Characterization of carbonic anhydrase isozyme CA2, which is the CAH2 gene product, in Chlamydomonas reinhardtii.

From high-CO2 (5% CO2) grown unicellular green alga, Chlamydomonas reinhardtii, carbonic anhydrase (CA) was isolated by affinity chromatography and characterized. Isolated CA was identified as an isozyme (CA2) which is the product from the second gene CAH2 by peptide sequencing. The CA2 was inactivated by dithiothreitol. This treatment caused dissociation of CA2 into the large (38 kDa) and small subunits (4243 Da). The molecular mass of the CA2 holoenzyme measured by low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC was 87.9 kDa. These results and gene structure indicate that CA2 is a heterotetramer consisting of two large and two small subunits linked by disulfide bonds like CA1, which is the CAH1 gene product. The specific activity of CA2 purified by anion-exchange HPLC was 3300 units per mg protein, which was approximately 1.6 times higher than that of CA1. Therefore, it was concluded that two structurally related isozymes, CA1 and CA2, are present in the wild type cells of C. reinhardtii and differentially regulated by the atmospheric CO2 concentration.     

Post-translational processing of the highly processed,secreted periplasmic carbonic anhydrase of Chlamydomonas is largely conserved in transgenic tobacco

The periplasmic carbonic anhydrase (CA) gene CAH1 of Chlamydomonas reinhardtii codes for a highly processed secreted glycoprotein. The primary translation product of the CAH1 gene is targeted to the ER, where it is proteolytically processed to yield two different subunits, glycosylated, assembled into an active heterotetramer, and secreted. After replacing the target leader sequence with that from tobacco anionic peroxidase, expression of this gene in transgenic tobacco plants was investigated. SDS-PAGE gels of the purified protein from tobacco, showed that it migrated as a series of discrete bands (two large and one small) with slightly faster mobility than the comparable bands in the purified algal protein. The expressed protein in the plant was active, and staining with thymol and sulfuric acid confirmed that it was also glycosylated. The periplasmic CA1 (peri-CA1) also was found to be enriched in the intercellular fluid of transgenic tobacco, indicating it was secreted. The specific activity of the enzyme and its sensitivity to sulfonamide inhibitors were similar to that of the native algal enzyme. These results suggest that the post translational processing of Chlamydomonas peri-CA1 is largely conserved in a higher plant.     

CO2 acquisition in Chlamydomonas acidophila is influenced mainly by CO2, not phosphorus,availability

The extremophilic green microalga Chlamydomonas acidophila grows in very acidic waters (pH 2.3–3.4), where CO₂ is the sole inorganic carbon source. Previous work has revealed that the species can accumulate inorganic carbon (Ci) and exhibits high affinity CO₂ utilization under low-CO₂ (air-equilibrium) conditions, similar to organisms with an active CO₂ concentrating mechanism (CCM), whereas both processes are down-regulated under high CO₂ (4.5 % CO₂) conditions. Responses of this species to phosphorus (Pi)-limited conditions suggested a contrasting regulation of the CCM characteristics. Therefore, we measured external carbonic anhydrase (CA_ext) activities and protein expression (CAH1), the internal pH, Ci accumulation, and CO₂-utilization in cells adapted to high or low CO₂ under Pi-replete and Pi-limited conditions. Results reveal that C. acidophila expressed CA_ext activity and expressed a protein cross-reacting with CAH1 (the CA_ext from Chlamydomonas reinhardtii). Although the function of this CA remains unclear, CA_ext activity and high affinity CO₂ utilization were the highest under low CO₂ conditions. C. acidophila accumulated Ci and expressed the CAH1 protein under all conditions tested, and C. reinhardtii also contained substantial amounts of CAH1 protein under Pi-limitation. In conclusion, Ci utilization is optimized in C. acidophila under ecologically relevant conditions, which may enable optimal survival in its extreme Ci- and Pi-limited habitat. The exact physiological and biochemical acclimation remains to be further studied.     

