Inhibition of Bovine Carbonic Anhydrase by New Sulfonamide Compounds

Inhibitory effects of three new derivatives of 2-acetylamino-1,3,4-thiadiazole-5-sulfonamide on bovine carbonic anhydrase have been investigated. The new compounds are 2-(3-chloropropionylamino)-1,3,4-thiadiazole-5-sulfonamide, 2-(2,2-dichloroacetylamino)-1,3,4-thiadiazole-5-sulfonamide, and 2-(3-phenylpropionylamino)-1,3,4-thiadiazole-5-sulfonamide. The new compounds inhibit the esterase activity of carbonic anhydrase noncompetitively and have inhibition constants and I ₅₀ values very similar to those for 2-acetylamino-1,3,4-thiadiazole-5-sulfonamide, the latter being clinically used in the treatment of glaucoma.     

原核生物的碳酸酐酶

碳酸酐酶（CA）催化CO2和HCO3^-之间的相互转化反应,其显著特征是存在α,β,γ三种分子结构不同且独立进化的分子类别,最近的研究表明CA不仅广泛存在于真核界所有高度进化的生物体中,而且还广泛存在于古细菌界和细菌界代谢多样化的原核生物种类中,这表明该酶在原核生物中的作用比先前所认识的更广泛和更基本,鉴于CA在原核生物生理学上的重要性,有必要对近年来原核生物CA的进化分类,分子结构特性,酶学特性及其在原核生物中的分布以及可能的生理功能等方面的研究进展作一介绍。     

碳酸酐酶在红细胞分化发育过程中的功能研究

目前红系分化调控相关的研究主要集中在细胞因子、转录因子、lncRNA及表观遗传方面,为了对红系分化调控机制进行更加深入的解析,研究了碳酸酐酶在红系分化中的功能。碳酸酐酶可以高效催化二氧化碳的水合,但它在红细胞发育过程中的功能尚不清楚。利用脐带血来源的CD34⁺细胞在体外进行红细胞诱导分化,在分化过程中通过慢病毒介导的基因敲降的方法能够降低碳酸酐酶1和碳酸酐酶2的表达,并使用流式细胞仪检测红细胞的生成和分化效率。研究结果表明,与对照组相比,碳酸酐酶1的表达缺陷使红细胞的晚期分化明显受阻,而碳酸酐酶2的表达缺陷则将红细胞的分化阻滞在早期阶段。研究结果表明,虽然作用窗口不同,但碳酸酐酶1和碳酸酐酶2在红系分化的过程中均发挥着重要的调控作用,这一发现对将来在体外红细胞生成具有指导意义。     

Product Inhibition Study on Carbonic Anhydrase Using Spectroscopy and Calorimetry

Kinetic and thermodynamic studies have been made on the effect of the p -nitrophenol product on the activity of bovine carbonic anhydrase in 50 mM Tris buffer pH 7.5, at 300 K using UV spectrophotometry and isothermal titration calorimetry (ITC). A competitive inhibition was observed for p -nitrophenol as a product of the enzymatic reaction. A graphical fitting method was used for determination of the binding constant and enthalpy of inhibitor binding using ITC data. The dissociation binding constant was 0.10 mM by the microcalorimetric method, which is in good agreement with the value of 0.11 mM for the inhibition constant obtained from the spectrophotometric method.     

Hypoxia Inducible Carbonic Anhydrase IX,Marker of Tumour: Hypoxia,Survival Pathway and Therapy Target

Over 50 genes are inducible by hypoxia, via hypoxia inducible factor 1a (HIF-1a). Carbonic anhydrase IX (CAIX) is one of the most inducible and most uniformly induced genes and because of its stability and membrane location provides a reliable histochemical marker of hypoxia. Recent studies have shown the importance of pH in cell death under hypoxia, thus mechanisms of pH regulation are likely to be vital pathways for survival. Carbonic anhydrases have a widespread role in normal tissues in regulating pH, with 14 isoforms described, so inhibition may have substantial normal tumour toxicity. Selective non-membrane permeable inhibitors are available and may synergise with chemotherapy agents more active in acid conditions. CAIX has a major role in regulating hydrogen ion (H+) flux and blockade of CAIX results in increased cell death under hypoxia, indicating that it is one mechanism of hypoxic adaptation. As it is commonly expressed in tumours with the worst prognosis it is a potential target for therapy.     

