Carbonic anhydrase II in new world monkeys

Carbonic anhydrase II electrophoretic patterns were investigated in 3113 animals belonging to 12 genera and 24 species of New World primates. Polymorphism was detected in 13 species. A total of 24 different alleles was postulated to explain the variability found; the genusAotus showed the highest (eight) number of such alleles. Three genera of the family Callitrichidae (Callithrix, Saguinus, andCebuella) presented five alleles that were not found among the Cebidae. Important markers at the generic level were observed inCallicebus (CA2 ^*6 andCA2 ^*12),Cebus (CA2 ^*10, CA2^*16, andCA2 ^*21), andAotus (CA2^*3, CA2^*4, CA2^*5, CA2^*9, CA2^*15, CA2^*17, CA2^*22, CA2^*23). CA2^*13 seems to be the most common allele among the Cebidae; six genera of this family showed frequencies higher than 70% of it.     

碳酸酐酶的生理功能、多样性及其在CO2捕集中的应用

碳酸酐酶（carbonic anhydrase）作为一种活性中心含有锌离子的金属酶,能够可逆催化CO2生成碳酸氢盐的水合反应,该反应在生物体内承担着多样的生理学功能,具有高度的生物学意义。除广泛存在于真核生物以外,该酶在淡水、海水、嗜常温、嗜热、厌氧、好氧、致病、产酸、自养、异养等多种原核微生物中也有广泛的分布,并参与光合作用、呼吸作用和以CO2作为底物的反应,维持生理pH以及离子转运等生理过程。近年来,随着温室效应的日益加剧．生物固定CO2作为该酶的一种全新应用引起了研究者的广泛关注。回顾了碳酸酐酶作为催化剂参与CO2固定过程的历史、现状和最新发现,同时展望了未来应用的趋势。     

Purification, Properties, and Localization of Two Carbonic Anhydrases from Amaranthus cruentus Leaves

The method has been developed for obtaining two purified forms of carbonic anhydrase (CA, A and B forms) from amaranth (Amaranthus cruentus L.) leaves. The method includes precipitation with ammonium sulfate, fractionation by ion-exchange chromatography on DEAE Sephadex A-50, gel filtration on AcA-34 ultragel, and ion-exchange chromatography on DEAE cellulose. The molecular weights of A and B forms were different and equaled to 151 and 251 kD, respectively. The results suggest that SH groups and zinc play important roles in the catalytic activity of both CA forms. Both forms exhibited a high hydratase activity and did not represent allosteric enzymes. However, the catalytic properties of A form, evaluated from the pH dependence of kinetic parameters, differed from those of B form, which was apparently caused by dissimilar structures of these forms. Furthermore, the A form was localized in chloroplast membranes of bundle sheath cells, whereas B form was a soluble enzyme located in the cytoplasm of mesophyll cells.     

CO2 is the main inorganic C species entering photosynthetically active leaf protoplasts of the freshwater macrophyte Ranunculus penicillatus ssp. pseudofluitans

Submerged aquatic macrophytes growing in water where free CO₂ is unavailable (above pH 8·2) must use mechanisms to supply external dissolved inorganic carbon in a form available to chloroplasts (CO₂). Active transport of HCO₃^– across the plasmalemma has not been proven to be widespread in aquatic macrophytes and catalytic conversion of HCO₃^– to CO₂ is the usual supply mechanism in submerged macrophytes. The interaction of leaf form and function in this respect was investigated in the linear, submerged leaves of Ranunculus penicillatus (Dumort.) Bab ssp. pseudofluitans (Syme) S.Webster. Viable protoplasts were isolated using a mixture of cell wall degrading enzymes optimized for this species. Protoplast viabilities greater than 80% after 5 h of isolation were achieved. Photosynthetic rates of isolated protoplasts were comparable with that of intact plant tissue. Results of carbon isotopic disequilibrium experiments showed that CO₂ was the preferred species of dissolved inorganic carbon for photosynthesis by protoplasts and that HCO₃^– which predominates in the plant’s natural environment mainly contributes by supplying CO₂ outside the cells.     

