The effect of ethanol on erythrocyte carbonic anhydrase isoenzymes activity: an in vitro and in vivo study

The ethanol is a widely consumed as sedative-hypnotic drug throughout the world. In this study, the effects of ethanol were investigated on carbonic anhydrase (CA) enzyme activities both in vitro in human erythrocyte and in vivo in Sprague-Dawley rat erythrocyte. For in vitro study, the human carbonic anhydrase-I (HCA-I) and -II (HCA-II) are purified by Sepharose 4B-L-tyrosine-sulphanilamide affinity chromatography. In vivo CA enzyme activity was determined colorimetrically by using CO(2)-hydration method of Wilbur and Anderson. Rat blood samples were taken from each rat before and after the ethanol administration at different times (1 h, 3 h, and 5 h). Rat erythrocyte CA activity was significantly inhibited by pharmacological dosage of the ethanol (2 mL.kg(- 1)) for up to 3 h (p < 0.001) following intraperitoneally administration. The ethanol showed in vitro inhibitory effects on HCA-I and HCA-II hydratase activity, determined by colorimetrically using the CO(2)-hydratase method. The inhibitor concentrations causing up to 50% inhibition (IC(50)) were 2.09 M for HCA-I (r(2):0.9273) and 1.83 M for HCA-II (r(2):9749). In conclusion, it was demonstrated that carbonic anhydrase enzyme in erythrocytes was significantly inhibited by the ethanol both in in vitro and in vivo.     

Decreased total carbonic anhydrase esterase activity and decreased levels of carbonic anhydrase 1 isozyme in erythrocytes of type II diabetic patients

In this exploratory study, we investigated total erythrocyte carbonic anhydrase (CA) estrase activity as well as CA I isozyme concentration in patients with diabetes mellitus type II (DM) and healthy individuals of Howard University Hospital community. Total estrase activity of CA was measured spectrophotometrically using p-nitrophenol acetate before and after inhibition with acetazolamide. CA I isozyme was measured by radial immunodiffusion using monoclonal antibody (CA I) in agarose plates. The study involved 20 consented participants; 10 normal (N) and 10 (DM), 21 to 84 years of age. The study was approved by the Howard University Institution Review Board. The CA activity was measured following lysis of cells as U/min/mL and CA I concentration as mg/l. We observed CA activity as 46.3±4(N) and 25±2.1 (DM) whereas CA I concentration as 1896±125 (N) and 1104 ±63 (DM). We speculate that the change in the CA activity may of fundamental importance in the regulation of intracellular; pH_i for the basic control of metabolism in diabetes mellitus. Further, we propose that CA activity is a good candidate for a biomarker of diabetes mellitus for the early detection of insulin resistance because the CA activity variation was proportional to the severity of the diabetes. Jehan Ornasir—these studies were undertaken as a partial requirement of her M.S. Degree, Graduate School, Howard University, Washington, DC, USA     

1H‐indazole molecules reduced the activity of human erythrocytes carbonic anhydrase I and II isoenzymes

Carbonic anhydrase (CA) is an important metabolic enzyme family closely related to many physiological and pathological processes. Currently, carbonic anhydrase inhibitors are the target molecules in the treatment and diagnosis of many diseases. In present study, we investigated the inhibitory effects of some indazole molecules on the CA‐I and CA‐II isoenzymes isolated from human erythrocytes. We showed that human CA‐I and CA‐II activities were reduced by of some indazoles at low concentrations. IC₅₀ values, K_i constants, and inhibition types for each indazole molecule were determined. The indazoles showed K_i constants in a range of 0.383 ± 0.021 to 2.317 ± 0.644 mM, 0.409 ± 0.083 to 3.030 ± 0.711 mM against CA‐I and CA‐II, respectively. Each indazole molecule exhibited a noncompetitive inhibition effect. Bromine‐ and chlorine‐bonded indazoles were found to be more potent inhibitory effects on carbonic anhydrase isoenzymes. In conclusion, we conclude that these results may be useful in the synthesis of carbonic anhydrase inhibitors.     

