A 38-kilodalton low-CO2-inducible polypeptide is associated with the pyrenoid in Chlorella vulgaris

In the green alga Chlorella vulgaris UAM 101, a CO₂-concentrating mechanism (CCM) is induced when cells are transferred from high (5%) to low (0.03%) CO₂ concentrations. The induction of the CCM is correlated with de-novo synthesis of several polypeptides that remain to be identified. The internal carbonic anhydrase (CA; EC 4.2.1.1) activity increased 6- to 7-fold within 6 h of acclimation to air. When crude homogenates were further separated into soluble and insoluble fractions, nearly all of the CA activity was associated with the membrane fraction. Immunoblot analysis of cell homogenates probed with antibodies raised against the 37-kDa subunit of periplasmic CA of Chlamydomonas reinhardtii showed a cross-reaction with a single 38-kDa polypeptide in both high- and low-CO₂-grown cells. The up-regulation of the expression of the 38-kDa polypeptide was closely correlated with the increase in internal CA activity. Furthermore, its subcellular location was also correlated with the distribution of the activity. Immunoblot analysis of pyrenoid fractions showed that the 38-kDa polypeptide was concentrated in the pyrenoids from low-CO₂-grown cells but was not present in pyrenoids from high-CO₂-grown cells. In addition, immunogold labeling experiments showed that the protein was mainly associated with membranes crossing the pyrenoid, while it was absent from the pyrenoid matrix. These studies have identified a putative intracellular CA polypeptide associated with the pyrenoid in Chlorella vulgaris, suggesting that this structure may play an important role in the operation of the CCM and the acclimation to low CO₂ conditions. Received: 16 July 1997 / Accepted: 26 April 1998     

Effect of external CO2 concentrations on protein synthesis in the green algae Scenedesmus obliquus (Turp.) Kütz and Chlorella vulgaris (Kosikov)

Unicellular algae grown under low-CO₂ conditions (0.03% CO₂) have developed a means of concentrating CO₂ at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase. Cells with the CO₂-concentrating mechanism (CCM) acquire the ability to accumulate inorganic carbon to a level higher than that obtained by simple diffusion. To identify proteins which are involved in the organization of the CCM, cells of Scenedesumus obliquus and Chlorella vulgaris grown in high CO₂ (5% CO₂ in air) were transferred to low-CO₂ (0.03%) conditions in the presence of ³⁵SO _inf4 ^sup2? and, thereafter, polypeptides labeled with ³⁵S were detected. Under low-CO₂ conditions the inducton of 36-, 39-, 94- and 110- to 116kDa polypeptides were particularly observed in S. obliquus and 16-, 19-, 27-, 36-, 38- and 45-kDa polypeptides were induced in C. vulgaris. Western blots with antibodies raised against 37-kDa subunits of the periplasmic carbonic anhydrase (CA) of Chlamydomonas reinhardtii showed immunoreactive bands with the 39-kDa polypeptide in the whole-cell homogenates from S. obliquus and with 36 and 38-kDa polypeptides in both high- and low-CO₂grown cells of C. vulgaris. Anti-pea-chloroplast CA antibodies cross-reacted with a single polypeptide of 30 kDa in the whole-cell homogenates but not with thylakoid membranes. The CA activity was associated with soluble and membrane-bound fractions, except thylakoid membranes.     

Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO₂ concentrations. The synthesis of these polypeptides is correlated with the induction of the CO₂-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO₂-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO₂ conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O₂/CO₂ ratio than by the CO₂ concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.Abbreviations pCA (periplasmic) carbonic anhydrase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TAP Trisacetate phosphate medium The authors thank Prof. M. Spalding (Iowa State University, USA) for providing antisera to LIP-21 and LIP-36. We thank Prof. S. Bartlett and Dr. J. Moroney (Louisiana State University, USA) for providing antibodies to C. reinhardtii, Rubisco and 37-kDa pCA, respectively. This work was supported by the Instituto Tecnologico de Canarias.     

