

Abstract Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme the expression of which is often considered a marker for hypoxia, an indicator of poor prognosis, and associated with acidification of the tumor microenvironment. Many studies have shown that CAIX expression is induced by hypoxia exposing its catalytic domain to the interstitial milieu. Several recent studies have suggested that hypoxic conditions may also permit activation of CAIX, perhaps by causing a conformational change which exposes the catalytic pocket. Our goal was to assess the effect of anoxic conditions on CAIX activity in MDA-MB-231 cells, previously exposed to hypoxia which increases CAIX expression in the absence of other membrane-bound carbonic anhydrase (CA) family members. We have taken advantage of membrane inlet mass spectrometry (MIMS) to directly analyze CA activity in intact cells by measuring the 18O exchange between CO2 and H2O. This method distinguishes between intracellular and extracellular CA activity. MDA-MB-231 cells were exposed to 1% oxygen for 16 hours after which they were isolated under normoxic or anoxic conditions. CA activity was then measured, again under normoxic or anoxic conditions. These data show biphasic depletion of 18O from CO2 under both normoxic and anoxic assay conditions. The first phase (which occurs over the first 20-40 seconds) represents a rapid diffusion of CO2 into cells where it is exposed to intracellular CAII. A catalytic cycle depletes 18O followed by efflux of CO2 from the cell. There was no difference in this phase between cells prepared and assayed under normoxic or anoxic conditions. The second phase (from 200-500 sec) is dominated by the hydration-dehydration reaction of CO2/HCO3− catalyzed by exofacial CA activity (CAIX). In cells exposed to anoxia, the slope of the second phase was greater than that observed with cells exposed to normoxic conditions indicating elevated CAIX activity. While this provided evidence that oxygen limitation might influence CAIX activity, the Ki values for two impermeant CA inhibitors, Cpd 5C and a polymeric sulfonamide, did not differ between anoxic and normoxic cells. We conclude from these data that the catalytic site of CAIX is exposed and functional under both anoxic or normoxic conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2056. doi:10.1158/1538-7445.AM2011-2056

Membrane Transport of CO 2 and H 2S: No Facilitator Required

The observation that some membranes and epithelia have no demonstrable gas permeability suggested that membrane channels may be involved in CO2 transport. Both aquaporins and Rhesus proteins were reported to serve as pathways for CO2 and H2S. In contrast, we show here that membrane lipid has such high CO2 and H2S permeabilities that the presence of a protein channel does not enhance the flux. Therefore we reconstituted aquaporins into lipid bilayers and used scanning microelectrodes to monitor pH in the immediate vicinity of planar lipid bilayers. The lower limits of lipid bilayer permeabilities to CO2 and to H2S were equal to 3.2±1.6 cm/s[1] and 0.5±0.4 cm/s[2], respectively. We also observed that the CO2 flux through the lipid bilayer decreases several fold when the rate of CO2 formation from HCO3- was not augmented by carbonic anhydrase (CA). Experiments with epithelial cell monolayers grown on permeable support revealed the same result. Inhibition of CA transformed these otherwise highly CO2 permeable cell monolayers into CO2 barriers. Finally we tested the CO2 permeability of the epithelium of the mammalian bladder. It was impermeable to CO2 even after uroplakin knock-out. We found that the lack of intrinsic intracellular CA activity of these epithelial cells hampers the CO2 exchange between blood and urine. [1] A. Missner, P. Kugler, S. M. Saparov, K. Sommer, J. C. Matthai, M. L. Zeidel, P. Pohl, J.Biol.Chem. 2008, 283 25340-25347. [2.] J. C. Mathai, A. Missner, P. Kügler, S. M. Saparov, M. L. Zeidel, J. K. Lee, P. Pohl, Proc.Natl.Acad.Sci.U.S.A. 2009, 106 16633-16638.