A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: impact on calculation of secondary structure content.

The CD spectra of human carbonic anhydrase I and II and bovine carbonic anhydrase III were recorded and analyzed. The 3D structures of these isoenzymes are known, showing very similar secondary structure and polypeptide-chain fold. The tryptophan content, however, differs between the isoenzymes, i.e., isoenzymes I, II, and III possess 6, 7, and 8 tryptophans, respectively. All of the tryptophans except the additional tryptophans in isoenzymes II and III, i.e., W245 and W47, are conserved. Despite the fact that X-ray structure determinations showed that the isoenzymes had highly similar secondary structure, the contents of alpha-helix and beta-sheet structure differed considerably when using different CD algorithms for estimation of the fractions of various secondary structural elements. This shows that aromatic amino acids also interfere in the wavelength region (far-UV) used to calculate the amount of secondary structure. Such interference is especially problematic when analyzing proteins like carbonic anhydrase, which consist mainly of beta-structure that gives rise to weak ellipticity bands, compared to the bands arising from alpha-helical structure.     

Preparative affinity electrophoresis: application to human erythrocyte carbonic anhydrase

A modification of affinity electrophoresis for preparative purposes is described. This method has been applied to the purification of human erythrocyte carbonic anhydrases B and C. During conventional affinity chromatography some hemoglobin contamination occurs. By introduction of an electrophoretic purification step after the immobilization of carbonic anhydrase to the affinity gel, the hemoglobin impurity is reduced about eight and two times in the preparations of the B and C enzymes, respectively, compared to the enzymes purified by affinity chromatography.     

A single gel for determining genetic variants of equine erythrocyte carbonic anhydrase (CA) and catalase (Cat)

We describe a method for agarose IEF under acid conditions in which a single gel can be used to diagnose from equine red cell lysates genetic variants for carbonic anhydrase (CA) and catalase (Cat). Family and population data for 4801 horses of 27 breeds and seven trap sites of Great Basin feral horses are presented to support the presence of a sixth CA allele, CAE, which has been recognized previously, but not described by published data. Allelic frequencies for the two systems suggest it may be appropriate to use this gel for parentage verification programmes or to obtain population data for studies of the genus Equus.     

The use of prontosil for the visualization of carbonic anhydrase bands in polyacrylamide gels. Application to the carbonic anhydrase isozymes of erythrocytes and white skeletal muscle of the rabbit

Prontosil, a carbonic anhydrase inhibitor of orange-red colour, is used to visualize carbonic anhydrase bands during isoelectric focusing in polyacrylamide gels. 5–60 ng of the sulfonamide Prontosil are added to the 100–200 μl samples before application to the gels. Bound Prontosil moves into the gel together with carbonic anhydrase and stains the enzyme bands formed there, while unbound Prontosil remains on top of the gels. The method is specific, no proteins other than carbonic anhydrase were observed to be stained, and it requires no special equippment. It was applied to chloroform/ethanol extracts of erythrolysates and while muscle homogenates from rabbits. Densitometric evaluation of the Prontosil-stained bands obtained with these extracts showed that rabbit red cells contain roughly equla amounts of carbonic anhydrase isoenzymes B and C while in rabbit white skeletal muscle isoenzyme C is predominant and little B enzyme occurs. These results confirm previous findings obtained by affinity chromatography of erythrolysates and muscle homogenates.     

7-Aryl-triazolyl-substituted sulfocoumarins are potent,selective inhibitors of the tumor-associated carbonic anhydrase IX and XII

Sulfocoumarins behave as interesting inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Here, we report a new series of 7-substituted derivatives which were obtained by the click chemistry approach from 7-propargyloxy-sulfocoumarin and aryl azides incorporating halogens, hydroxy, methoxy and carboxyl moieties in their molecules. The new compounds were screened for the inhibition on four physiologically relevant human CA (hCA) isoforms, the cytosolic hCA I and II and the transmembrane tumor-associated hCA IX and XII. The new compounds did not inhibit the cytosolic isoforms but were low nanomolar inhibitors of the tumor-associated ones hCA IX and 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.     