微藻碳酸酐酶的特性及其环境调控

本文概述微藻碳酸酐酶的性质、种类、分布、分离纯化和环境调控的研究进展,并对未来有关微藻碳酸酐酶研究中需要探讨的问题作了展望。     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

岩溶环境因子对细菌胞外碳酸酐酶表达及活性的影响

以从西南岩溶生态系统分离出的一株细菌（编号GLRT102Ca）为例,研究了温度、pH、金属离子和阴离子等主要岩溶环境因子对其胞外碳酸酐酶（CA）表达及活性的影响。结果照乐,任实验温度（10℃～50℃）和pH（5.5～9.0）范围内,实验菌株均能表达出不同程度的胞外CA活性,其中20℃～30℃以及中性偏碱性条件下的胞外酶活性较高。钙、镁、锌、钻等4种金属离子以及SO4^2-、H2PO4^-、NO3^-、NO2^-、Cl^-、Br^-、I^-和HCO3^-等8种阴离子往实验浓度范围内一般都能促进胞外酶活性的表达,但表达较高活性所需的离子浓度因不同离子而异。以上结果为进一步研究微生物碳酸酐酶的岩溶作用提供了一定的理论依据。     

Production,purification, and characterization of a fusion protein of carbonic anhydrase from Neisseria gonorrhoeae and cellulose binding domain from Clostridium thermocellum

Carbon dioxide capture technologies have the potential to become an important climate change mitigation option through sequestration of gaseous CO₂. A new concept for CO₂ capture involves use of immobilized carbonic anhydrase (CA) that catalyzes the reversible hydration of CO₂ to HCO₃^? and H⁺. Cost‐efficient production of the enzyme and an inexpensive immobilization system are critical for development of economically feasible CA‐based CO₂ capture processes. An artificial, bifunctional enzyme containing CA from Neisseria gonorrhoeae and a cellulose binding domain (CBD) from Clostridium thermocellum was constructed with a His₆ tag. The chimeric enzyme exhibited both CA activity and CBD binding affinity. This fusion enzyme is of particular interest due to its binding affinity for cellulose and retained CA activity, which could serve as the basis for improved technology to capture CO₂ from flue gasses. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

杜氏盐藻两种碳酸酐酶基因启动子的克隆和功能研究

将克隆得到的杜氏盐藻DCA7和CA基因的启动子区与bar基因和NOS polyA终止子片段融合,分别构建成pMDDC-B和pMDC-B转基因杜氏盐藻表达载体。用基因枪法将两种表达载体转化人杜氏盐藻细胞,通过除草剂草丁膦筛选培养获得转化藻株,对转化藻株进行分析。对转化杜氏盐藻藻株的筛选培养结果表明：pMDDC-B和pMDC-B载体中的外源bar基因能在杜氏盐藻细胞中稳定或瞬时表达。同时在氦气压力为690kPa条件下,微弹轰击2次比微弹轰击1次或3次的效果更好。对pMDDC-B转化杜氏盐藻得到的稳定表达的转化藻株进行的PCR和Southern印迹分析的结果表明：外源的bar基因确已整合到杜氏盐藻基因组中。Northern印迹分析表明：DCA7基因启动子驱动bar基因在杜氏盐藻细胞中的表达效率受氯化钠浓度梯度调控。推测首次克隆得到的DCA7基因启动子可能是一种活性高、安全性好的高渗诱导性启动子;杜氏盐藻DCA7和CA基因启动子区的GT高度重复序列,可能与杜氏盐藻高度耐盐的分子机制有关。     

The active uptake of carbon dioxide by the marine diatoms Phaeodactylum ticornutum and Cyclotella sp.

Mass spectromelry has been used to investigate the uptake of CO₂ by two marine diatoms, Phaeodactylum tricornutum and Cyclotella sp. The time course of CO₂ formation in the dark after addition of 100 mmol m^?3 dissolved inorganic carbon (DIC) to cell suspensions showed that external carbonic anhydrase (CA) was not present in cells of P. tricornutum but was present in Cyclotella sp. In the absence of external CA, or when it was inhibited by 5 mmol m^?3 acetazolamide, cells of both species preincubated with 100 mmol m^?3 DIG rapidly depleted almost all of the free CO₂ (3·2mmol m^?31 at pH7·5) from the suspending medium within seconds of illumination and prior to the onset of steady-state photosynthesis. Addition of bovine CA quickly restored the HCO₃^?–CO₂ equilibrium in the medium, indicating that the initial depletion of CO₂ resulted from the selective uptake of CO₂ rather than uptake of all DIG species. Transfer of cells to the dark caused a rapid increase in the CO₂ concentration in the medium, largely as a result of the efflux of unfixed inorganic carbon from the cells. The measured CO₂ uptake rates for both species accounted for 50% of the total DIG uptake at HCO₃^?–CO₂ equilibrium, indicating that HCOHCO₃^? was also being taken up. These results indicate that both Phaeodactylum tricornutum and Cyclotella sp. have the capacity to transport CO₂ actively against concentration and pH gradients.     

