Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii which Is Distinct from the Periplasmic Form of the Enzyme

A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system.     

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.     

Mass Spectrometric Measurement of Intracellular Carbonic Anhydrase Activity in High and Low C(i) Cells of Chlamydomonas: Studies Using O Exchange with C/O Labeled Bicarbonate

By measuring ¹⁸O exchange from doubly labeled CO₂ (¹³C¹⁸O¹⁸O), intracellular carbonic anhydrase activity was studied with protoplasts and chloroplasts isolated from Chlamydomonas reinhardtii grown either on air (low inorganic carbon [C_i]) or air enriched with 5% CO₂ (high C_i). Intact low C_i protoplasts had a 10-fold higher carbonic anhydrase activity than did high C_i protoplasts. Application of dextran-bound inhibitor and quaternary ammonium sulfanilamide, both known as membrane impermeable inhibitors of carbonic anhydrase, had no influence on the catalysis of ¹⁸O exchange, indicating that cross-contamination with extracellular carbonic anhydrase was not responsible for the observed activity. This intracellular in vivo activity from protoplasts was inhibited by acetazolamide and ethoxyzolamide. Intracellular carbonic anhydrase activity was partly associated with intact chloroplasts isolated from high and low C_i cells, and the latter had a sixfold greater rate of catalysis. The presence of dextran-bound inhibitor had no effect on chloroplast-associated carbonic anhydrase, whereas 150 micromolar ethoxyzolamide caused a 61 to 67% inhibition of activity. These results indicate that chloroplastic carbonic anhydrase was located within the plastid and that it was relatively insensitive to ethoxyzolamide. Carbonic anhydrase activity in crude homogenates of protoplasts and chloroplasts was about six times higher in the low C_i than in high C_i preparations. Further separation into soluble and insoluble fractions together with inhibitor studies revealed that there are at least two different forms of intracellular carbonic anhydrase. One enzyme, which was rather insoluble and relatively insensitive to ethoxyzolamide, is likely an intrachloroplastic carbonic anhydrase. The second carbonic anhydrase, which was soluble and sensitive to ethoxyzolamide, is most probably located in an extrachloroplastic compartment.     

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.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

Consumption of Carbonyl Sulphide by Chlamydomonas reinhardtii with Different Activities of Carbonic Anhydrase (CA) Induced by Different CO2 Growing Regimes

The green alga, Chlamydomonas reinhardtii, was grown under high and low CO₂ regimes inducing significantly different activities of the extracellular carbonic anhydrase (CA). In close relation to the CA activities, the algae exhibited different consumption rates of the climatically relevant atmospheric trace gas, carbonyl sulphide (COS), thus indicating that CA is responsible for uptake of COS from the medium.     

Dissolved inorganic carbon concentration mechanism in Chlamydomonas moewusii

The dissolved inorganic carbon concentrating mechanism(s) of Chlamydomonas moewusii CC 55 was compared with C. reinhardtii strain 137. C. moewusii is similar to C. reinhardtii with respect to maximal rates of photosynthetic oxygen evolution, CO₂ fixation, respiration, and the ability to efficiently concentrate inorganic carbon. C. moewusii has a low, but measurable amount of external carbonic anhydrase (CA) that was not inhibited by acetazolamide (AZ), an inhibitor of periplasmic carbonic anhydrase (pCA) in C. reinhardtii. The K_0.5(CO₂) for air-grown C. moewusii is about 1 μM and the algal cells accumulated dissolved inorganic carbon (DIC) to a level of about 1 mM in 60 s. AZ did not inhibit CO₂ fixation and the DIC accumulation by air-grown cells of C. moewusii. The K_0.5(CO₂) for both species remains constant from pH 6.5 to 9.5 while K_0.5(HCO₃^-) increased logarithmically, which indicates that CO₂ is the apparent inorganic carbon species that enters the cells in both algae. Antiserum prepared against the 37 kDa peptide of pCA from C. reinhardtii was immunoreactive with polypeptides of 26, 28, and 32 kDa in C. moewusii. The periplasmic carbonic anhydrase (pCA) activity is a part of the dissolved inorganic carbon concentrating mechanism in C. reinhardtii, but C  moewusii accomplished inorganic carbon accumulation without an AZ-sensitive pCA.     