Purification, characterization and cDNA cloning of soluble carbonic anhydrase from Chlorella sorokiniana grown under ordinary air

Soluble carbonic anhydrase (CA, EC 4.2.1.1) inducible by low levels of CO₂ was purified from the unicellular green alga Chlorella sorokiniana grown at alkaline pH. The purified CA had a specific activity of 2,300 units (mg protein)⁻¹. The molecular mass of the CA was found to be 100 kDa by non-dissociating (native)-polyacrylamide gel electrophoresis and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 50-kDa subunit was recognized by concanavalin A. These results suggest that the protein has a dimeric form with two 50-kDa subunits that are glycosylated in an asparagine-linked manner. The native CA was revealed by isoelectric focusing to be a very acidic protein with an isoelectric point of 4.2. About 60% of the CA activity was inhibited by 0.5 M NaCl. The enzyme was inactivated over 95% by preincubation with 50 mM dithiothreitol but not with 1 mM dithiothreitol. After partial amino acid sequence analysis, a cDNA clone of the CA was isolated and characterized. The cloned cDNA fragment encoded a 348-amino-acid polypeptide (36,709 Da) including an NH₂-terminal hydrophobic signal peptide composed of 35 amino acids (3,725 Da). Conserved regions of sequences found in animal CAs, in the periplasmic (pCA) and the intracellular CAs of Chlamydomonas, and in the plasma-membrane-bound CA of Dunaliella (Dca) were also found in this Chlorella CA. The signal sequence was significantly homologous to the pCA and the Dca. The internal signal sequence between the large and the small subunits reported for pCA was not found in this Chlorella CA. The soluble CA of this alga was an α-type CA with salt-sensitive, periplasm-locating and acidic properties and very different from pCA and Dca with their salt-sensitive/neutral and salt-resistant/acidic properties, respectively. Received: 25 May 1998 / Accepted: 9 July 1998     

Characterization,bioinformatic analysis and dithiocarbamate inhibition studies of two new α-carbonic anhydrases,CAH1 and CAH2, from the fruit fly Drosophila melanogaster

Carbonic anhydrases (CAs) are essential and ubiquitous enzymes. Thus far, there are no articles on characterization of Drosophila melanogaster α-CAs. Data from invertebrate CA studies may provide opportunities for anti-parasitic drug development because α-CAs are found in many parasite or parasite vector invertebrates. We have expressed and purified D. melanogaster CAH1 and CAH2 as proteins of molecular weights 30 kDa and 28 kDa. CAH1 is cytoplasmic whereas CAH2 is a membrane-attached protein. Both are highly active enzymes for the CO₂ hydration reaction, being efficiently inhibited by acetazolamide. CAH2 in the eye of D. melanogaster may provide a new animal model for CA-related eye diseases. A series of dithiocarbamates were also screened as inhibitors of these enzymes, with some representatives showing inhibition in the low nanomolar range.     

CAH1 and CAH2 as key enzymes required for high bicarbonate tolerance of a novel microalga Dunaliella salina HTBS

Outdoor microalgal cultivation with high concentration bicarbonate has been considered as a strategy for reducing contamination and improving carbon supply efficiency. The mechanism responsible for algae's strong tolerance to high bicarbonate however, remains not clear. In this study, we isolated and characterized a strain and revealed its high bicarbonate tolerant mechanism by analyzing carbonic anhydrase (CA). The strain was identified as Dunaliella salina HTBS with broad temperature adaptability (7–30 °C). The strain grew well under 30% CO₂ or 70 g L⁻¹ NaHCO₃. In comparison, two periplasm CAs (CAH1 and CAH2) were detected with immunoblotting analysis in HTBS but not in a non-HCO₃⁻—tolerant strain. The finding was also verified by an enzyme inhibition assay in which only HTBS showed significant inhibition by extracellular CA inhibitor. Thus, we inferred that the extracellular CAH1 and CAH2 played a multifunctional role in the toleration of high bicarbonate by HTBS.     

Isolation of cDNA clones of genes induced upon transfer of Chlamydomonas reinhardtii cells to low CO2

Unicellular algae grow well under limiting CO₂ conditions, aided by a carbon concentrating mechanism (CCM). In C. reinhardtii, this mechanism is inducible and is present only in cells grown under low CO₂ conditions. We constructed a cDNA library from cells adapting to low CO₂, and screened the library for cDNAs specific to low CO₂-adapting cells. Six classes of low CO₂-inducible clones were identified. One class of clone, reported here, represents a novel gene associated with adaptation of cells to air. A second class of clones corresponds to the air-inducible periplasmic carbonic anhydrase I (CAH1). These clones represent genes that respond to the level of CO₂ in the environment.     

