Carbonic Anhydrase Enzyme Activities Research Articles

Carbonic anhydrase I reduction in colonic mucosa of patients with active ulcerative colitis.

Ulcerative colitis (UC) is associated with low intracolonic pH and unbalanced transmucosal ionic exchanges. Along the gastrointestinal tract carbonic anhydrase isoenzyme I (CA-I) is specifically expressed in colon epithelium and is involved in mucosal control of ion, fluid, and acid-base balance. Since altered CA-I expression may play some role in UC, CA-I was measured at the mRNA and protein level and carbonic anhydrase (CA) enzyme activity was determined in colon biopsies of 14 UC patients (6 remission, 4 mild, 4 moderate UC) and of 12 healthy subjects. Patients with mild or moderate UC showed a significant reduction of CA-I mRNA and protein and of total CA activity in the inflamed mucosa compared to controls. Patients with UC in remission showed a pattern of CA-I expression and CA activity similar to controls. This is the first report showing a reduction in the expression of CA-I in active UC.

Buffer capacity in the blood of the hemoglobinless Antarctic fish Chionodraco hamatus

Blood acid-base homeostasis of the hemoglobinless Antarctic teleost Chionodraco hamatus was analyzed by measuring the titratable buffer capacity (β) and carbonic anhydrase (CA) activity in blood smaples. Results were compared with those obtained in the red-blooded temperate fish Anguilla anguilla. Data show that the titratable blood buffer capacity of icefish, in the range of red-blooded teleost species, was significantly higher than that of A. anguilla. Furthermore, plasma inorganic phosphate and reactive sulfhydryl content was significantly higher in the Antarctic species. No enzymatic activity of CA was detected in blood samples of Antarctic fish, suggesting the absence of a blood CA-isozyme.

The effects of some pesticides on human and bovine erythrocyte carbonic anhydrase enzyme activitiesin vitro

Abstract The influence of Penncozeb [Mangan‐zinc ethylenediamine‐bisdithicarbamate], Dithane, [Manganese etylenebisdithiocarbamate], Cupravit [Cuper oksichloride], Bayleton [1‐(4‐ chlorophenoxy)‐3,3‐dimethyl‐1‐(1H‐1.2.4‐Thiazol‐l‐yl)‐2.2‐butanon], Baythroid [Cyano‐(4 fluoro‐3‐phenoxyphenyl)‐methyl‐3‐(2.2‐dichloroethyl ‐2. 2 metyl ‐ cyclo‐propancarboxylate], Mavrik [‐cyano‐3‐phenoxybenzyl N‐(2‐chloro—trifluoro ‐ p ‐tolyl], [alstar [2‐methyl biphenyl‐3‐ylmethyl(z)‐(IRS,3RS)‐3‐(2‐chloro‐3.3.3 ‐triflouroprop ‐1‐enyl)=Dimethylcyclo propenecarboxyate], Endosulfan [6.7.8.9.10.10‐hekzachloro‐1.5.5a.6.9.9a ‐ hekzahydro‐6. 9‐methano‐2.3.4 ‐ benzole dioxi atehiepin 3‐oxide] has been investigated on human erythrocyt carbonic anhydrase isozymes (HCA‐I, HCA‐II) and bovine erythrocyte carbonic anhydrase (BCA) in vitro. Among the chemicals; Baythroid, Talstar, Mavrik were determined to have inhibition effect, on bovine CA and human CA isoenzymes. Dithane had effect of inhibition only on BCA. The I50 values of chemicals c...

Endolithic algae within corals - life in an extreme environment

Endolithic algae living inside skeletons of living corals are exposed to very low light intensities. Assays carried out on the coral Porites compressa revealed drastic diurnal fluctuations in pH and O 2 levels within the skeleton, dominated mainly by coral photosynthesis by day and respiration at night. Enzymatic activity of carbonic anhydrase and catalase, as well as photosynthetic adaptations to low light level and low metabolic activity rates, enable the algae to grow in this unique habitat.