Inhibition properties of some flavonoids on carbonic anhydrase I and II isoenzymes purified from human erythrocytes

Carbonic anhydrases (CAs, E.C.4.2.1.1) play a critical role in many important physiological events and treatment of some diseases. Flavonoids or phenolic compounds have been discovered as novel CAs inhibitors instead of the traditional sulfonamides, with different binding to CAs, pro‐drug activities, and new inhibition mechanisms. Here, we investigated the inhibition effects of some flavonoids including malvin, callistephin, oenin, pelargonin, silychristin, and 1‐(4‐methoxyphenyl)‐2‐methyl‐3‐nitro‐1‐H‐indol‐6‐ol (ID‐8) against hCA I and II, which purified from human erythrocytes by affinity column chromatography. Both hCA isoenzymes were inhibited by flavonoids, with IC₅₀ and K_i values in the range of 2.34 nM to 346.5 μM and 51.01–99.55 μM for hCA I and 86.60–750.00 μM for hCA II, respectively. These results showed that flavonoids especially malvin and oenin effectively inhibited hCA I and II isoenzymes. Hence, they may be used as an effective CA inhibitor in medical applications for treatment of certain diseases such as glaucoma, in the future.     

Reflections on Edsall's carbonic anhydrase: paradoxes of an ultra fast enzyme

John Edsall's investigations of human erythrocyte carbonic anhydrase, a zinc metalloenzyme that powerfully catalyzes the reversible hydration of carbon dioxide, highlighted a conundrum regarding the correct hydration product. The measured kinetic parameters could not be reconciled with the choice of carbonic acid, since its bimolecular recombination rate with enzyme would exceed the diffusion limit. The alternate choice of bicarbonate obviated the recombination rate problem but required that the active site deprotonation exceed the diffusion-limited maximum rate by an even greater extent. This paradox was resolved in favor of bicarbonate when the unsuspected role of buffer species indirectly deprotonating the enzyme was finally proposed, spurring numerous investigations to verify the hypothesis. Edsall's laboratory also reported the accidental discovery of the first competitive inhibitor, imidazole. This opened new avenues to understanding the binding of the CO₂ substrate and stimulated many investigations on this inhibitor. Paramagnetic NMR and crystallographic studies demonstrated that the only other known competitive inhibitor, phenol, apparently shared this unusual binding site. Despite enormous progress since Edsall's retirement, particularly the use of site-directed mutagenesis approaches, the precise interactions of carbon dioxide and bicarbonate with specific active site moieties remain as elusive today as when Edsall first considered these questions.     

Selected trace elements and esterase activity of carbonic anhydrase levels in lambs with pneumonia

The levels of, zinc, copper, Fe, Zn, Cu, Mn, Mg, K, Na, and Cl and the activity of carbonic anhydrase were determined in lambs with pneumonia. A significant decrease of p<0.01 level in Zn concentration, in Cu level (p<0.001) and significant increases in K and Na levels (p<0.05) and of the Cu/Zn ratio (p<0.001) were observed in the study group. The carbonic anhydrase activity was decreased in the study group, but the decrease was not statistically significant (p>0.05). Also, nonsignificant decreases of Fe, Mg, and Cl and increase of the Mn concentration were also observed in the lambs with pneumonia (p>0.05). Our results suggest that the significant element changes reported here and the Cu/Zn ratio, but not the activity of carbonic anhydrase, can be used as indicators of pneumococcal infection.     

(3,4-Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives as carbonic anhydrase isoenzymes inhibitors

In this study, we have synthesised (3,4-dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and a series of its derivatives (5, 13–16) and tested the ability of these compounds to inhibit two metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I and hCA II. The synthesised compounds showed inhibitory effect on hCA I and hCA II isozymes. The results showed that synthesised compounds (5, 13–16) demonstrated the best inhibition activity against hCA I (IC₅₀: 3.22–54.28 μM) and hCA II (IC₅₀: 18.52–142.01 μM). The compound 14 showed the highest inhibiton effect against hCA I (IC₅₀: 3.22 μM; K_i: 1.19?±?1.4 μM). On the other hand, the compound 13 showed the highest inhibiton effect against hCA II (IC₅₀: 18.52 μM; K_i: 3.25?±?1.13 μM).     