Mitochondrial carbonic anhydrases are needed for optimal photosynthesis at low CO2 levels in Chlamydomonas

Chlamydomonas reinhardtii can grow photosynthetically using CO₂ or in the dark using acetate as the carbon source. In the light in air, the CO₂ concentrating mechanism (CCM) of C. reinhardtii accumulates CO₂, enhancing photosynthesis. A combination of carbonic anhydrases (CAs) and bicarbonate transporters in the CCM of C. reinhardtii increases the CO₂ concentration at Ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) in the chloroplast pyrenoid. Previously, CAs important to the CCM have been found in the periplasmic space, surrounding the pyrenoid and inside the thylakoid lumen. Two almost identical mitochondrial CAs, CAH4 and CAH5, are also highly expressed when the CCM is made, but their role in the CCM is not understood. Here, we adopted an RNAi approach to reduce the expression of CAH4 and CAH5 to study their possible physiological functions. RNAi mutants with low expression of CAH4 and CAH5 had impaired rates of photosynthesis under ambient levels of CO₂ (0.04% CO₂ [v/v] in air). These strains were not able to grow at very low CO₂ (<0.02% CO₂ [v/v] in air), and their ability to accumulate inorganic carbon (C_i = CO₂ + HCO3−) was reduced. At low CO₂ concentrations, the CCM is needed to both deliver C_i to Rubisco and to minimize the leak of CO₂ generated by respiration and photorespiration. We hypothesize that CAH4 and CAH5 in the mitochondria convert the CO₂ released from respiration and photorespiration as well as the CO₂ leaked from the chloroplast to HCO3- thus “recapturing” this potentially lost CO₂.

Mitochondrial carbonic anhydrases CAH4 and CAH5 in Chlamydomonas reinhardtii are involved in maintaining optimal photosynthesis.     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.     

Absorption characteristics and kinetics of CO2 capture into N-methyldiethanolamine aqueous solution catalyzed by the immobilized carbonic anhydrase

Abstract

Carbonic anhydrase (CA) is the most effective CO₂ hydratase catalyst, but the poor storage stability and repeatability of CA limit its development. Therefore, CA was immobilized on the epoxy magnetic composite microspheres to enhance the CO₂ absorption into N-methyldiethanolamine (MDEA) aqueous solution in this work. In the presence of immobilized CA, the CO₂ absorption rate of MDEA solution (10?wt%) (0.63?mmol·min^?1) was greatly improved by almost 40%, and their reaction equilibrium time was shortened from 150?min to 90?min compared with that into MDEA solution. The results indicated that the absorption of CO₂ into MDEA solution had been significantly enhanced by using CA. After the 7th reuse recycle, the activity of the immobilized CA was still closed to its initial value at 313.15?K. Moreover, enzyme catalytic kinetics of immobilized CA was investigated using the p-nitrophenyl acetate (p-NPA) as substrate. The values of Michaelis–Menten constant (K_m) and the maximum velocity (V_max) of the immobilized CA were calculated to be 27.61?mmol/L and 20.14?×?10^?3?mmol·min^?1·mL^?1, respectively. Besides, the kinetics of CO₂ reaction into MDEA with or without CA were also compared. The results showed that CO₂ absorption into CA/MDEA aqueous solution obeyed the pseudo first order regime and the second order kinetics rate constant (k₂) was calculated to be 929?m³·kmol^?1·s^?1, which was twice higher than that of MDEA aqueous solution without immobilized CA (k₂=414 m³·kmol^?1·s^?1) at 313.15?K.     

Active transport of CO2 by three species of marine microalgae

The occurrence of an active CO₂ transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H¹³CO₃^? caused a rapid initial drop, followed by a slower decline in the extracellular CO₂ concentration. Addition of bovine CA to the medium raised the CO₂ concentration by restoring the HCO₃^?–CO₂ equilibrium, indicating that cells were taking up CO₂ and were maintaining the CO₂ concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO₂ concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of ¹⁸O from ¹³C¹⁸O₂ into water. Exchange of ¹⁸O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane‐impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane‐permeable CA‐inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO₂ uptake by active transport for use as a carbon source for photosynthesis.     

The drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

Partial characterization of a new isoenzyme of carbonic anhydrase isolated from Chlamydomonas reinhardtii

A new isoenzyme of carbonic anhydrase has been isolated and purified from Chlamydomonas reinhardtii. This carbonic anhydrase is composed of two nonidentical subunits with apparent molecular masses of 39 and 4.5 kDa and is located in the periplasmic space. This is the second periplasmic carbonic anhydrase found in C. reinhardtii. Two genes, CAH1 and CAH2, which code for carbonic anhydrase, have been recently described by Fujiwara et al. (Fujiwara, S., Fukuzawa, H., Tachiki, A., and Miyachi, S. (1990) Proc. Natl. Acad, Sci. U.S.A. 87, 9779-9783). The CAH1 gene codes for a periplasmic carbonic anhydrase which is induced under low CO2 conditions and is well characterized. The carbonic anhydrase characterized in this report was isolated from a mutant that is unable to synthesize the CAH1 gene product. Amino acid sequencing demonstrates that this newly isolated carbonic anhydrase is the CAH2 gene product. This is the first report of another functional carbonic anhydrase in C. reinhardtii.     

