Effects of dopaminergic compounds on carbonic anhydrase isozymes I,II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K_i values of the molecules 3–6 were in the range of 41.12–363 μM against hCA I, of 0.381–470 μM against hCA II and of 0.578–1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K_A values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 μM against hCA I, of 0.381-470 μM against hCA II and of 0.578-1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Immobilized carbonic anhydrase: preparation,characteristics and biotechnological applications

Carbonic anhydrase (CA) is an essential metalloenzyme in living systems for accelerating the hydration and dehydration of carbon dioxide. CA-catalyzed reactions can be applied in vitro for capturing industrially emitted gaseous carbon dioxide in aqueous solutions. To facilitate this type of practical application, the immobilization of CA on or inside solid or soft support materials is of great importance because the immobilization of enzymes in general offers the opportunity for enzyme recycling or long-term use in bioreactors. Moreover, the thermal/storage stability and reactivity of immobilized CA can be modulated through the physicochemical nature and structural characteristics of the support material used. This review focuses on (i) immobilization methods which have been applied so far, (ii) some of the characteristic features of immobilized forms of CA, and (iii) biotechnological applications of immobilized CA. The applications described not only include the CA-assisted capturing and sequestration of carbon dioxide, but also the CA-supported bioelectrochemical conversion of CO₂ into organic molecules, and the detection of clinically important CA inhibitors. Furthermore, immobilized CA can be used in biomimetic materials synthesis involving cascade reactions, e.g. for bone regeneration based on calcium carbonate formation from urea with two consecutive reactions catalyzed by urease and CA.     

Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues

Carbonic anhydrase (CA) III was demonstrated immunocytochemically in epithelium in some regions of salivary gland ducts, colon, bronchi, and male genital tract and in adipocytes, in addition to skeletal muscle and liver where the isozyme was previously localized. Basal cells beneath the submandibular gland's excretory ducts in guinea pig stained for CA III. Carbonic anhydrase III occurred alone in some and with CA II in other sites but was often absent from CA-II-containing types of cells. This was exemplified by CA III's abundance in CA-II-positive proximal colon and its sparsity in the CA-II-rich distal colon of the mouse. Striated ducts in guinea pig, but not mouse salivary glands, stained darker for CA and appeared accordingly to function more actively in ion transport compared with excretory ducts. Carbonic anhydrase content varied among genera in liver and pancreas and between mouse species and strains in salivary glands and kidney. Newly observed murine sites of CA II activity included Auerbach's plexus and a population of leukocytes infiltrating the lamina propria in small intestine, and several types of cells in the male genital tract. In immunoblot tests, antisera to CA III showed no cross reactivity with antisera to CA II, but those to CA II disclosed weak cross reactivity with CA III.     

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。     

Differential regulation of hepatic carbonic anhydrase isozymes in the streptozotocin-diabetic rat

Most work with the male rat liver carbonic anhydrase isozymes in the past decade has centered on the cytosolic CA III and the mitochondrial CA V. This paper reports that the relative activity of both isozymes is altered in streptozotocin-diabetes. Carbonic anhydrase activity of perfused liver homogenates and disrupted, isolated mitochondria was measured by the mass spectrometric 18O decay technique at 37 degrees C. The contributions of the different isozymes were determined based on intracellular location and sensitivity to acetazolamide inhibition. Diabetes resulted in a twofold increase in the activity of CA V but a halving in the activity of CA III. This is the first time that liver CA V has been shown to be altered by physiological stress. The total carbonic anhydrase activity in the diabetic rat liver was unaltered compared with control rats; however, CA III never accounted for more than 50% of this activity. Since CA isozymes I, II, and IV together account for 30% of the CA activity in control rats and 70% in diabetic rats it is concluded that one or more of these isozymes is subject to regulation in the diabetic male rat. The increase in CA V during diabetes is in accord with this isozyme having an important function in provision of substrate for hepatic gluconeogenesis and ureagenesis.     

Rapid separation of carbonic anhydrase isozymes using cellulose acetate membrane electrophoresis

A method is described for the rapid separation of carbonic anhydrase(CA) isozymes by cellulose acetate membrane electrophoresisin which CA activity is detected using the pH-indicating dye,bromcresol purple. This method can detect bovine erythrocyteCA in a 0.3 mm³ sample applied at a concentration of 100 ngcm^–3 (total of 30 pg applied) while at higher concentrationsthree isozymes were observed. It was found, using a potentiometrictechnique, that intact cells of Anabaena flos-aquae (Cyanophyceae)and Chlorella ellipsoidea had no detectable activity while C.saccharophila and Chlamydomonas reinhardtii (Chlorophyceae)had external CA activity. CA activity of the extracts suggestedthe presence of internal CA in all species. After electrophoresisit was found that C. saccharophila and C. reinhardtii had twoisozymes while A. flos-aquae and C. ellipsoidea had only a singledetectable band. Spinach had up to five detectable isozymesthat were difficult to resolve. Incubation of spinach extractwith the CA inhibitor ClO₄^– (500 mol m^–3) inhibitedCA activity by 90% using the potentiometric technique, but afterelectrophoresis had no detectable effect. This technique isuseful in identifying isozymes that are substantially differentin electrical charge and in monitoring CA isozyme activity duringenzyme purification. Key words: Carbonic anhydrase, isozymes, cyanobacteria, microalgae, spinach     

Selective tissue distribution of carbonic anhydrase isozymes in the ox.