A New Chloroplast Protein Is Induced by Growth on Low CO(2) in Chlamydomonas reinhardtii

The biosynthesis of a 36 kilodalton polypeptide of Chlamydomonas reinhardtii was induced by photoautotrophic growth on low CO₂. Fractionation studies using the cell-wall-deficient strain of C. reinhardtii, CC-400, showed that this polypeptide was different from the low CO₂-induced periplasmic carbonic anhydrase. In addition, the 36 kilodalton polypeptide was found to be localized in intact chloroplasts isolated from low CO₂-adapting cultures. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO₂-grown C. reinhardtii cells.     

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     

Effect of red and blue light on acclimation of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> to CO<Subscript>2</Subscript>-limiting conditions

Effects of red (RL) and blue (BL) light on acclimation of the unicellular green alga Chlamydomonas reinhardtii to the low level of ambient CO₂ were studied. C. reinhardtii cells grown at 5% CO₂ and under white light (170 μmol/(m²s)) had a relatively low activity of extracellular carbonic anhydrase (CA), a low affinity for dissolved inorganic carbon, and a low rate of photosynthesis under CO₂-limiting conditions. These cells readily started acclimation to the low CO₂ concentration when they were exposed to atmospheric air (～ 0.03% CO₂) under RL or BL (150 μmol/(m² s) each). The acclimation was manifested in a significant increase in the CO₂-limited rate of photosynthesis, the affinity for dissolved inorganic carbon, and the extracellular CA activity with no difference between RL-and BL-cells. Independently of light quality, the acclimation was completed for 5–7 h after cell exposure to air. As is evident from RL-and BL-dependent changes in the sum of chlorophylls and chlorophyll a/b ratio, transfer of C. reinhardtii cells to air and RL or BL triggered also the process of algal photosynthetic adaptation to light quality. However, this process did not interfere with acclimation to low CO₂ because started 4 h later. On the basis of similarity in the low CO₂-induced changes under RL and BL, it is concluded that acclimation of C. reinhardtii to CO₂-limiting conditions does not depend on light quality.     

The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO₂ hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K_m of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (K_i = 1.22 × 10^?3M), arsenate (K_i = 1.17 × 10^?3M), and molybdate (K_i = 1.34 × 10^?7M), with these inhibitors having no effect on the CO₂ hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO₂ hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO₂ hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.     

Acid-base disequilibrium in the venous blood of rainbow trout (Oncorhynchus mykiss)

Acid-base equilibria/disequilibria were evaluated in vivo in post-branchial arterial blood and pre-branchial venous blood of freshwater rainbow trout (Oncorhynchus mykiss). This was accomplished using arterial and venous extracorporeal circuits in conjunction with a stopped-flow apparatus. After the abrupt stoppage of circulating post-branchial blood within the stopped-flow apparatus, pH increased slowly ([Delta]pH = +0.032 ± 0.004 pH units; n = 15), thus confirming the existence of an acid-base disequilibrium state in the arterial blood of rainbow trout. The slow downstream pH changes were unaffected by prior treatment of fish with the carbonic anhydrase inhibitor benzolamide (1.2 mg kg^-1; [Delta]pH = +0.032 ± 0.01 pH units; n = 5) but were eliminated after intra-vascular injection of 10 mg kg^-1 bovine carbonic anhydrase ([Delta]pH = -0.011 ± 0.003 pH units; n = 8). These results demonstrate that the acid-base disequilibrium in the arterial blood reflects a total absence of extracellular carbonic anhydrase activity. Similar stopped-flow experiments revealed the existence of a reduced, yet significant, acid-base disequilibrium in the venous blood circulating within the caudal vein ([Delta]pH = +0.004 ± 0.003 pH units; n = 15). Selective inhibition of extracellular carbonic anhydrase using benzolamide did not significantly influence the magnitude of the venous pH disequilibrium ([Delta]pH = +0.007 ± 0.007 pH units; n = 8) whereas intra-vascular injection of carbonic anhydrase eliminated the pH disequilibrium. These results demonstrate that extracellular carbonic anhydrase, although reported to be present within the skeletal muscle of rainbow trout, does not accelerate post-capillary pH changes in the venous circulation.     