Photosynthetic inorganic carbon utilization of gametophytes and sporophytes of Undaria pinnatifida (Phaeophyceae)

X. Zhang, H. Hu and T. Tan. 2006. Photosynthetic inorganic carbon utilization of gametophytes and sporophytes of Undaria pinnatifida (Phaeophyceae). Phycologia 45: 642–647. DOI: 10.2216/05-28.1The characteristics of inorganic carbon assimilation by photosynthesis were investigated in male and female gametophytes and juvenile sporophytes of Undaria pinnatifida. Gametophytes and sporophytes have detectable extracellular and intracellular carbonic anhydrase (CA) activity, and the CA inhibitor, acetazolamide (AZ), significantly inhibited their photosynthesis O2 evolution. In pH-drift experiments, it was found that gametophytes did not raise the final pH of seawater above 9.00 (CO2 concentrations of about 2.2 μM), indicating a low ability to utilize inorganic carbon. In contrast, sporophytes rapidly raised pH to over 9.53 and depleted the free CO2 concentration to less than 0.16 μM. The apparent photosynthetic affinity for CO2 was almost the same for gametophytes and sporophytes, whereas gametophytes had a much lower affinity for HCO3− than sporophytes. Two inhibitors of band 3 anion exchange protein (DIDS and SITS) inhibited the photosynthesis of gametophytes but not that of sporophytes. It was indicated that both gametophytes and sporophytes were capable of using HCO3−, which involved the external CA activity, and a direct HCO3− use also occurred in the former, but the latter showed a greater capacity of HCO3− use than the former. In addition, male and female gametophytes did not show great differences in the inorganic carbon uptake mechanism underlying photosynthesis.

Inhibition of the putative anion transporter (PAT‐1, Slc26a6) impairs the ability of villous epithelial cells to regulate intracellular pH (pH i ) during peptide transport

The upper villus is exposed to acid challenge from H+ influx via the H+ dipeptide cotransporter PEPT1. Intracellular carbonic anhydrase activity facilitates diffusive H+ movement to help maintain a transmembrane ion gradient allowing for maximal absorption through PEPT1. Other studies have revealed that carbonic anhydrase II (CAII) can interact with the apical membrane Cl−/HCO3− exchanger PAT-1. Therefore, we hypothesized that CAII functions with PAT-1, the predominant Cl−/HCO3− exchanger of the upper duodenal villus, to form a HCO3− transport metabolon at the apical membrane to regulate pHi during PEPT1 transport activity. Measurements of pHi by BCECF microfluoroscopy were conducted in wild-type (WT) and PAT-1 knockout (KO) upper duodenal villous epithelium exposed to the dipeptide glycyl-sarcosine (gly-sar) in Kreb's HCO3− Ringers. During gly-sar exposure, inhibition of luminal Cl−/HCO3− exchange in WT epithelium with niflumic acid resulted in greater acid influx (NFA = −0.83 ± 0.94; control = −7.05± 0.89 mM/min, n = 3; p<0.05). Furthermore, a greater acid influx was observed in the PAT-1 KO as compared to WT during gly-sar exposure (−0.07 ± 0.79 vs. −9.54 ± 3.40 mM/min, n = 3, p<0.05). These observations suggest that PAT-1 provides protection against intracellular acidification in the upper villous epithelium, perhaps by operating in reverse mode as a base-loading mechanism for the cell. Supported by NIH.