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 Carbonic Anhydrase Inhibition on Leptin Secretion by Rat Adipose Tissue

It is well known that the role of leptin in the body is to regulate food intake and energy expenditure but the process of leptin secretion by adipose tissue and the components involved in this process are still obscure. Carbonic anhydrase III (CA III) is the most abundant protein of the rat adipose tissue and its amount decreases with obesity. The effect of the inhibition of CA III on leptin secretion by rat epididymal adipose tissue was examined. Dorzolamide, a CA inhibitor, caused a decrease in dexamethasone and insulin-induced leptin secretion suggesting a possible role for CA III in the mechanism of leptin secretion.     

Histochemical Demonstration of Carbonic Anhydrase Activity

Fresh tissue slices fixed in chilled acetone for 1 hour and washed in distilled water for 10-30 minutes were incubated for 30-45 minutes at 37°C. in the freshly prepared incubating mixture: filtrate of a mixture of 8% sodium bicarbonate, 100 ml., and MnCl₂·4H₂O, 1 g. After washing in distilled water for 1 hour, they were dehydrated and embedded in paraffin. Sections were cut 15-20μU, deparaffinized, rinsed in absolute alcohol and placed in a 0.1% solution of potassium periodate for 48 hours at 37°C. The mounted sections were counterstained (if desired), dehydrated in alcohol, cleared in xylene (not carbol-xylene) and mounted in balsam. Many brown granules were produced on the sites of enzyme activity by this procedure. The results obtained seem to be in good agreement with previous findings by biochemical determinations.     

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.     

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...     

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 of Chlamydomonas: Purification and Studies on its Induction Using Antiserum against Chlamydomonas Carbonic Anhydrase

Carbonic anhydrase (EC 4.2.1.1 [EC] ; CA) was purified by affinitychromatography from cells of the unicellular green alga Chlamydomonasreinhardtii which had been grown photoautotrophically in ordinaryair. Antiserum raised in rabbit against this purified CA crossreactedwith Chlamydomonas CA but not with spinach leaf CA nor bovineerythrocyte CA. When the CO₂ concentration provided to the algalcells was decreased from 4% to the ordinary air level (0.04%),CA activity and the content of CA protein determined by theimmunodiffusion test showed parallel increases. In contrast,when the CO₂ concentration was raised from air level to 4% CO₂CA activity and its content expressed on the basis of culturevolume remained rather constant. These results indicate thatsynthesis of the CA protein is induced when the CO₂ concentrationis lowered from 4 to 0.04% during algal growth. On the otherhand, the synthesis of CA stops when CO₂ concentration is raisedfrom air level to 4%. (Received June 30, 1984; Accepted October 8, 1984)     

Carbonic Anhydrase and the Regulation of Photosynthesis

THE role of CO₂ in the regulation of photosynthetic and respiratory metabolism in plants is little understood in the unicellular alga Chlorella pyrenoidosa; for example, after autotrophic growth in high CO₂ (5·5% by volume), transfer to a CO₂ concentration about ten times less than the concentration in air results initially in low rates of photosynthesis characterized by the virtual absence of the Calvin cycle¹ of CO₂ fixation². An induction period of about 2 h is necessary before normal photosynthetic rates are established. Cells grown in air (0.03% CO₂) do not show this effect and photosynthesize at comparatively high rates even in very low concentrations of CO₂.     

Crystal Structure of Human Carbonic Anhydrase C

The three dimensional structure of human carbonic anhydrase C has been determined at 2.0 Å resolution. The active site has been identified by the binding of inhibitors and the location of the zinc ion.     

碳酸酐酶与临床医学

碳酸酐酶是一类含锌的蛋白酶,共发现了10种同工酶及三种碳酸酐酶相关蛋白,广布于人体各组织,能可逆性地催化C02的水合反应,参与调节pH、离子运输等多种生理过程,碳酸酐酶的缺乏和异常将可能会导致一系列的疾病。     

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.