Adaptation to CO(2) Level and Changes in the Phosphorylation of Thylakoid Proteins during the Cell Cycle of Chlamydomonas reinhardtii

The photosynthetic performance of synchronously grown Chlamydomonas reinhardtii alternated rhythmically during the cell cycle. The activity of the “CO₂ concentrating mechanism” including the ability to accumulate CO₂ internally and the activity of carbonic anhydrase peaked after 6 to 9 hours of light and reached minimum after 6 to 9 hours of dark. Consequently, the apparent photosynthetic affinity to extracellular CO₂ alternated rhythmically. At the end of the dark period the cells behaved as if they were adapted to high CO₂ even though they were continuously aerated with air. Results from experiments in which the light or dark periods were extended bear on the interaction between the internal (cell cycle or biological clock) and the external (light) signal. The observed rhythmical alterations in photosynthetic V_max may result from changes in PSII activity. The latter may be partly explained by the capacity for phosphorylation of thylakoid proteins, which reached maximum after 9 hours of light and decreased toward the dark period.     

Carbonic anhydrase activity of Prochloron sp. isolated from an ascidian host

The prokaryotic algal symbiont of ascidians, Prochloron sp., was found to exhibit carbonic anhydrase activity which is largely associated with the cell surface. This extracellular carbonic anhydrase activity was inhibited, while the intracellular activity was not affected, by chloride or bromide. Acetazolamide and ethoxyzolamide inhibited carbonic anhydrase activity with I₅₀ values of 7×10^-4 and 3×10^-4M, respectively. These I₅₀ values are similar to those observed for intracellular carbonic anhydrases of Synechococcus sp. PCC7942, Chlamydomonas reinhardii and spinach.Abbreviations AZA acetazolamide - CA carbonic anhydrase - chl chlorophyll - EZA ethozyzolamide - I₅₀ concentration of an inhibitor required to cause 50% inhibition - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - U unit     

Properties of a Mutant from Synechocystis PCC6803 Resistant to Acetazolamide, an Inhibitor of Carbonic Anhydrase

A spontaneous mutant of the cyanobacterium Synechocystis PCC6803 was isolated for its resistance to acetazolamide, an inhibitor of carbonic anhydrase. The mutant showed a deficiency in oxygen exchange between CO₂ and H₂O, a lower level of stable internal CO₂ pool and a decreased capacity to adapt its photosynthetic affinity under limited inorganic carbon regime. The initial rate of uptake of inorganic carbon was identical to that of wild-type cells. It is demonstrated that the mutation affects the carbonic anhydrase activity. This could result from either of two impairments: a deficiency in the enzyme activity detectable by mass spectrometric determinations, or a modification of the cellular compartment in which the enzyme is located, preventing its activity.     

The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from Arabidopsis leaves

Supernatant obtained after high-speed centrifugation of disrupted thylakoids that had been washed free from extrathylakoid carbonic anhydrases demonstrated carbonic anhydrase activity that was inhibited by the specific inhibitors acetazolamide and ethoxyzolamide. A distinctive feature of the effect of Triton X-100 on this activity also suggested that the source of the activity is a soluble protein. Native electrophoresis of a preparation obtained using chromatography with agarose/mafenide as an affinity sorbent revealed one protein band with carbonic anhydrase activity. The same protein was revealed in a mutant deficient in soluble stromal carbonic anhydrase β-CA1, and this indicated that the newly revealed carbonic anhydrase is not a product of the At3g01500 gene. These data imply the presence of soluble carbonic anhydrase in the thylakoid lumen of higher plants.     

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.     

