Mechanism of Inorganic Carbon Uptake in Chlorella saccharophila: The Lack of Involvement of Carbonic Anhydrase

The acid-tolerant green alga Chlorella saccharophila maintainedphotosynthesis and accumulated intracellular pools of inorganiccarbon over a a range of external pH from 4.0 to 7.5. This accumulationwas unaffected by treatment of cells with 10 mol m^–3 acetazolamide(AZA). Cells grown at alkaline pH had extracellular carbonicanhydrase (CA), but CA activity was repressed when cells weregrown at pH 5.0. Acid-grown cells retained a high affinity forCO₂, both at acid and alkaline pH, and the ability to accumulateinorganic carbon. Rates of photosynthesis of acid-grown cellsand alkaline-grown AZA-treated cells at pH 8.0 were 2.5-foldhigher than the rate of CO₂ supply from the uncatalysed dehydrationof , indicating that the cells can take up as a source of substrate for photosynthesis. Isotopic disequilibrium experiments with acid-grown cells maintainingsteady-state photosynthesis at pH 7.5 demonstrate that ¹⁴C from¹⁴CO₂ was taken up more rapidly than from H¹⁴. This uptake takes place against a concentration gradient. Theseresults demonstrate that C. saccharophila cells have activetransport systems for the uptake of both CO₂ and and both operate without the mediation of CA. Key words: Bicarbonate transport, carbon dioxide, carbonic anhydrase, Chlorella saccharophila, inorganic carbon accumulation     

Effect of External pH on the Internal pH of Chlorella saccharophila

The overall internal pH of the acid-tolerant green alga, Chlorella saccharophila, was determined in the light and in the dark by the distribution of 5,5-dimethyl-2-[¹⁴C]oxazolidine-2,4-dione ([¹⁴C]DMO) or [¹⁴C]benzoic acid ([¹⁴C]BA) between the cells and the surrounding medium. [¹⁴C]DMO was used at external pH of 5.0 to 7.5 while [¹⁴C]BA was used in the range pH 3.0 to pH 5.5. Neither compound was metabolized by the algal cells and intracellular binding was minimal. The internal pH of the algae obtained with the two compounds at external pH values of 5.0 and 5.5 were in good agreement. The internal pH of C. saccharophila remained relatively constant at pH 7.3 over the external pH range of pH 5.0 to 7.5. Below pH 5.0, however, there was a gradual decrease in the internal pH to 6.4 at an external pH of 3.0. The maintenance of a constant internal pH requires energy and the downward drift of internal pH with a drop in external pH may be a mechanism to conserve energy and allow growth at acid pH.     

Quantification of the Contribution of CO2, HCO3-, and External Carbonic Anhydrase to Photosynthesis at Low Dissolved Inorganic Carbon in Chlorella saccharophila

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.     

A New Method for Purification of Carbonic Anhydrase Isozymes by Affinity Chromatography

A new affinity gel for purification of carbonic anhydrase isozymes was prepared using EUPERGIT C-250L derivatized with p-aminobenzenesulfonamide, an inhibitor of carbonic anhydrase. The binding capacity of the affinity gel was determined at different temperatures, pH values, elution buffers, and ionic strengths. Human carbonic anhydrase isozymes (HCA I and HCA II) and bovine carbonic anhydrase (BCA) were purified in high yields from erythrocytes.     

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.     

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.     

Arginine utilization by Chlorella autotrophica and Chlorella saccharophila

Chlorella saccharophila can utilize the amino acids arginine, glutamate. ornithine and proline as sole sources of nitrogen for growth. By comparison C. autotrophica utilized only arginine and ornithine. Following osmotic shock of Chlorella autotrophica from 50 to 150% artificial seawater rapid synthesis of proline (the main osmoregulatory solute in this alga) occurred in cells grown on arginine or citrulline. However, little proline synthesis occurred in ornithine-grown cells. Distribution of radiolabelled carbon from [¹⁴C]-arginine assimilation following osmotic shock of C. autotrophica agrees with the following pathway of arginine utilization: arginine→citrulline→ornithine→glutamate semialdehyde→pyrroline-5-carboxylate→proline. These 4 steps are catalysed by arginine deiminase (EC 3.5.3.6), citrullinase (EC 3.5.1.20), ornithine transaminase (EC 2.6.1.13) and pyrroline-5-carboxylate reductase (EC 1.5.1.2), respectively. Of these 4 enzymes, only arginine deiminase and pyrroline-5-carboxylate reductase were detected in the crude extract of the 2 Chlorella species. Arginine deiminase did not require specific cations for optimal activity. The deimi-nase showed maximal activity at pH 8.0 and followed Michaelis-Menten kinetics with an apparent K_m for L-arginine of 0.085 m M for the C. autotrophica enzyme and 0.097 m M for that of C. saccharophila. The activity of arginine deiminase was not influen-ced by growing C. saccharophila on arginine. Ornithine competitively inhibited arginine deiminase with an apparent K, of 2.4 m M for the C. autotrophica enzyme, and 3.8 m M for that of C. saccharophila . Arginine utilization by Chlorella is discussed in relation to that of other organisms.     