Inorganic carbon acquisition in red tide dinoflagellates

Carbon acquisition was investigated in three marine bloom-forming dinollagellates-Prorocentrum minimum, Heterocapsa triquetra and Ceratium lineatum. In vivo activities of extracellular and intracellular carbonic anhydrase (CA), photosynthetic O2 evolution, CO2 and HCO3- uptake rates were measured by membrane inlet mass spectrometry (MIMS) in cells acclimated to low pH (8.0) and high pH (8.5 or 9.1). A second approach used short-term 14C-disequilibrium incubations to estimate the carbon source utilized by the cells. All three species showed negligible extracellular CA (eCA) activity in cells acclimated to low pH and only slightly higher activity when acclimated to high pH. Intracellular CA (iCA) activity was present in all three species, but it increased only in P. minimum with increasing pH. Half-saturation concentrations (K1/2) for photosynthetic O2 evolution were low compared to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetics. Moreover, apparent affinities for inorganic carbon (Ci) increased with increasing pH in the acclimation, indicating the operation of an efficient CO2 concentration mechanism (CCM) in these dinoflagellates. Rates of CO2 uptake were comparably low and could not support the observed rates of photosynthesis. Consequently, rates of HCO3- uptake were high in the investigated species, contributing more than 80% of the photosynthetic carbon fixation. The affinity for HCO3- and maximum uptake rates increased under higher pH. The strong preference for HCO3- was also confirmed by the 14C-disequilibrium technique. Modes of carbon acquisition were consistent with the 13C-fractionation pattern observed and indicated a strong species-specific difference in leakage. These results suggest that photosynthesis in marine dinoflagellates is not limited by Ci even at high pH, which may occur during red tides in coastal waters.

An investigation of the role of carbonic anhydrase in aquatic and aerial gas transfer in the African lungfishProtopterus dolloi

Experiments were performed on bimodally breathing African lungfish Protopterus dolloi to examine the effects of inhibition of extracellular vs total (extracellular and intracellular) carbonic anhydrase (CA) activity on pulmonary and branchial/cutaneous gas transfer. In contrast to previous studies on Protopterus, which showed that the vast majority of CO(2) is excreted into the water through the gill and/or skin whereas O(2) uptake largely occurs via the lung, P. dolloi appeared to use the lung for the bulk of both O(2) uptake (91.0+/-2.9%) and CO(2) excretion (76.0+/-6.6%). In support of the lung as the more important site of CO(2) transfer, aerial hypercapnia (P(CO(2))=40 mmHg) caused a significant rise in partial pressure of arterial blood CO(2) (Pa(CO(2))) whereas a similar degree of aquatic hypercapnia was without effect on Pa(CO(2)). Intravascular injection of low levels (1.2 mg kg(-1)) of the slowly permanent CA inhibitor, benzolamide, was without effect on red blood cell CA activity after 30 min, thus confirming its suitability as a short-term selective inhibitor of extracellular CA. Benzolamide treatment did not affect CO(2) excretion, blood acid-base status or any other measured variable within the 30 min measurement period. Injection of the permeant CA inhibitor acetazolamide (30 mg kg(-1)) resulted in the complete inhibition of red cell CA activity within 10 min. However, CO(2) excretion (measured for 2 h after injection) and arterial blood acid-base status (assessed for 24 h after injection) were unaffected by acetazolamide treatment. Intra-arterial injection of bovine CA (2 mg kg(-1)) caused a significant increase in overall CO(2) excretion (from 0.41+/-0.03 to 0.58+/-0.03 mmol kg(-1) h(-1)) and an increase in air breathing frequency (from 19.0+/-1.3 to 24.7+/-1.8 breaths min(-1)) that was accompanied by a slight, but significant, reduction in Pa(CO(2)) (from 21.6+/-1.6 to 19.6+/-1.8 mmHg). The findings of this study are significant because they (i) demonstrate that, unlike in other species of African lungfish that have been examined, the gill/skin is not the major route of CO(2) excretion in P. dolloi, and (ii) suggest that CO(2) excretion in Protopterus may be less reliant on carbonic anhydrase than in most other fish species.

Low carbon dioxide permeability of the apical epithelial membrane of guinea‐pig colon