Photosynthetic response of three climber plant species to osmotic stress induced by polyethylene glycol (PEG) 6000

We examined the photosynthesis response to osmotic stress in three climber plant species, Pharbitis nil (Linn.) Choisy, Lonicera japonica Thunb, and Parthenocissus tricuspidata (Sieb.et Zucc.) Planch. All climber plants were exposed to osmotic stress induced by polyethylene glycol (PEG) 6000 at 4 levels (slight, moderate, severe osmotic and the control) for 30?days. Photosynthesis response was determined by measuring leaf photosynthesis, chlorophyll fluorescence, carbonic anhydrase activity and stable carbon isotope ratios. P. nil maintained high photosynthetic activity under long-term moderate osmotic stress due to both stable photosystem II photochemical efficiency and high carbonic anhydrase activity. L. japonica maintained high photosynthetic activity under long-term moderate stress due to high carbonic anhydrase activity rather than photosystem II photochemical efficiency. P. tricuspidata tolerated only short-term moderate osmotic stress and long-term slight osmotic stress because its response was mainly stomatal limitation, with the lowest photosynthetic activity and hardly any carbonic anhydrase activity. Carbonic anhydrase activity was inversely correlated with stable carbon isotope ratios. The regulation by carbonic anhydrase was probably the reason for P. nil and L. japonica to tolerate long-term moderate osmotic stress. The selection on the species should consider the differential adaptation mechanism to osmotic stress during the development of drought-resistant plants.     

Induction of Inorganic Carbon Accumulation in the Unicellular Green Algae Scenedesmus obliquus and Chlamydomonas reinhardtii

The induction of a dissolved inorganic carbon (DIC) accumulating mechanism in the two algal species Scenedesmus obliquus (WT) and Chlamydomonas reinhardtii (137 c+) was physiologically characterized by monitoring DIC uptake kinetics at a low and constant DIC concentration (120-140 micromolar), after transfer from high-DIC culturing conditions. A potentiometric titration method was used to measure and calculate algal DIC uptake. Full acclimation to low-DIC conditions was obtained within a period of 90 min, after which time the DIC uptake had been increased 7 to 10 times. Experiments were also conducted in the presence of inhibitors against DIC accumulation. The inhibitor of extracellular carbonic anhydrase (CA), acetazolamide (50 micromolar), inhibited the adaptation partly, while the inhibitor of both extra- and intracellular CA, ethoxyzolamide (50 micromolar) totally inhibited the acclimation. Cycloheximide (10 micrograms per milliliter), which inhibits protein synthesis on cytoplasmic ribosomes, and vanadate (180 micromolar), which inhibits the plasmamembrane bound ATPase, also inhibited the acclimation totally. These results taken together suggest that the algae are dependent on intracellular CA, plasmamembrane bound ATPase, and de novo protein synthesis for DIC accumulation. Also, these components are more important than extracellular CA for the overall function of the DIC-accumulating mechanism.     

A role for mitochondrial carbonic anhydrase in limiting CO2 leakage from low CO2-grown cells of Chlamydomonas reinhardtii

A model is presented which quantifies a possible role for the carbonic anhydrase in the mitochondrial matrix of Chlamydomonas reinhardtii which incorporates the observation that the expression of this enzyme is increased under growth conditions in which the expression of the carbon dioxide-concentrating mechanism is increased. It is assumed that the inorganic carbon enters the cytosol from the medium, and leaves the cytosol to the plastids, as HCO₃⁻ and that there is negligible carbonic anhydrase activity in the cytosol. The role of the mitochondrial carbonic anhydrase is suggested to be the conversion to HCO₃^– of the CO₂ produced in the mitochondria in the light from tricarboxylic acid cycle activity and from decarboxylation of glycine in any photorespiratory carbon oxidation cycle activity which is not suppressed by the carbon concentrating mechanism. If there is a HCO₃⁻ channel in the inner mitochondrial membrane then almost all of the inorganic carbon leaves the mitochondria as HCO₃⁻, thus limiting the potential for CO₂ leakage through the plasmalemma. This mechanism could increase inorganic C supply to ribulose bisphosphate carboxylase-oxygenase by some 10% at the energetic expense of less than 1% of the total ATP generation by plastids plus mitochondria.