Engineering the genetic components of a whole-cell catalyst for improved enzymatic CO2 capture and utilization

Carbonic anhydrase (CA) is a diffusion-limited enzyme that rapidly catalyzes the hydration of carbon dioxide (CO₂). CA has been proposed as an eco-friendly yet powerful catalyst for CO₂ capture and utilization. A bacterial whole-cell biocatalyst equipped with periplasmic CA provides an option for a cost-effective CO₂-capturing system. However, further utilization of the previously constructed periplasmic system has been limited by its relatively low activity and stability. Herein, we engineered three genetic components of the periplasmic system for the construction of a highly efficient whole-cell catalyst: a CA-coding gene, a signal sequence, and a ribosome-binding site (RBS). A stable and halotolerant CA (hmCA) from the marine bacterium Hydrogenovibrio marinus was employed to improve both the activity and stability of the system. The improved secretion and folding of hmCA and increased membrane permeability were achieved by translocation via the Sec-dependent pathway. The engineering of RBS strength further enhanced whole-cell activity by improving both the secretion and folding of hmCA. The newly engineered biocatalyst displayed 5.7-fold higher activity and 780-fold higher stability at 60°C compared with those of the previously constructed periplasmic system, providing new opportunities for applications in CO₂ capture and utilization.     

Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Summary Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H₂ at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H₂ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H₂ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H₂ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H₂ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H₂ases.     

Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His₆PheA1 and His₆PheA2 were purified and its catalytic activity characterized. His₆PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His₆PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His₆PheA1 and His₆PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.     

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO2

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO₂ concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1–4 days. In contrast, C. littorale cells which had been grown in 5% CO₂ (5% CO₂-grown cells) could grow in 40% CO₂ without any lag period. When air-grown cells were transferred to 40% CO₂, the quantum efficiency of PS II (Φ_II) decreased greatly, while no decrease in Φ_II was apparent when the 5% CO₂-grown cells were transferred to 40% CO₂. In contrast to air-grown cells, 5% CO₂-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO₂-grown cells to air, photosynthetic susceptibility to 40% CO₂ was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO₂. The intracellular pH value (pH_i) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO₂ for 1–2 h, but no such decrease in pH_i was apparent in the presence of EZA. Both air- and 5% CO₂-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO₂ concentrations, grew in 40% CO₂ without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO₂. No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of carbonic anhydrase and anion exchange in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish

The purpose of this study was to investigate the characteristics of carbonic anhydrase (CA) and the Cl⁻/HCO₃⁻ exchanger (Band 3; AE1) in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish, in order to further understand the strategies of blood CO₂ transport in lower vertebrates and gain insights into the evolution of the vertebrate erythrocyte proteins, CA and Band 3. A significant amount of CA activity was measured in the erythrocytes of bowfin (70 mmol CO₂ min⁻¹ ml⁻¹), although it appeared to be lower than that in the erythrocytes of teleost fish. The turnover number (K_cat) of bowfin erythrocyte CA was intermediate between that of the slow type I CA isozyme in agnathans and elasmobranchs and the fast type II CA in the erythrocytes of the more recent teleost fishes, but the inhibition properties of bowfin erythrocyte CA were similar to the fast mammalian CA isozyme, CA II. In contrast to previous findings, a plasma CA inhibitor was found to be present in the blood of bowfin. Bowfin erythrocytes were also found to possess a high rate of Cl⁻/HCO₃⁻ exchange (6 nmol HCO₃⁻ s⁻¹ cm⁻²) that was sensitive to DIDS. Visualization of erythrocyte membrane proteins by SDS-PAGE revealed a major band in the 100 kDa range for the trout, which would be consistent with the anion exchanger. In contrast, the closest major band for the membranes of bowfin erythrocytes was around the 140 kDa range. Taken together, these results suggest that the strategy for blood CO₂ transport in bowfin is probably similar to that in most other vertebrates despite several unique characteristics of erythrocyte CA and Band 3 in these primitive fish.