Alpha Carbonic Anhydrase 5 Mediates Stimulation of ATP Synthesis by Bicarbonate in Isolated Arabidopsis Thylakoids
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ABSTRACT:
SUBMITTER: Fedorchuk T
PROVIDER: S-EPMC8427869 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bicarbonate transport plays a role in aqueous humor (AH) secretion. The authors examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine nonpigmented ciliary epithelium (NPE).Cytoplasmic pH (pH(i)) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC), and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique.Anion exchanger AE2, CAII, and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO(2)/HCO(3)(-)-free HEPES buffer, exposure to a CO(2)/HCO(3)(-)-containing buffer caused rapid acidification followed by a gradual increase in pH(i). Subsequent removal of CO(2)/HCO(3)(-) with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pH(i). The rate of gradual alkalinization after the addition of HCO(3)(-)/CO(2) was inhibited by sodium-free conditions, DIDS, and the CA inhibitors acetazolamide and methazolamide but not by the Na-H exchange inhibitor dimethylamiloride or low-chloride buffer. The phase of gradual acidification after removal of HCO(3)(-)/CO(2) was inhibited by DIDS, acetazolamide, methazolamide, and low-chloride buffer. DIDS reduced baseline pH(i). In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44%, respectively.The results suggest the NPE uses a Na(+)-HCO(3)(-) cotransporter to import bicarbonate and a Cl(-)/HCO(3)(-) exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII, and CAIV are enriched in the NPE layer of the ciliary body, and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye.
| S-EPMC2744210 | biostudies-literature
Project description:The Haemophilus influenzae beta-carbonic anhydrase (HICA) allosteric site variants V47A and G41A were overexpressed and purified to homogeneity. These variants have k(cat)/K(m) values similar to that of the wild-type enzyme and exhibit a similar dramatic decrease in catalytic activity at pH <8.0. However, both HICA-G41A and -V47A were serendipitously found to bind sulfate ion or bicarbonate ion near pairs of Glu50 and Arg64 residues located on the dimerization interface. In the case of HICA-V47A, bicarbonate ions simultaneously bind to both the dimerization interface and the allosteric sites. For HICA-G41A, two of 12 chains in the asymmetric unit bind bicarbonate ion exclusively at the dimerization interface, while the remaining 10 chains bind bicarbonate ion exclusively at the allosteric site. We propose that the new anion binding site along the dimerization interface of HICA is an "escort" site that represents an intermediate along the ingress and egress route of bicarbonate ion to and from the allosteric binding site, respectively. The structural evidence for sulfate binding at the escort site suggests that the mechanism of sulfate activation of HICA is the result of sulfate ion competing for bicarbonate at the escort site, preventing passage of bicarbonate from the bulk solution to its allosteric site.
| S-EPMC2861718 | biostudies-literature
Project description:cea05-01_carbonic-anhydrase - carbonic anhydrase (1 or 2) simple or double mutants - Identification of genes differentially expressed in leaves of single CA1 and CA2 T-DNA insertional mutants and in the corresponding double mutant vs wild type - CA1 (At3g01500) and CA2 (At5g14740) T-DNA insertional mutant lines, the double (CA1+CA2) mutant and wild type Arabidopsis seeds were sown in soil in a phytotron. Leaves were harvested 40 days later for RNA extraction
2012-03-25 | GSE36625 | GEO
Project description:cea05-01_carbonic-anhydrase - carbonic anhydrase (1 or 2) simple or double mutants - Identification of genes differentially expressed in leaves of single CA1 and CA2 T-DNA insertional mutants and in the corresponding double mutant vs wild type - CA1 (At3g01500) and CA2 (At5g14740) T-DNA insertional mutant lines, the double (CA1+CA2) mutant and wild type Arabidopsis seeds were sown in soil in a phytotron. Leaves were harvested 40 days later for RNA extraction 6 dye-swap - gene knock out
2012-03-25 | E-GEOD-36625 | biostudies-arrayexpress
Project description:Understanding the mechanisms that drive acid-base regulation in organisms is important, especially for organisms in aquatic habitats that experience rapidly fluctuating pH conditions. Previous studies have shown that carbonic anhydrases (CAs), a family of zinc metalloenzymes, are responsible for acid-base regulation in many organisms. Through the use of phylogenetic tools, this present study attempts to elucidate the evolutionary history of the α-CA superfamily, with particular interest in the emerging model aquatic organism Daphnia pulex. We provide one of the most extensive phylogenies of the evolution of α-CAs, with the inclusion of 261 amino acid sequences across taxa ranging from Cnidarians to Homo sapiens. While the phylogeny supports most of our previous understanding on the relationship of how α-CAs have evolved, we find that, contrary to expectations, amino acid conservation with bacterial α-CAs supports the supposition that extracellular α-CAs are the ancestral state of animal α-CAs. Furthermore, we show that two cytosolic and one GPI-anchored α-CA in Daphnia genus have homologs in sister taxa that are possible candidate genes to study for acid-base regulation. In addition, we provide further support for previous findings of a high rate of gene duplication within Daphnia genus, as compared with other organisms.