We have investigated the apical membrane permeability for CO2 of intact epithelia of proximal and distal colon of the guinea pig. The method used was the mass spectrometric 18O-exchange technique previously described. In a first step, we determined the intraepithelial carbonic anhydrase (CA) activity by studying vital isolated colonocytes before and after lysis with Triton X-100. Intraepithelial CA activity was found to be 41,000 and 900 for proximal and distal colon, respectively. Then 18O-exchange measurements were done with stripped intact epithelial layers, which on their apical side were exposed to the reaction solution containing 18O-labelled CO2 and HCO3-. The mass spectrometric signals in these measurements are determined by the intracellular epithelial CA activity, and by the apical membrane permeabilities for CO2 and HCO3-, P(CO2) and P(HCO3). From the signals, we calculated the two permeabilities while inserting the CA activities obtained from isolated colonocytes. From layers of intact colon epithelium, the apical P(CO2) was determined to be 1.5 x 10(-3) cm s(-1) for proximal and 0.77 x 10(-3) cm s(-1) for distal colon. These values are > or =200 times lower than the P(CO2) of the human red cell membrane as studied with the same technique (0.3 cm s(-1)). We conclude that the apical membrane offers a significant resistance towards CO2 diffusion, which implies that a major drop in CO2 partial pressure (pCO2) will occur across the apical membrane when luminal pCO2 is higher than basolateral or capillary pCO2. In view of the very high pCO2 that can occur in the colonic lumen, this property of the apical membrane constitutes a significant protection of the cell against the high acid load associated with high pCO2.

Impacts of Elevated CO2Concentration on Biochemical Composition, Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

Abstract: To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations, Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2 concentrations within the range 3-186 ümol/L and the biochemical composition, carbonic anhydrase (CA), and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate, and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186 ümol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in Chlorella pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186 ümol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2 enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent. (Managing editor: Ping HE)

A study of the activity and ecological significance of carbonic anhydrase from soil and its microbes from different karst ecosystems of Southwest China

The origin and distribution of carbonic anhydrase (CA), which could accelerate karst processes, are explored in this paper. The soil samples used in the study were collected from four different kinds of karst ecosystems of Southwest China. The results indicate that CA activity could be detected in surface layer soils, and that CA activity varied obviously among the soils in different karst ecosystems. Of the four kinds of karst site, the mean CA activity of the surface soil in Misuga, Liupanshui, which had the lowest vegetation cover, was the lowest with 0.02 U g −1 dry soil. Nongla and Jinfu mountain, where there is abundant vegetable species diversity and dense vegetation, had higher mean CA activity in the surface soil with 3.83 U g −1 dry soil and 3.13 U g −1 dry soil, respectively. Moreover, there was certain difference in CA activities of soils among different kinds of karst landscape in the same kind of karst ecosystem. On the other hand, the higher CA activity could be detected in the soils in the vicinity of the plant roots, and CA activity in soil exhibited both obvious seasonal and vertical changes. These facts implied that plant roots and soil microorganisms might serve as important sources of CA. Besides, the results indicate that CA activity is found in bacteria, actinomycetes and fungi. Actinomycetes had higher intracellular CA activity, while fungi had higher extracellular CA activity. This study shows that higher microbial CA activities are found in Jinfu Mountain, while lower microbial CA activities are found in Misuga, Liupanshui.

The gene NCE103 (YNL036w) from Saccharomyces cerevisiae encodes a functional carbonic anhydrase and its transcription is regulated by the concentration of inorganic carbon in the medium

Carbonic anhydrase (CA) catalyses the rapid interconversion between CO(2) and HCO(3) (-). Despite its wide distribution among living organisms, the presence of CA in fungi has been controversially discussed. Using mass spectrometric analysis of (18)O exchange from doubly labelled CO(2), we were able to measure CA activity in intact cells of Saccharomyces cerevisiae. Intracellular CA activity was lacking in the Deltance103 mutant, indicating that NCE103 encodes a functional CA. This was proven by overexpressing and purification of the NCE103 gene product showing a specific activity of around 6900 units per mg protein. Interestingly, the in vivo CA activity was 10-20 times higher in cells grown on low inorganic carbon (Ci; air containing 0.035% CO(2)) than in high-Ci cells (grown on 5% CO(2)). The enhanced CA activity of low-Ci cells was inducible after transferring high-Ci cells to air. Northern blot analysis revealed that that expression of NCE103 is transcriptionally regulated by low Ci which was also demonstrated by fusing the NCE103 promoter to beta-galactosidase as a reporter gene. Inactivation of NCE103 results in a high CO(2) requiring mutant indicating that a functional CA is an important prerequisite for S. cerevisiae to grow under low-Ci conditions.