Three new aromatic sulfonamide inhibitors of carbonic anhydrases I,II, IV and XII

4-Sulfamoyl-N-(3-morpholinopropyl)benzamide (I-1), N-(3-morpholinopropyl)benzene-1,4-disulfonamide (I-2) and N-(4-diethylaminoethoxybenzyl)benzene-1,4-bis(sulfonamide (I-3), were prepared and assayed as inhibitors of four carbonic anhydrase (CA) isoenzymes hCA I, hCA II, hCA IV and hCA XII. These compounds exhibited nanomolar half maximal inhibitory concentration (IC₅₀) ranging from 58 to 740 nmol/L. All three aromatic sulfonamides show different activities for the isoenzymes studied with lowest affinity against isoenzyme hCA XII.     

Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: molecular basis of isozyme-drug discrimination.

Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the "130's segment" and the residue-63 loop (these may affect the nearby catalytic proton shuttle, His-64). Similar to human CAIV, the C-terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.     

Competitive Inhibitory Effects of Acetazolamide upon Interactions with Bovine Carbonic Anhydrase II

Sulfonamide drugs mediate their main therapeutic effects through modulation of the activity of membrane and cytosolic carbonic anhydrases. How interactions of sulfonamide drugs impact structural properties and activity of carbonic anhydrases requires further study. Here the effect of acetazolamide on the structure and function of bovine carbonic anhydrase II (cytosolic form of the enzyme) was evaluated. The Far-UV CD studies indicated that carbonic anhydrase, for the most part, retains its secondary structure in the presence of acetazolamide. Fluorescence measurements using iodide ions and ANS, along with ASA calculations, revealed that in the presence of acetazolamide minimal conformational changes occurred in the carbonic anhydrase structure. These structural changes, which may involve spatial reorientation of Trp 4 and Trp 190 or some other related aminoacyl residues near the active site, considerably reduced the catalytic activity of the enzyme while its thermal stability was slightly increased. Our binding results indicated that binding of acetazolamide to the protein could occur with a 1:1 ratio, one mole of acetazolamide per one mole of the protein. However, the obtained kinetic results supported the existence of two acetazolamide binding sites on the protein structure. The occupation of each of these binding sites by acetazolamide completely inactivates the enzyme. Advanced analysis of the kinetic results revealed that there are two substrate (p-NPA) binding sites whose simultaneous occupation is required for full enzyme activity. Thus, these studies suggest that the two isoforms of CA II should exist in the medium, each of which contains one substrate binding site (catalytic site) and one acetazolamide binding site. The acetazolamide binding site is equivalent to the catalytic site, thus, inhibiting enzyme activity by a competitive mechanism.     

Carbonic Anhydrase in Subchloroplast Particles of Pea Plants

The partitioning of carbonic anhydrase (CA) activity in chloroplasts isolated from 10–14-day-old pea (Pisum sativum L.) seedlings was investigated. The effect of CA inhibitors on the kinetics of chlorophyll fluorescence in photosystem II (PSII) preparations was also studied. The activity of CA was detected in fractions of soluble proteins and in the polypeptide complexes of the PSI and PSII. Isolated particles of photosystems retained a high photochemical activity similar to that of intact chloroplasts and the high level of polyunsaturated fatty acids. The association of CA with the particles of PSII (PSII-CA) was also tested by Western-blot analysis using antibodies against PSII-CA (Cah3) from Chlamydomonas reinhardtii. The PSII particles isolated with Triton X-100 (T-20) showed a higher activity of the enzyme as calculated on a protein basis than the DT-20 particles isolated with digitonin and Triton X-100. This difference seems to be related to the higher degree of nativity of the chloroplast T-20 fragments as compared to DT-20 particles. The higher level of chlorophyll per reaction center as well as the higher content of chlorophyll b and lipid fatty acids as calculated on protein basis, in particular of E-16:113 acid, which stabilizes the oligomeric structure of the light-harvesting complex of the PSII, also confirms this suggestion. The activity of CA was not detected in the DT-20 preparations treated with Tris–HCl to eliminate manganese ions. This is likely to indicate that one of the extrinsic polypeptides of PSII exhibits CA activity. Specific inhibitors of CA (acetazolamide and imidazole) inhibited the photoinduced yield of chlorophyll fluorescence (F). This might be determined by damaging the water-oxidizing system or its interaction with the PSII reaction centers. The functional role of PSII-CA for ₂-concentrating in carboxylation sites as well as its role in the coupling of light and dark reactions in chloroplasts is discussed.     