Glycolate Excretion and the Oxygen to Carbon Dioxide Net Exchange Ratio during Photosynthesis in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells were grown in high (5% v/v) or low (0.03% v/v) CO₂ concentration in air. O₂ evolution, HCO₃⁻ assimilation, and glycolate excretion were measured in response to O₂ and CO₂ concentration. Both low- and high-CO₂-grown cells excrete glycolate. In low-CO₂-grown cells, however, glycolate excretion is observed only at much lower CO₂ concentrations in the medium, as compared with high-CO₂-adapted cells. It is postulated that the activity of the CO₂-concentrating mechanism in low-CO₂-grown cells is responsible for the different dependence of glycolate excretion on external CO₂ concentration in low- versus high-CO₂-adapted cells.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

Purification and characterisation of an intracellular carbonic anhydrase from the unicellular green alga Coccomyxa

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified and characterised from the unicellular green alga Coccomyxa sp. Initial studies showed that cultured Coccomyxa cells contain an intracellular CA activity around 100 times higher than that measured in high-CO₂-grown cells of Chlamydomonas reinhardtii CW 92. Purification of a protein extract containing the CA activity was carried out using ammonium-sulphate precipitation followed by anion-exchange chromatography. Proteins were then separated by native (non-dissociating) polyacrylamide gel electrophoresis, with each individual protein band excised and assayed for CA activity. Measurements revealed CA activity associated with two discrete protein bands with similar molecular masses of 80 +5 kDa. Dissociation by denaturing polyacrylamide gel electrophoresis showed that both proteins contained a single polypeptide of 26 kDa, suggesting that each 80-kDa native protein was a homogeneous trimer. Isoelectric focusing of the 80-kDa proteins also produced a single protein band at a pH of 6.5. Inhibition studies on the purified CA extract showed that 50% inhibition of CA activity was obtained using 1 M azetazolamide. Polyclonal antibodies against the 26-kDa CA were produced and shown to have a high specific binding to a single polypeptide in soluble protein extracts from Coccomyxa cells. The same antiserum, however, failed to cross-react with soluble proteins isolated from two different species of green algae, Chlamydomonas reinhardtii and Chlorella vulgaris. Correspondingly, antisera directed against pea chloroplastic CA, extracellular CA from C. reinhardtii and human CAII, showed no cross-hybridisation to the 26-kDa polypeptide in Coccomyxa. The 26-kDa protein was confirmed as being a CA by N-terminal sequencing of two internal polypeptide fragments and alignment of these sequences with that of previously identified CA proteins from several different species.Abbreviations CA carbonic anhydrase - CCM CO₂-concentrating mechanism - IEF isoelectric focusing - Rubisco ribulose-l,5-bisphosphate carboxylase/oxygenase We would like to thank Drs. Cecilia Forsman, Inga-Maj Johansson and Nalle Jonsson for their valuable advice concerning the isolation of CA. This work was supported by the Swedish Natural Research Council and Seth M. Kempes Memorial foundation.     

The relationship between carbonic anhydrase activity and glycolate excretion in the blue-green alga Coccochloris peniocystis

Summary The rate of glycolate excretion by Coccochloris peniocystis Kütz. cells incubated under conditions of low bicarbonate concentration and high light intensity was linear for only the initial 15 min of incubation and no additional glycolate accumulated in the medium after 20 min. Excretion was maximal in cells grown on 5% CO₂ in air when transferred to an incubation medium containing no added bicarbonate. The inhibitor INH (isonicotinyl hydrazide) had no measurable effect on the amount of glycolate released whereas HPMS (-hydroxy-2-pyridyl methanesulfonate) stimulated excretion 3-fold. Cells transferred to air from growth on 5% CO₂ in air increased in carbonic anhydrase activity, while a decrease occurred in the cells' ability to excrete glycolate. Cells grown on air and switched to 5% CO₂ in air showed an increase in their ability to excrete glycolate with a concomitant decrease in carbonic anhydrase activity. Diamox, a specific inhibitor of carbonic anhydrase, was found to stimulate excretion with both airgrown and 5% CO₂-grown cells which had been off 5% CO₂ for approximately 30 min. The rate of carbon fixation by 5% CO₂-grown cells put on air was found to rise over a 110 min period, corresponding to both the induction period of carbonic anhydrase and the period of decline in the ability of the cells to excrete glycolic acid. These results suggest that the absence of carbonic anhydrase in 5% CO₂-grown cells causes a stimulation of glycolate excretion when these cells are transferred to a low bicarbonate medium, because of an increased rate of glycolate formation due to the oxidation of ribulose diphosphate by molecular oxygen at low internal CO₂ concentrations.Abbreviations INH isonicotinyl bydrazide - HPMS -hydroxy-2-pyridyl methanesulfonate     

Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta)

In order to broaden our understanding of the eukaryotic CO₂-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO₂-concentrating mechanism when grown under limiting CO₂ conditions. Using mass-spectrometric measurements of ¹⁸O exchange from doubly labelled CO₂, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex. Received: 13 May 2000 / Accepted: 16 August 2000     

Photosynthesis and the intracellular inorganic carbon pool in the bluegreen alga Anabaena variabilis: Response to external CO2 concentration

The apparent photosynthetic affinity of A. variabilis to CO₂ is greatly affected by the CO₂ concentration in the medium during growth. Halfmaximal rate of photosynthetic O₂ evolution is achieved at 10 M and 100 M inorganic carbon (C_inorg) in cells grown at low-CO₂ (air) and high CO₂ (5% v/v CO₂ in air), respectively, whilst the maximum rate of photosynthesis is similar in both cases. Both high- and low-CO₂-grown Anabaena accumulate C_inorg within the cell; however, the rate of accumulation and the steady-state internal C_inorg concentration reached is much higher in low as compared with high-CO₂-grown cells. It is suggested that Anabaena cells actively accumulate C_inorg. Measurements of the kinetics of C_inorg transport indicate that the affinity of the transport mechanism for C_inorg is similar (K_m(C_inorg(150 M) in both high- and low-CO₂-grown cells. However, V _max is 10-fold higher in the latter case. It is suggested that this higher V _max for transport is the basis of the superior capability to accumulate C_inorg and the higher apparent photosynthetic affinity for external C_inorg in low-CO₂-grown Anabaena. Carbonic anhydrase activity was not detectable in Anabaena, yet both photosynthetic affinity to C_inorg in the medium (but not V _max) and the rate of accumulation of C_inorg were inhibited by the carbonic-anhydrase inhibitor ethoxyzolamide.Abbreviations C_inorg inorganic carbon - PEP phosphoenol pyruvate - RuBP ribulose-1,5-bisphosphate CIW-DPB Publication No. 682     

A 36 Kilodalton Limiting-CO(2) Induced Polypeptide of Chlamydomonas Is Distinct from the 37 Kilodalton Periplasmic Carbonic Anhydrase

Chlamydomonas reinhardtii possesses a CO₂-concentrating mechanism, induced by limiting CO₂, which involves active transport and accumulation of inorganic carbon within the cell. Synthesis of several proteins is induced by limiting CO₂, but, of those, only periplasmic carbonic anhydrase has an identified function in the system. No proteins involved in active transport have yet been identified, but induced, membrane-associated polypeptides, such as the 36 kilodalton polypeptide focused on in this paper, would seem to be candidates for such involvement. The 36 kilodalton polypeptide was shown to be synthesized de novo upon transfer of cells to limiting CO₂. It was purified using SDS-PAGE and used to produce polyclonal antibodies. Antibodies were used to confirm the air-specific nature of the polypeptide, its strict association with membrane fractions, and the time course of its induction. Using the antibodies, a single, 36 kilodalton polypeptide was found to be specifically immunoprecipitated from in vitro translation products of poly(A⁺) RNA from cells only after exposure to limiting CO₂. The absence of translatable mRNA for this polypeptide in CO₂-enriched cells indicated that regulation occurs at the level of message abundance. The antibodies were also used to demonstrate the distinction between the limiting-CO₂ induced 36 kilodalton polypeptide and the similarly sized, limiting-CO₂ induced periplasmic carbonic anhydrase.     