By affinity chromatography the isozymic distribution of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been studied in extract from various bovine tissues. Carbonic anhydrase II forms isolated from erythrocyte, kidney and brain are indistinguishable by specific activity, amino acid composition, fingerprint, electrophoretic and immunological behaviour. By these criteria they differ from carbonic anhydrase I isolated from rumen epithelium.     

Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. IV. Degradation by heat and proteolysis of normal and variant carbonic anhydrase isozymes of Macaca nemestrina

Studies were undertaken on the heat denaturation and proteolytic degradation by alpha-chymotrypsin of the normal red cell carbonic anhydrase isozyme, CA II, and two electrophoretic variants of carbonic anhydrase I, CA Ia and CA Ib, of the pigtail macaque. The heat degradation results showed a difference of about 40-fold in the rate constants between CA Ia and CA Ib, which is due to the marked thermostability of CA Ib compared to CA Ia. The enthalpies and entropies of activation were calculated from the heat denaturation constants. These values were compared, on enthalpy-entropy compensation plots, with those values previously determined for the human CA I and CA II isozymes. They were highly correlated and clearly fell into two distinct clusters, separated by about 200 kJ mol-1; one group comprising the macaque and human CA I isozymes and the other the CA II isozymes. The proteolytic degradation results showed that CA Ia is degraded about 2.5 times more rapidly than CA Ib by alpha-chymotrypsin. Thus, the characteristic 3/1 ratio of CA Ib/CA Ia in mature red cells could be accounted for by the greater susceptibility of CA Ia to degradation at some stage in red cell development.     

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.     

Hormonal manipulation and neonatal imprinting of carbonic anhydrase isozymes in rat liver

Thyroidectomy has a feminizing effect on the carbonic anhydrase isozymes II and III in male rat liver. These isozymes are known to be sexually differentiated but no treatment so far administered has produced total feminization and this is also true for thyroidectomy. Neonatal castration, however, does produce this result, suggesting the possibility of androgen imprinting for these isozymes during the neonatal period.     

Immunocytochemical localization of carbonic anhydrase isozymes I, II, and III in rat skeletal muscle

Specific antisera were raised against the three carbonic anhydrase (CA) isozymes, CAI, CAII, and CAIII, and were used to determine the fiber distribution of these isozymes in skeletal muscle. Fiber types were determined by ATPase staining, and the CA isozymes were detected using a peroxidase-anti-peroxidase (PAP) technique. All three isozymes were present in type I fibers; CAII and CAIII were exclusive to these fibers, and CAI were also present in some small type 2A fibers.     

The cytoplasmic isozymes of carbonic anhydrase in the gastrointestinal tract of reindeer

The cytoplasmic isozymes of carbonic anhydrase: CA I and CA II, in the gastrointestinal tract tissues of reindeer were identified by electrophoresis and substrate-inhibitory tests. The study of the tissue distribution and composition of the cytoplasmic isozymes has shown, that isozyme CA II is found in the forestomachs and the colon mucosal tissue, whereas isozyme CA II predominates in the abomasum and small intestinal mucosa tissue.     

Expression of carbonic anhydrase isozymes II and III in developing bovine parotid gland

Two cytosolic carbonic anhydrase isozymes (CA-II and CA-III) were studied by immunohistochemistry in bovine parotid glands during fetal development. In a 3-month-old fetus of crown-rump length (CRL) 17 cm, the expression of CA-II in undifferentiated epithelial cells was observed, whereas immunostaining for CA-III remained negative. At 26 cm CRL (4–5 months old), weak expression of CA-III in large ductal epithelial cells was noted. The accumulation of secreted granules in primary acinar cells was initially observed at this stage. In a newborn calf, anti-CA-II reactivity almost disappeared from most duct segments. The time-dependent expression and distribution of the isozymes in parotid glands may reflect different biological functions of these structurally closely related isozymes. Bovine parotid acinar cells of fetuses would thus appear to possess all the cellular structures and immunohistochemical properties at 4 and 5 months of gestation. CA-II subsequently disappeared from duct segments and nearly all acinar cells in adults were present at or just after birth.     

Inhibition of carbonic anhydrase isozymes with benzene sulfonamides incorporating thio,sulfinyl and sulfonyl glycoside moieties

A series of benzene sulfonamides incorporating thio, sulfinyl or sulfonyl glycoside moieties were synthesized. These glycoconjugates were investigated for their ability to inhibit the enzymatic activity of four human carbonic anhydrases (hCA): isozymes I, II and tumour-associated isozymes IX and XII. The oxidation state of the sulfur in the carbohydrate tail moiety did not influence either enzyme inhibition potency or isozyme selectivity even though presenting opportunities for differing interactions with the target isozymes.