Influence of Chirality of Benzimidazole Amine Hybrids on Inhibition of Human Erythrocytes Carbonic Anhydrase I,II and Acetylcholinesterase

Novel chiral benzimidazole amine hybrids ( 4a – 4d ) were synthesized from commercially available amine [(R)- (+)-phenylethylamine, (−) (S)-(-)-phenylethylamine, (−) (R)-(-)-cyclohexylethylamine, (S)-(+)-cyclohexylethylamine] and 2-(chloromethyl)-N-tosyl-1H-benzimidazole. The synthesized compounds ( 4a – 4d ) were characterized by IR, NMR, and LC/MS analysis. The inhibitory effect of 4a – 4d on human erythrocytes carbonic anhydrase I (hCA-I), II (hCA-II), and acetylcholinesterase (AChE) activity was investigated. For hCA-I, the IC₅₀ values of 4a – 4d were found to be 4.895 μM, 1.750 μM, 0.173 μM, and 0.620 μM, respectively, and for hCA-II, the IC₅₀ values of 4a – 4d were found to be 0.469 μM, 0.380 μM, 0.233 μM, 0.635 μM, respectively. Furthermore, IC₅₀ values of 4a – 4d on AChE were found as 87.5 nM, 100 nM, 26.92 nM, and 100 nM, respectively. In addition, molecular docking analysis was performed to evaluate the affinity of 4a – 4d against hCA-I, hCA-II, and AChE and explain their binding interactions.     

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.     

碳酸酐酶在中肋骨条藻光合作用中的作用

探讨了在正常空气条件下生长的中肋骨条藻(Skeletonema costatum)的碳酸酐酶(CA)在其光合固碳中的作用.在中肋骨条藻的胞内和胞外均有CA活性,但胞外CA活性很低.CA抑制剂AZ(乙酰唑磺胺)对中肋骨条藻的光合放氧速率没有明显影响,而CA抑制剂EZ(乙氧苯唑胺)对其光合放氧速率有强烈的抑制作用.EZ的抑制作用使细胞最大光合速率、饱和光强和无机碳亲和力下降,无机碳的补偿点和光呼吸提高,使强光下光抑制作用增强.这些结果表明:中肋骨条藻的胞外CA在其光合作用中所起的作用较小,而其胞内CA通过催化胞内碳库中的HCO-3快速转化成CO2,提高胞内CO2的有效供给,从而提高细胞光合固碳能力和对逆境(高O2、强光和低CO2)的适应能力.     

A protein is involved in accessibility of the inhibitor acetazolamide to the carbonic anhydrase(s) in the cyanobacterium Synechocystis PCC 6803

A gene, zam (for resistance to acetazolamide), controlling resistance to the carbonic anhydrase inhibitor acetazolamide, is described. It has been cloned from a spontaneous mutant, AZA^r-5b, isolated from the cyanobacterium Synechocystis PCC 6803, for its resistance to this drug (Bédu et al., Plant Physiol 93: 1312–1315, 1990). This mutant, besides its resistance to acetazolamide, displayed an absence of catalysed oxygen exchange activity on whole cells, suggestive of a deficiency in carbonic anhydrase activity. The gene was isolated by screening a genomic library of AZA^r-5b, and selecting for the capacity to transfer the AZA^r phenotype to wild-type cells. A system leading to forced homologous recombination in the host chromosome, using a platform vector, was devised in order to bypass direct selection difficulties. The putative encoded protein, 782 amino acids long, showed some homology with four eukaryotic and prokaryotic proteins involved in different cellular processes, one of them suppressing a phosphatase deficiency. The mutated allele of AZA^r-5b showed an in-frame 12 nucleotide duplication, which should not interfere with translation, and might result from transposition of a mobile element. Integration into a wild-type genome of either the spontaneous mutated allele or one inactivated by insertional mutagenesis conferred the character of resistance, but not the deficiency in oxygen exchange, indicating that the two phenotypic aspects of AZA^r-5b corresponded to two independent mutations. A working hypothesis explaining the phenotypes of the mutants is that the presence of the Zam protein would be necessary for the inhibitor to reach (one of) the two carbonic anhydrases present in this strain. This, however, would be a secondary action, the physiological role of the protein still being cryptic.