3H]Acetazolamide binding to carbonic anhydrase in normal and transformed cells

The binding of [ 3H]acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor, to soluble and particulate forms of CA was investigated. Sources for the assays were purified CA II, adult rat cortical, oligodendrocyte and neuronal enriched preparations; cultured murine glial cells, rat C-6 glioma, rat hepatoma and human glioblastoma cells. CA enzyme activity in the same preparations was also assayed by following change in pH during incubation. A gel permeation chromatographic method was developed to assess [ 3H]AZ binding to soluble CA, while glass fiber filter vacuum filtration was used for paniculate CA binding. Saturable specific binding of [ 3H]AZ to rat cortical soluble and particulate CA preparations was demonstrated. Computer-assisted data analysis estimated the binding parameters of [ 3H]AZ to soluble rat cortical CA to be B max = 0.38 ± 0.13 pmol/mg protein and K d = 34.7 ± 17.5 nM. The rat cortical particulate fraction B max was 2.05 ± 0.28 pmol/mg protein with a K d of 107.1 ± 24.2 nM. Purified bovine CA-II bound 1.15 ± 0.19 pmol [ 3H]AZ/mg protein with a K d of 54.0 ± 3.4 nM. The pH optima for [ 3H]AZ binding to soluble and particulate CA was between 6.5 and 7.5. Binding was linear with respect to protein up to 1.0 mg/mL. The particulate fraction bound 3–4 times more [ 3H]ligand per unit protein than the soluble fraction. Interestingly, no detectable CA enzyme activity or [ 3H]AZ binding was observed in the soluble or particulate fractions of human glioblastoma, rat C-6 glioma or rat hepatoma cells. Binding of [ 3H]AZ to other soluble enzymes or proteins was negligible. In competition binding experiments, a rank order of inhibition of [ 3H]AZ binding to rat cortical CA by established CA inhibitors was: dichlorphenamide > acetazolamide ⩾ benzolamide > methazolamide > hydrochlorothiazide ⩾ sulfanilamide. [ 3H]AZ binding was not affected by other classes of pharmacologic characterizing agents. The binding of [ 3H]AZ to the CA enzyme molecule is highly specific and sensitive and may prove useful in vitro or in situ as a probe for this enzyme.

Biochemistry and physiology of brain ammonia.

Biochemistry and physiology of brain ammonia.

Brush border Mg 2+-HCO 3−-ATPase, supernatant carbonic anhydrase and other enzyme activities isolated from rat intestinal mucosa: Effect of adrenalectomy and aldosterone administration

The possible role of Mg 2+ -HCO 3 -ATPase, carbonic anhydrase and several other enzymes in rat intestinal mucosa us mediators of the action of aldosterone has been examined. The small-intestinal tract was cut into seven segments, 15 cm each in length and the mucosa was scraped off, homogenized in 50 mM d-mannitol-2 mM Tris-HCl buffer (pH 7.1), differentially fractionated and a crude brush border was obtained. The mucosa from the colon and rectum was combined and used as the large-intestinal sample. Five days after the adrenalectomy, activities of brush border Mg 2+ -HCO 3 -ATPase and supernatant carbonic anhydrase from the upper small intestine decreased to about 60 and 40% of normal values, respectively. Activities of Na +-K + -ATPase, β-glycerophosphatase and succinate dehydrogenase were all decreased. Two and 4 h after i.p. injection of aldosterone (40 μg/kg) to adrenalectomized rats, all enzyme activities increased except for Na +-K +-ATPase in the upper small intestine. In contrast, Mg 2+-HCO 3 −-ATPase and carbonic anhydrase activities were unchanged 3 h after i.p. injection of dexamethasone (200 μg and 1 mg/kg). The activation of both Mg 2+ -HCO 3 -ATPase and carbonic anhydrase by a single injection of aldosterone was blocked by pretreatment with cycloheximide (1 mg/kg). These results suggest that aldosterone may induce the synthesis of enzyme proteins in the intestinal mucosa.

Rapid isolation of mammalian Mu¨ller cells

A procedure has been developed for the rapid isolation of relatively pure populations of glia, Mu¨ller cells, from mammalian retina and is reported here for the rabbit. The retina, cleaned of vitreous, is dissociated by 4 interacting operations — enzymatic digestion of extracellular matrix by means of hyaluronidase, collagenase and papain, removal of divalent cations, acidification and mild trituration. The resultant admixture consists of receptors, neurons and glia; 10% of the cells are Mu¨ller cells. These glial elements can be brought to 95% purity by rapid centrifugation on convex 0–30% Percoll gradients. Of the resultant glia, 80% exclude trypan blue. The Mu¨ller cells are enriched in two glial specific enzymes, glutamine synthetase and carbonic anhydrase and they retain a significant fraction of the membrane bound carbonic anhydrase enzyme activity. Light and scanning electron microscopy show the cells to be well preserved and covered extensively with microvilli. In the outer nuclear zone, the cells are plicated and end in bulbs tufted with microvilli. The procedure we describe allows studies of a new preparation of intact, relatively undamaged, adult, isolated mammalian Mu¨ller cells to better understand the functioning of glial cells.

Determination of Ultratrace Zinc by Enzymatic Activity of Carbonic Anhydrase. II. Use of Carbonate Hydro-lyase Activity

Abstract A method for the determination of trace zinc has been investigated using hydro-lyase activity of carbonic anhydrase. It was proved theoretically and experimentally that the activation rate of the apo-carbonic anhydrase with zinc is proportional to the amount of zinc present in the solution. Hydro-lyase activity was measured electrochemically using sodium hydrogencarbonate as a substrate. The limit of detection was 0.89 ng/ml or 40 pg of zinc, lower than that obtained by the esterase activity method. Application of the present method to the determination of zinc in real samples has been examined in order to evaluate its analytical feasibility.

Effects of radiocontrast media on ion transport in the toad urinary bladder.