Synthesis,characterization and carbonic anhydrase inhibitory activity of novel benzothiazole derivatives

N-protected amino acids were reacted with substituted benzothiazoles to give the corresponding N-protected amino acid-benzothiazole conjugates (60–89%). Their structures were confirmed by proton nuclear magnetic resonance (¹H NMR), carbon-13 nuclear magnetic resonance (¹³C NMR), IR and elemental analysis. Their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activities were determined against two cytosolic human isoforms (hCA I and hCA II), one membrane-associated (hCA IV) and one transmembrane (hCA XII) enzyme by a stopped-flow CO₂ hydrase assay method. The new compounds showed rather weak, micromolar inhibitory activity against most of these enzymes.     

The inhibitory effect of boric acid on hypoxia-regulated tumour-associated carbonic anhydrase IX

Carbonic anhydrases (EC 4.2.1.1) catalyse the reversible hydration of CO₂ into bicarbonate and protons. As a hypoxia-sensitive and tumour-associated isoform, isoform CA IX, is significantly overexpressed in various malignancies, being a validated target for new anticancer/antimetastatic drugs. A multitude of studies has shown that CA IX inhibition decreases cancer cell proliferation and metastasis through pHe/pHi modulation and enhancement of ferroptosis among others. Numerous studies demonstrated increased efficacy of cytotoxic drugs combined with CA inhibitors (CAIs) in various cancer types. We tested the inhibitory effect of boric acid (BA), an inorganic Lewis acid, on CA IX as well as other isoforms (CA I, II, and XII). BA acted as a millimolar in vitro CAI, decreased proliferation of two cancer cell lines, although not strong correlations between the in vitro inhibition and in vivo effects were observed. The mechanism of antiproliferative action of BA should be investigated in more detail.     

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-l-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K_m and V_max values for the same substrate by Lineweaver–Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K_m values at optimum pH and 20°C were 0.625?mM, 0.541?mM, 0.785?mM and 0.862?mM with p-nitro phenyl acetate, for all CA localizations. The respective V_max values at optimum pH and 20°C were 0.875?μmol/L?min, 0.186?μmol/L?min, 0.214?μmol/L?min and 0.253?μmol/L?min with the same substrate. The K_i and I₅₀ values for the inhibitors sulphanilamide, KSCN, NaN₃ and acetazolamide were determined for all the CA localizations.     

Sequence of the high-activity equine erythrocyte carbonic anhydrase: N-terminal polymorphism (acetyl-ser/acetyl-thr) and homologies to similar mammalian isozymes

The amino acid sequence of the high-activity equine erythrocyte carbonic anhydrase (CA-II) has been determined. Two different N-termini are noted, the C₁ form having an N-acetyl-serine and the C₂ form an N-acetyl-threonine. The sequence of the equine enzyme is most homologous to the human CA-II isozyme, with 224 of the 259 residues being identical.This investigation was supported in part by United States Public Health Service Grant CA-1786 from the National Cancer Institute.     

Modification of carbonic anhydrase activity by antisense and over-expression constructs in transgenic tobacco

The activity and location of carbonic anhydrase has been modified by transformation of tobacco with antisense and over-expression constructs. Antisense expression resulted in the inhibition of up to 99% of carbonic anhydrase activity but had no significant impact on net CO₂ assimilation. Stomatal conductance and susceptibility to water stress appeared to increase in response to the decline in carbonic anhydrase activity. An over-expression construct designed to increase cytosolic carbonic anhydrase abundance resulted in a significant increase in net activity, a small increase in stomatal conductance but little impact on CO₂ assimilation. Chloroplastic carbonic anhydrase activity was enhanced by the expression of an additional construct which targeted the polypeptide to the organelle. The increase in chloroplastic carbonic anhydrase appeared to be accompanied by a concomitant increase in Rubisco activity.