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Carbonic Anhydrase II (carbonic + anhydrase_ii)
Kinds of Carbonic Anhydrase II Selected AbstractsMolecular physiology of SLC4 anion exchangersEXPERIMENTAL PHYSIOLOGY, Issue 1 2006Seth L. Alper Plasmalemmal Cl,,HCO3, exchangers regulate intracellular pH and [Cl,] and cell volume. In polarized epithelial cells, they contribute also to transepithelial secretion and reabsorption of acid,base equivalents and of Cl,. Members of both the SLC4 and SLC26 mammalian gene families encode Na+ -independent Cl,,HCO3, exchangers. Human SLC4A1/AE1 mutations cause either the erythroid disorders spherocytic haemolytic anaemia or ovalocytosis, or distal renal tubular acidosis. SLC4A2/AE2 knockout mice die at weaning. Human SLC4A3/AE3 polymorphisms have been associated with seizure disorder. Although mammalian SLC4/AE polypeptides mediate only electroneutral Cl,,anion exchange, trout erythroid AE1 also promotes osmolyte transport and increased anion conductance. Mouse AE1 is required for DIDS-sensitive erythroid Cl, conductance, but definitive evidence for mediation of Cl, conductance is lacking. However, a single missense mutation allows AE1 to mediate both electrogenic SO42,,Cl, exchange or electroneutral, H+ -independent SO42,,SO42, exchange. In the Xenopus oocyte, the AE1 C-terminal cytoplasmic tail residues reported to bind carbonic anhydrase II are dispensable for Cl,,Cl, exchange, but required for Cl,,HCO3, exchange. AE2 is acutely and independently inhibited by intracellular and extracellular H+, and this regulation requires integrity of the most highly conserved sequence of the AE2 N-terminal cytoplasmic domain. Individual missense mutations within this and adjacent regions identify additional residues which acid-shift pHo sensitivity. These regions together are modelled to form contiguous surface patches on the AE2 cytoplasmic domain. In contrast, the N-terminal variant AE2c polypeptide exhibits an alkaline-shifted pHo sensitivity, as do certain transmembrane domain His mutants. AE2-mediated anion exchange is also stimulated by ammonium and by hypertonicity by a mechanism sensitive to inhibition by chelation of intracellular Ca2+ and by calmidazolium. This growing body of structure,function data, together with increased structural information, will advance mechanistic understanding of SLC4 anion exchangers. [source] Characterization of carbonic anhydrase from Neisseria gonorrhoeaeFEBS JOURNAL, Issue 6 2001Björn Elleby We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s,1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O-exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa of 6.5. The results of a kinetic analysis of a His66,Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar Ki values for the inhibitors NCO,, SCN, and N3, as HCA II, while CN, and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO,, SCN,, CN, and N3, resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO2 hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8,2.9 m GdnHCl was observed. [source] Whole genome analysis for liver metastasis gene signatures in colorectal cancerINTERNATIONAL JOURNAL OF CANCER, Issue 9 2007Dong Hyuk Ki Abstract Liver metastasis is one of the major causes of death in colorectal cancer (CRC) patients. To understand this process, we investigated whether the gene expression profiling of matched colorectal carcinomas and liver metastases could reveal key molecular events involved in tumor progression and metastasis. We performed experiments using a cDNA microarray containing 17,104 genes with the following tissue samples: paired tissues of 25 normal colorectal mucosa, 27 primary colorectal tumors, 13 normal liver and 27 liver metastasis, and 20 primary colorectal tumors without liver metastasis. To remove the effect of normal cell contamination, we selected 4,583 organ-specific genes with a false discovery rate (FDR) of 0.0067% by comparing normal colon and liver tissues using significant analysis of microarray, and these genes were excluded from further analysis. We then identified and validated 46 liver metastasis-specific genes with an accuracy of 83.3% by comparing the expression of paired primary colorectal tumors and liver metastases using prediction analysis of microarray. The 46 selected genes contained several known oncogenes and 2 ESTs. To confirm that the results correlated with the microarray expression patterns, we performed RT-PCR with WNT5A and carbonic anhydrase II. Additionally, we observed that 21 of the 46 genes were differentially expressed (FDR = 2.27%) in primary tumors with synchronous liver metastasis compared with primary tumors without liver metastasis. We scanned the human genome using a cDNA microarray and identified 46 genes that may play an important role in the progression of liver metastasis in CRC. © 2007 Wiley-Liss, Inc. [source] Formation of osteoclast-like cells from peripheral blood of