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii with a Carbonic Anhydrase-Directed Photoaffinity Label

A carbonic anhydrase (CA)-directed photoaffinity reagent, 125I-labeled p-aminomethylbenzenesulfonamide-4-azidosalicylamide,was synthesized and shown to derivatize periplasmic CA in the unicellular green alga Chlamydomonas reinhardtii. The photoderivatization of purified C. reinhardtii periplasmic CA or intact C. reinhardtii cells with the reagent resulted in the modification of the large (37 kD) subunit of the enzyme. Photoderivatization of proteins in lysed C. reinhardtii cells also resulted in the specific labeling of a polypeptide of 30 kD. Centrifugation of the cell extract prior to photoaffinity labeling revealed that the labeled peptide was present predominantly in a particulate fraction. The photoaffinity-labeled 30-kD polypeptide was not observed in extracts from a mutant of C. reinhardtii that is believed to be deficient in an intracellular form of CA. These results provide evidence that the 30-kD polypeptide, which is photoaffinity labeled in lysed C. reinhardtii cells, is an intracellular form of CA.     

Factors Controlling Induction of Carbonic Anhydrase and Efficiency of Photosynthesis in Chlorella vulgaris llh Cells

When Chlorella vulgaris llh cells which had been grown in airenriched with 2–4% CO₂ (high-CO₂ cells) were bubbled withair containing ca. 400 ppm CO₂, illumination at an intensityas low as the light compensation point (350 lux) was sufficientto increase the photosynthetic rate under limiting CO₂ concentrations.The same treatment induced carbonic anhydrase (CA) activity.The induction of CA activity and increase in photosyntheticrate at limiting CO₂ concentrations were observed in the presenceof 10 µM DCMU which completely inhibits photosynthesis.These results indicate that photosynthetic electron transportis not involved in CA induction in Chlorella vulgaris llh cells.The parallelism between the changes in CA activity and the rateof photosynthesis under limiting CO₂ concentrations agree withthe previous conclusion that the transport of CO₂ from outsideto the site of CO₂ fixation is facilitated by CA and hence lowersthe apparent K_m(CO₂) for photosynthesis. (Received December 24, 1982; Accepted May 10, 1983)     

Carbonic Anhydrase in Chlamydomonas reinhardtii II. Requirements for Carbonic Anhydrase Induction

Carbonic anhydrase (CA) activity in wild type cells of Chlamydomonasreinhardtii was low when cells were cultured under 2% CO₃ inthe light. When the gas phase was changed to air, CA activityincresaed as much as 20 fold over the next 24 hours. In contrast,CA activity did not change markedly in cells of the mutantspet 20-8 (PS II-negative), lip 10-2 (photophosphorylation-negative),and F60 (phosphoribulokinase-negative), when they were subjectedto the same induction regimen. DCMU (10^–5 M) and cydoheximide(3 µg/ml) severely inhibited the induction in wild typecells. No induction occured when CO₂ concentration was loweredin darkness. ³Present adress: Photoconversion Research Branch, Solar EnergyResearch Institute, Golden, Colorado 80401, USA. (Received June 7, 1982; Accepted December 25, 1982)     

Carbonic Anhydrase in Marine Algae

An electrometric system for determination of carbonic anhydrase activity was constructed. Enzyme activity was assayed in homogenates of marine macroscopic Chlorophyta, Rhodophyta, and Phaeophyta. Plants surveyed included Ulva expansa (Setchell) Setchell and Gardner, Codium fragile (Suringar) Hariot, Enteromorpha sp., Chaetomorpha torta (Farlow) McClatchie (Chlorophyta); Laurencia papillosa (Greville). Plocamium coccineum var. pacificum (Kylin) Dawson, Pterocladia capillacea (Gmelin) Bornet and Thuret. Gigartina armata J. Agardh (Rhodophyta); Eisenia arborea Areschoug and Macrocystis pyrifera (Linnaeus) C. A. Agardh (Phaeophyta). Activity was present in all algae; in the Phaeophyta this could be demonstrated only after dialysis. p-Chloromercuriphenylsulfonic acid (10⁻⁴m) decreased activity in 1 species, Plocamium; this inhibition could be almost completely overcome with the addition of 10⁻³m dithiothreitol. In 2 green and 2 red algae assayed for sensitivity to acetazolamide (Diamox), inhibition was complete at 10⁻⁴m concentration of inhibitor. Dithiothreitol at a concentration of 10⁻³m did not enhance activity in any of the homogenates, and was not necessary for enzyme expression.     