The effects of the ionic contrast media, diatrizoate and iothalamate, on the sodium transport system of the toad urinary bladder, a biological model for the distal nephron of the mammalian kidney, were examined. It was found that 15–60 mM concentrations of diatrizoate suppress sodium transport in this tissue in a concentration-dependent fashion. They have this effect only from the serosal (blood) side of the tissue and not from the mucosal (urinary) side. By means of radioisotopic bidirectional sodium fluxes we showed that the short-circuit current (SCC) remains a valid measure of the net sodium transport in this tissue in the presence of contrast media. The neutral (nonionic) contrast medium metrizamide has no suppressant effects on the SCC system. The double-ring gallbladder contrast media (iodipamide, iocarmate, and iodoxamate) have somewhat more potent sodium transport suppressant effects than the single-ring media. When vasopressin is applied to the toad bladder after the contrast media the SCC is strongly stimulated, and in some cases rises to match the SCC of paired control tissue not subjected to contrast media. We take this as evidence that the contrast media do not poison the specific sodium transport enzyme, Na-K-ATPase, and further, that the contrast media are not metabolic inhibitors of the energy-producing reactions linked to sodium transport in this tissue. It was found that numerous other large anions that are not contrast media (but rather are from unrelated pharmacologic classes such as antibiotics, anionic dyes, bile salts, and dextran sulfates) have similar effects on the sodium transporting system of the toad bladder. The authors interpret these effects in terms of a mechanical model for regulation of sodium transport in the toad urinary bladder at the lateral intercellular space, after the active sodium pump, rather than at the classical locus, the sodium permeability of the apical (mucosal) cell membrane. In separate studies the authors found that the ionic contrast media will inhibit acidification in the toad urinary bladder. In pH-stat experiments the authors have shown that iodipamide will inhibit acidification when applied to the serosal side of the bladder, but not when it is applied to the mucosal side. Again, there is a concentration dependence, with larger concentrations of iodipamide having a greater inhibition effect on the acidification system. The authors have tested the effects of the contrast media on purified carbonic anhydrase enzyme activity and found that in the concentrations used to inhibit acidification in the toad bladder, there is no effect on the enzyme activity. Thus, they conclude that contrast media inhibit acidification by some means other than inhibition of carbonic anhydrase. The exact site of action of the contrast media remains to be determined, but the authors favor the idea that the anionic contrast media inhibit escape of bicarbonate from the serosal side of the epithelial tissue. It remains to be shown whether contrast media cause a temporary renal tubular acidosis in whole organisms.

Determination of Ultratrace Zinc by Enzymatic Activity of Carbonic Anhydrase

Abstract A method for the determination of trace zinc has been investigated by using enzymatic activity of carbonic anhydrase. The recovery of enzymatic activity of apo-carbonic anhydrase was proportional to the amount of zinc in solution theoretically and experimentally, when it was monitored spectroscopically using the substrate, p-nitrophenyl acetate. The amount of zinc in the sample solutions was therefore determined from the curve, zinc concentration vs. the enzymatic activity. The limit of detection is 10 ng/ml or 2 ng. The method has been applied to the analysis of zinc in fruit juices and water samples.

Calcium transport across avian uterus. III. Comparison of laying and nonlaying birds.

Transepithelial calcium fluxes were measured across isolated uterus of laying, nonlaying, and molting quail under conditions where no electrochemical difference existed across the tissue. Net uterine transfer of calcium in molting and nonlaying birds occurs in the secretory direction and is approximately one-fifth the value obtained for laying birds. The carbonic anhydrase enzyme activity of uteri from laying birds is twice that of uteri from molting birds and five times greater than that of uteri from nonlaying birds. When measured in the presence and absence of 2-mercaptoethanol or dithiothreitol, no statistical difference exists in uterine carbonic anhydrase activities of laying birds or the inhibitory effect of acetazolamide. These results indicate that nonlaying and molting quail secrete calcium at a rate much lower than that of laying quail and that net uterine transfer of calcium exhibits varing degrees of dependence on bicarbonate ion in laying, nonlaying, and molting birds. Carbonic anhydrase data sugges that the activtiy of this enzyme in the quail uterus may be related to uterine calcium secretion.

Binding of Antibiotics to the Human Intracellular Erythrocyte Proteins Hemoglobin and Carbonic Anhydrase

The interaction between human erythrocyte lysates and antibiotics was studied, and the effect of intracellular components on the activity and binding of the drugs was determined. Lysates inhibited antibacterial activity of penicillin G, dicloxacillin, tetracycline, and minocycline to about the same extent as did human plasma. Dicloxacillin activity was the most inhibited, followed by the activities of penicillin G, minocycline and tetracycline. All four antibiotics bound to human hemoglobin, as determined by gel filtration methods. Heme-free globin was also effective in binding the antibiotics. In addition, minocycline and tetracycline were bound to another erythrocytic protein, which, on the basis of electrophoretic mobility, molecular size, and localization, has been identified as carbonic anhydrase. Experiments with pure preparations of carbonic anhydrase revealed that the C isozyme is the major binder of the tetracyclines and that zinc is required for binding. Tetracyclines did not inhibit enzymatic activity of carbonic anhydrase.