periodontitis patients occurs without supplementation of macrophage colony-stimulating factorJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 7 2008Stanley T. S. Tjoa Abstract Aim: To determine whether peripheral blood mononuclear cells (PBMCs) from chronic periodontitis patients differ from PBMCs from matched control patients in their capacity to form osteoclast-like cells. Material and Methods: PBMCs from 10 subjects with severe chronic periodontitis and their matched controls were cultured on plastic or on bone slices without or with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor- ,B ligand (RANKL). The number of tartrate-resistant acid phosphatase-positive (TRACP+) multinucleated cells (MNCs) and bone resorption were assessed. Results: TRACP+ MNCs were formed under all culture conditions, in patient and control cultures. In periodontitis patients, the formation of TRACP+ MNC was similar for all three culture conditions; thus supplementation of the cytokines was not needed to induce MNC formation. In control cultures, however, M-CSF or M-CSF/RANKL resulted in higher numbers compared with cultures without cytokines. Upregulations of osteoclast marker mRNA cathepsin K and carbonic anhydrase II confirmed the osteoclastic character. Bone resorption was only observed when PBMCs were cultured in the presence of M-CSF and RANKL. Conclusion: Our data indicate that PBMCs from periodontitis patients do not need priming by M-CSF to become osteoclast-like cells, suggesting that PBMCs from periodontitis patients are present in the circulation in a different state of activity. [source] Coupled atomic charge selectivity for optimal ligand-charge distributions at protein binding sitesJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 16 2006Sathesh Bhat Abstract Charge optimization as a tool for both analyzing and enhancing binding electrostatics has become an attractive approach over the past few years. An interesting feature of this method for molecular design is that it provides not only the optimal charge magnitudes, but also the selectivity of a particular atomic center for its optimal charge. The current approach to compute the charge selectivity at a given atomic center of a ligand in a particular binding process is based on the binding-energy cost incurred upon the perturbation of the optimal charge distribution by a unit charge at the given atomic center, while keeping the other atomic partial charges at their optimal values. A limitation of this method is that it does not take into account the possible concerted changes in the other atomic charges that may incur a lower energetic cost than perturbing a single charge. Here, we describe a novel approach for characterizing charge selectivity in a concerted manner, taking into account the coupling between the ligand charge centers in the binding process. We apply this novel charge selectivity measure to the celecoxib molecule, a nonsteroidal anti-inflammatory agent binding to cyclooxygenase 2 (COX2), which has been recently shown to also exhibit cross-reactivity toward carbonic anhydrase II (CAII), to which it binds with nanomolar affinity. The uncoupled and coupled charge selectivity profiles over the atomic centers of the celecoxib ligand, binding independently to COX2 and CAII, are analyzed comparatively and rationalized with respect to available experimental data. Very different charge selectivity profiles are obtained for the uncoupled versus coupled selectivity calculations. © 2006 Wiley Periodicals, Inc. J Comput Chem, 2006 [source] Polymorphisms in eggshell organic matrix genes are associated with eggshell quality measurements in pedigree Rhode Island Red hensANIMAL GENETICS, Issue 1 2009I. C. Dunn Summary Novel and traditional eggshell quality measurements were made from up to 2000 commercial pedigree hens for a candidate gene association analysis with organic eggshell matrix genes: ovocleidin-116, osteopontin (SPP1), ovocalyxin-32 (RARRES1), ovotransferrin (LTF), ovalbumin and ovocalyxin-36, as well as key genes in the maintenance and function of the shell gland [estrogen receptor (ESR1) and carbonic anhydrase II (CAII)]. Associations were found for (i) ovalbumin with breaking strength and shell thickness; (ii) ovocleidin-116 with elastic modulus, shell thickness and egg shape; (iii) RARRES1 with mammillary layer thickness; (iv) ESR1 with dynamic stiffness; (v) SPP1 with fracture toughness and (vi) CAII with egg shape. The marker effects are as large as 17% of trait standard deviations and could be used to improve eggshell quality. [source] Emerging from pseudo-symmetry: the redetermination of human carbonic anhydrase II in monoclinic P21 with a doubled a axisACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010Arthur H. Robbins The crystal structure of human carbonic anhydrase II in the monoclinic P21 space group with a doubled a axis from that of the usually observed unit cell has recently been reported, with