The Effect of External pH on the Apparent CO2 Affinity of Chlorella saccharophila

The carbon dioxide compensation point of the unicellular greenalga, Chloretla saccharophila, was determined in aqueous mediumby a gas chromatographic method. Compensation points decreasedmarkedly from 63 cm³ m^–3 at an external pH of 4.0 to 3.2cm³ m^–3 at pH 8.0 and were not affected by the O₂ concentrationof the medium. The calculated CO² concentration required tosupport the half-maximum photosynthetic rate of the algal cellsranged from 6.0 mmol m_–3 at an external pH of 60 to 1.5mmol m^–3 at pH 8.0 and these values were not affectedby O₂ concentration. The K_m(CO₂) of nbulose-l,5-bisphosphatecarboxylase isolated from cells grown either at pH 4.0 or pH8.0 was determined to be 64 mmol m^–3. These results indicatethat loss of CO₂ by photorespiration does not occur in C. saccharophilacells at acid pH and the disparity between the apparent affinityfor CO₂ of the intact cells and that of the carboxylase indicatesthe operation of a ‘CO₂ concentrating mechanism’in this alga at acid pH. Key words: Acidophilic alga, bicarbonate transport, Chlorella saccharophila, compensation point, CO₂ affinity, PH, RuBP carboxylase     

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.     

Carbonic Anhydrase in Anoxygenic Phototrophic Bacteria

Microbiology - The genes encoding carbonic anhydrase (CA) were found in all anoxygenic purple bacteria. The genes of α- and β-CA were found in purple nonsulfur bacteria of the class...     

Carbonic Anhydrase Inhibitors: Inhibition of Isozymes I,II and IV by Sulfamide and Sulfamic Acid Derivatives

Abstract

Sulfamide and sulfamic acid are the simplest compounds containing the SO₂NH₂ moiety, responsible for binding to the Zn(II) ion within carbonic anhydrase (CA, EC 4.2.1.1) active site, and thus acting as inhibitors of the many CA isozymes presently known. Here we describe two novel classes of CA inhibitors obtained by derivatizations of the lead molecules mentioned above. The new compounds, possessing the general formula RSO₂NH-SO₂X (X = OH, NH₂), were obtained by reaction of sulfamide or sulfamic acid with alkyl/arylsulfonyl halides or aryl-sulfonyl isocyanates. A smaller series of derivatives has been obtained by reaction of aromatic aldehydes with sulfamide, leading to Schiff bases of the type ArCH=NSO₂NH₂. All the new compounds act as strong inhibitors of isozymes I, II and IV of carbonic anhydrase. Their mechanism of CA inhibition is also discussed based on electronic spectroscopic measurements on adducts with the Co(II)-substituted enzyme. These experiments led to the conclusion that the new inhibitors are directly coordinated (in a monodentate manner) to the metal ion within the enzyme active site, similarly to the classical inhibitors, the aromatic/heterocyclic sulfonamides.     

Up-Regulation of Carbonic Anhydrase Isozyme IV in CNS Myelin of Mice Genetically Deficient in Carbonic Anhydrase II

Abstract: Carbonic anhydrase (CA) II is the major CA isozyme in the brain, where it participates in acid-base homeostasis, fluid transport, and myelin synthesis. The CA II deficiency [CA(II)D] mutation in the mouse results in structural changes in the glial cells in the CNS and in decreased susceptibility to seizures, but no detectable changes in myelin yield and ultrastructure. We compared the CA isozymes in brain and spinal cord fractions, as well as in purified myelin, between CA(II)D and control mice. CA(II)D resulted in a much lower total CA specific activity in all tissues examined but in higher CA IV specific activities in soluble and membrane-associated fractions and pure myelin. Western blots of purified myelin showed a band corresponding to CA IV in CA(II)D mice. This band was weak or undetectable in myelin samples from normal mice. Immunocytochemical staining demonstrated CA IV in oligodendrocytes and myelinated tracts in normal mouse brains and stronger staining of the same structures in brains of CA(II)D mutants. We conclude that CA(II)D mutation in the mouse up-regulates CNS CA IV. We speculate that this up-regulation could mitigate the effect of CA(II)D on myelin formation and maintenance.     

碳酸酐酶Ⅳ与人类疾病

碳酸酐酶4（carbonic anhydrase 4,CAIV）是12种人类相关碳酸酐酶中的一员,主要通过糖基磷脂酰基醇锚定在细胞质膜上。哺乳动物的多个器官有CAIV的表达。CAIV高效催化CO2的水化和HCO3–的脱水反应,在尿液酸化、肺泡换气等生理反应中起重要作用。CAIV基因表达的变化、结构稳定性的破坏和活性的改变等均与人类多种疾病的发生发展密切相关。CAIV还可以作为药物治疗靶点应用于疾病治疗。为此,该文就CAIV与人类相关疾病发生的病理生理机制作一综述。     

Carbonic Anhydrase Polymorphism in Cattle and Swine

Two electrophoretically different carbonic anhydrase (Ca) isoenzymes have been demonstrated in cattle where their presence is presumed to be controlled by codominant allelic genes (Sartore & Bernoco 1966). Previous reports of Ca polymorphism in swine are not available.