Historical perspective on microalgal and cyanobacterial acclimation to low- and extremely high-CO2 conditions

Reports in the 1970s from several laboratories revealed that the affinity of photosynthetic machinery for dissolved inorganic carbon (DIC) was greatly increased when unicellular green microalgae were transferred from high to low-CO₂ conditions. This increase was due to the induction of carbonic anhydrase (CA) and the active transport of CO₂ and/or HCO₃ ⁻ which increased the internal DIC concentration. The feature is referred to as the `CO₂-concentrating mechanism (CCM)'. It was revealed that CA facilitates the supply of DIC from outside to inside the algal cells. It was also found that the active species of DIC absorbed by the algal cells and chloroplasts were CO₂ and/or HCO₃ ⁻, depending on the species. In the 1990s, gene technology started to throw light on the molecular aspects of CCM and identified the genes involved. The identification of the active HCO₃ ⁻ transporter, of the molecules functioning for the energization of cyanobacteria and of CAs with different cellular localizations in eukaryotes are examples of such successes. The first X-ray structural analysis of CA in a photosynthetic organism was carried out with a red alga. The results showed that the red alga possessed a homodimeric β-type of CA composed of two internally repeating structures. An increase in the CO₂ concentration to several percent results in the loss of CCM and any further increase is often disadvantageous to cellular growth. It has recently been found that some microalgae and cyanobacteria can grow rapidly even under CO₂ concentrations higher than 40%. Studies on the mechanism underlying the resistance to extremely high CO₂ concentrations have indicated that only algae that can adopt the state transition in favor of PS I could adapt to and survive under such conditions. It was concluded that extra ATP produced by enhanced PS I cyclic electron flow is used as an energy source of H⁺-transport in extremely high-CO₂ conditions. This same state transition has also been observed when high-CO₂ cells were transferred to low CO₂ conditions, indicating that ATP produced by cyclic electron transfer was necessary to accumulate DIC in low-CO₂ conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of Acetazolamide (Diamox), Ethoxzolamide and High Levels of CO2 on Carbonic Anhydrase, Photosystem Activity, and Oxygen Evolution in Euglena gracilis

Investigations using steady-state culture conditions indicate that carbonic anhydrase activity is correlated to the photosynthetic rate in Euglena in some but not all circumstances. When cultures grown with 5% CO₂ were changed to air growth, the photosynthetic rate was independent of the carbonic anhydrase activity. While experiments using the inhibitor acetazolamide indicated a close correlation between photosynthetic capacity and carbonic anhydrase activity, the inhibitor was found to be nonspecific. Acetazolamide altered photosystem activities directly as measured by the photoreduction of DCPIP in chloroplast preparations, whole-cell fluorescence transients of chlorophyll a, and by whole chain photoelectron flow. Ethoxzolamide, another inhibitor of carbonic anhydrase, was also found to inhibit photosystem activities, i.e., the photoreduction of DCPIP, and in vivo photoelectron flow, at high concentrations. Cells grown in 5% CO₂ were less sensitive to the effects of acetazolamide than cells exposed to air. The rate of electron flow in chloroplasts from cells grown with 5% CO₂ and exposed to 10 mM acetazolamide was 2.5-fold faster than that of chloroplasts from air-grown cells exposed to the same concentration of inhibitor. The whole cell chlorophyll a fluorescence transients of cultures grown with high CO₂ were completely different from those of air-grown cells and also showed fewer effects on exposure to acetazolamide. These results suggest a reevaluation of the hypothesis that carbonic anhydrase activity regulates photosynthesis. It is also apparent that results from air-grown and 5% CO₂-grown cultures cannot be directly compared in such studies.     

Effects of Carbonic Anhydrase Inhibitors on Proton Exchange and Photosynthesis in Pea Protoplasts

At concentrations of 100–200 M, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO₂-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 M CO₂) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO₂-dependent O₂evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO₂-dependent O₂evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C₃-plants are discussed.     