one of the two molecules in the asymmetric unit demonstrating rotational disorder [Robbins et al. (2010), Acta Cryst. D66, 628,634]. The structure has been redetermined, with the coordinates of both pseudo-symmetrically related molecules in the crystallographic asymmetric unit translated by x, = x± 1/4, and no rotational disorder is observed. This corresponds to a different choice of how the four molecules in the unit cell should be grouped into pairs that represent a single asymmetric unit. [source] Atomic resolution studies of carbonic anhydrase IIACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Craig A. Behnke Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X-ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide-containing inhibitor complex has been carried out and the structure was refined at 0.9,Å resolution with anisotropic atomic displacement parameters to an R value of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active-site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25,Å) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01,Å in some zinc,protein and zinc,ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron-density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X-ray structures, even if atomic resolution data are available. [source] Structure of a monoclinic polymorph of human carbonic anhydrase II with a doubled a axisACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Arthur H. Robbins The crystal structure of human carbonic anhydrase II with a doubled a axis from that of the usually observed monoclinic unit cell has been determined and refined to 1.4,Å resolution. The diffraction data with h = 2n + 1 were systematically weaker than those with h = 2n. Consequently, the scaling of the data, structure solution and refinement were challenging. The two molecules comprising the asymmetric unit are related by a noncrystallographic translation of ½ along a, but one of the molecules has two alternate positions related by a rotation of approximately 2°. This rotation axis is located near the edge of the central ,-sheet, causing a maximum distance disparity of 1.7,Å between equivalent atoms on the diametrically opposite side of the molecule. The crystal-packing contacts are similar to two sequential combined unit cells along a of the previously determined monoclinic unit cell. Abnormally high final Rcryst and Rfree values (20.2% and 23.7%, respectively) are not unusual for structures containing pseudo-translational symmetry and probably result from poor signal to noise in the weak h -odd data. [source] High-resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug designACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Katherine H. Sippel The crystal structure of human carbonic anhydrase II (CA II) complexed with the inhibitor acetazolamide (AZM) has been determined at 1.1,Å resolution and refined to an Rcryst of 11.2% and an Rfree of 14.7%. As observed in previous CA II,inhibitor complexes, AZM binds directly to the zinc and makes several key interactions with active-site residues. The high-resolution data also showed a glycerol molecule adjacent to the AZM in the active site and two additional AZMs that are adventitiously bound on the surface of the enzyme. The co-binding of AZM and glycerol in the active site demonstrate that given an appropriate ring orientation and substituents, an isozyme-specific CA inhibitor may be developed. [source] Preliminary joint neutron and X-ray crystallographic study of human carbonic anhydrase IIACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009S. Z. Fisher Carbonic anhydrases catalyze the interconversion of CO2 to HCO3,, with a subsequent proton-transfer (PT) step. PT proceeds via a proposed hydrogen-bonded water network in the active-site cavity that is stabilized by several hydrophilic residues. A joint X-ray and neutron crystallographic study has been initiated to determine the specific water network and the protonation states of the hydrophilic residues that coordinate it in human carbonic anhydrase II. Time-of-flight neutron crystallographic data have been collected from a large (,1.2,mm3) hydrogen/deuterium-exchanged crystal to 2.4,Å resolution and X-ray crystallographic data have been collected from a similar but smaller crystal to 1.5,Å resolution. Obtaining good-quality neutron data will contribute to the understanding of the catalytic mechanisms that utilize water networks for PT in protein environments. [source] Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase IIACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2006Monika Budayova-Spano Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H2O/OH,) and the proton-shuttling residue His64. This distance (,7.5,Å) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (,1.0,Å) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5,Å resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography. [source] | ||||||||||||||||