| S-EPMC4393933 | biostudies-literature
Project description:Transport metabolons have been discussed between carbonic anhydrase II (CAII) and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1), to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 µM), while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity.
| S-EPMC3208603 | biostudies-literature
Project description:Carbonic anhydrase IX (CA IX) is a transmembrane enzyme participating in adaptive responses of tumors to hypoxia and acidosis. CA IX regulates pH, facilitates metabolic reprogramming, and supports migration, invasion and metastasis of cancer cells. Extracellular domain (ECD) of CA IX can be shed to medium and body fluids by a disintegrin and metalloproteinase (ADAM) 17. Here we show for the first time that CA IX ECD shedding can be also executed by ADAM10, a close relative of ADAM17, via an overlapping cleavage site in the stalk region of CA IX connecting its exofacial catalytic site with the transmembrane region. This finding is supported by biochemical evidence using recombinant human ADAM10 protein, colocalization of ADAM10 with CA IX, ectopic expression of a dominant‑negative mutant of ADAM10 and RNA interference‑mediated suppression of ADAM10. Induction of the CA IX ECD cleavage with ADAM17 and/or ADAM10 activators revealed their additive effect. Similarly, additive effect was observed with an ADAM17‑inhibiting antibody and an ADAM10‑preferential inhibitor GI254023X. These data indicated that ADAM10 is a CA IX sheddase acting on CA IX non‑redundantly to ADAM17.
| S-EPMC9813547 | biostudies-literature
Project description:The crystal structure of α-carbonic anhydrase, an enzyme present in the periplasm of Helicobacter pylori, a bacterium that affects humans and that is responsible for several gastric pathologies, is described. Two enzyme monomers are present in the asymmetric unit of the monoclinic space group P21, forming a dimer in the crystal. Despite the similarity of the enzyme structure to those of orthologues from other species, the H. pylori protein has adopted peculiar features in order to allow the bacterium to survive in the difficult environment of the human stomach. In particular, the crystal structure shows how the bacterium has corrected for the mutation of an essential amino acid important for catalysis using a negative ion from the medium and how it localizes close to the inner membrane in the periplasm. Since carbonic anhydrase is essential for the bacterial colonization of the host, it is a potential target for antibiotic drugs. The definition of the shape of the active-site entrance and cavity constitutes a basis for the design of specific inhibitors.
| S-EPMC4528932 | biostudies-literature
Project description:Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryoelectron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCA's binding site overlaps with that of CsoS2, but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme but also as a central hub for mediating assembly through protein interactions.
| S-EPMC10614612 | biostudies-literature
Project description:Carbonic anhydrase IX (CA IX) is a hypoxia-induced cell surface enzyme expressed in solid tumors, and functionally involved in acidification of extracellular pH and destabilization of intercellular contacts. Since both extracellular acidosis and reduced cell adhesion facilitate invasion and metastasis, we investigated the role of CA IX in cell migration, which promotes the metastatic cascade. As demonstrated here, ectopically expressed CA IX increases scattering, wound healing and transwell migration of MDCK cells, while an inactive CA IX variant lacking the catalytic domain (ΔCA) fails to do so. Correspondingly, hypoxic HeLa cells exhibit diminished migration upon inactivation of the endogenous CA IX either by forced expression of the dominant-negative ΔCA variant or by treatment with CA inhibitor, implying that the catalytic activity is indispensable for the CA IX function. Interestingly, CA IX improves cell migration both in the absence and presence of hepatocyte growth factor (HGF), an established inducer of epithelial-mesenchymal transition. On the other hand, HGF up-regulates CA IX transcription and triggers CA IX protein accumulation at the leading edge of lamellipodia. In these membrane regions CA IX co-localizes with sodium bicarbonate co-transporter (NBCe1) and anion exchanger 2 (AE2) that are both components of the migration apparatus and form bicarbonate transport metabolon with CA IX. Moreover, CA IX physically interacts with AE2 and NBCe1 in situ, as shown here for the first time. Thus, our findings suggest that CA IX actively contributes to cell migration via its ability to facilitate ion transport and pH control at protruding fronts of moving cells.
| S-EPMC3270993 | biostudies-literature