Regulation of carbonic-anhydrase activity,inorganic-carbon uptake and photosynthetic biomass yield inChlamydomonas reinhardtii

The regulation of carbonic anhydrase by environmental conditions was determined forChlamydomonas reinhardtii. The depression of carbonic anhydrase in air-grown cells was pH-dependent. Growth of cells on air at acid pH, corresponding to 10 m CO₂ in solution, resulted in complete repression of carbonic-anhydrase activity. At pH 6.9, increasing the CO₂ concentration to 0.15% (v/v) in the gas phase, corresponding to 11 M in solution, was sufficient to completely repress carbonic-anhydrase activity. Photosynthesis and intracellular inorganic carbon were measured in air-grown and high-CO₂-grown cells using a silicone-oil centrifugation technique. With carbonic anhydrase repressed cells limited inorganic-carbon accumulation resulted from non-specific binding of CO₂. With air-grown cells, inorganic-carbon uptake at acid pH, i.e. 5.5, was linear up to 0.5 mM external inorganic-carbon concentration whereas at alkaline pH, i.e. 7.5, the accumulation ratio decreased with increase in external inorganic-carbon concentration. It is suggested that in air-grown cells at acid pH, CO₂ is the inorganic carbon species that crosses the plasmalemma. The conversion of CO₂ to HCO ₃ ^- by carbonic anhydrase in the cytosol results in inorganic-carbon accumulation and maintains the diffusion gradient for carbon dioxide across the cell boundary. However, this mechanism will not account for energy-dependent accumulation of inorganic carbon when there is little difference in pH between the exterior and cytosol.     

Arabidopsis thaliana carbonic anhydrase: cDNA sequence and effect of CO2 on mRNA levels

A full-length cDNA clone encoding carbonic anhydrase was isolated from an Arabidopsis thaliana (Columbia) leaf library. Comparison of the derived amino acid sequence obtained from this clone with those of pea and spinach reveals a considerable degree of identity. The carbonic anhydrase cDNA was used to probe the level of RNA encoding this protein in the leaves of plants grown in elevated CO₂ (660 ppm). We have found that under these conditions the steady-state level of carbonic anhydrase mRNA was increased in comparison with control plants grown in normal atmospheric concentrations of CO₂ (330 ppm). This raises the intruiging possibility that there exists in higher plants a mechanism for perceiving and responding to changes in environmental CO₂ concentrations at the genetic level.     

Carbonic Anhydrase and the Uptake of Inorganic Carbon by Synechococcus sp. (UTEX-2380)

We report the changes in the concentrations and ¹⁸O contents of extracellular CO₂ and HCO₃⁻ in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring ¹⁸O exchange between CO₂ and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO₂ hydration rate in different samples of cells that were grown on bubbled air (low-CO₂ conditions). This activity was only weakly inhibited by ethoxzolamide with an I₅₀ near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with CO₂-starved cells there is considerable generation of CO₂ from intracellular stores, a factor that can cause errors in measurement of net CO₂ uptake unless accounted for. It was demonstrated that use of ¹³C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO₂ readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although HCO₃⁻ was readily taken up by the cells, these experiments shown that there is no significant efflux of HCO₃⁻ from Synechococcus.     

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.     

Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii

We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO₂, but its synthesis was induced under low concentrations of CO₂ (air levels of CO₂). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO₂ to growth on air levels of CO₂, (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species.     

Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae

Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O₂ evolution, dissolved inorganic carbon accumulation, and photosynthetic CO₂ fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO₂-concentrating process. This COS inhibition can be attributed to inhibition of external HCO₃⁻ conversion to CO₂ and OH⁻ by the carbonic anhydrase component of an active CO₂ pump. At a low pH of 5 to 6, COS stimulated O₂ evolution during photosynthesis by algae with low CO₂ in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO₂. Although COS had less effect on HCO₃⁻ accumulation at pH 9 by a HCO₃⁻ pump in Scenedesmus, COS reduced O₂ evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO₂ and H₂S, its inhibition of the CO₂ pump activity and photosynthesis is not accurate, when measured by O₂ evolution, by NaH¹⁴CO₃ accumulation, or by ¹⁴CO₂ fixation.