Membrane-associated carbonic anhydrase activity in the brain of CA II-deficient mice

Membrane-associated carbonic anhydrase (CA) activity is of importance for transepithelial transport of ions and fluid. Histochemical studies have indicated its presence in the brain, but the data are difficult to evaluate because of interference from cytoplasmic CA isozymes, of which CA II is the predominant one. CA II-deficient mice offer a possibility to study the location of membrane-associated CA-activity, without interference from CA II. The location of CA activity in the brain of CA II-deficient and normal mice was studied by the cobalt-phosphate histochemical method, and that of CA I, CA II and CA III by an immunocytochemical method. The brains of both types of mice lacked cytoplasmic isozymes CA I and CA III, and the CA II-deficient mice also lacked CA II. In the normal mice, oligodendrocytes and choroid epithelium stained for CA II in the cytoplasm. In normal and CA (II)D-mice there was an intense membrane associated histochemical CA activity in neuronal processes. Neuronal perikarya were not stained. Endothelial membranes of brain capillaries showed strong histochemical CA-activity. Choroid epithelial cells had histochemical CA activity in the cytoplasm and along apical and baso-lateral cell membranes. The results suggest that membrane-associated CA-activity found along neuronal processes probably modulates pH of the extracellular fluid and thus neuronal activity. CA II and the membrane-associated CA of choroidal epithelium are probably involved in the secretion of cerebrospinal fluid.     

Membrane-associated carbonic anhydrase activity in the kidney of CA II-deficient mice.

Carbonic anhydrase II-deficient mice offer a possibility to study the localization along the nephron of membrane-associated carbonic anhydrase (CA) activity without interference from the cytoplasmic enzyme. We studied the localization of CA in kidneys from CA II-deficient and control mice by immunocytochemistry (CA II) and histochemistry. Cytoplasmic staining was found in convoluted proximal tubule, thick limb of Henle, and principal and intercalated cells of collecting duct in the control animals but was absent in the CA II-deficient mice. In cells with cytoplasmic staining the cell nuclei were stained. Intense histochemical activity was associated with apical and basolateral membranes of convoluted proximal tubule, first part of thin limb, thick limb, and basolateral membranes of late distal tubule. In collecting ducts of control animals, the basolateral cell membranes of intercalated cells were the only clearly stained membranes. In CA II-deficient animals one type of intercalated cell was stained most intensely at the apical membranes and another only at the basolateral. We suggest that the former corresponds to Type A intercalated cells secreting H+ ions to the luminal side and the latter to Type B cells secreting H+ ions to the basolateral side.     

Carboxyl methylation of cytosolic proteins in intact human erythrocytes. Identification of numerous methyl-accepting proteins including hemoglobin and carbonic anhydrase

Intact human erythrocytes incubated with L-[methyl-3H]methionine incorporated radioactivity into base-labile linkages with membrane and cytosolic proteins which are characteristic of protein methyl esters. Kinetic analysis of the methylation reactions in intact cells shows that individual erythrocytes contain approximately 38,000 and 115,000 protein methyl esters with biological half-lives of 150 min or less in the membrane and cytosolic protein fractions, respectively. Fractionation of the methylated cytosolic species by gel filtration chromatography at pH 6.5 followed by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 reveals that many different cytosolic proteins serve as methyl acceptors and that the degree of modification varies widely for individual proteins. For example, hemoglobin is modified to the extent of 3 methyl groups/10(6) polypeptide chains, while carbonic anhydrase contains 1 methyl group/approximately 16,500 polypeptide chains at steady state. Aspartic acid beta-[3H]methyl ester (Asp beta-[3H]Me) can be isolated from carboxypeptidase Y digests of cytosol proteins. By synthesizing and separating diastereomeric L-Leu-L-Asp beta Me and L-Leu-D-Asp beta Me dipeptides, we show that all of the Asp beta-[3H]Me recovered from cytosolic proteins is in the D-stereoconfiguration. Based on these data and on previous observations that erythrocytes contain a single methyltransferase which also methylates red cell membrane proteins at D-aspartyl residues both in vivo (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464) and in vitro (O'Connor, C. M., and Clarke, S. (1983) J. Biol. Chem. 258, 8485-8492), we propose that protein carboxyl methylation is part of a generalized mechanism for metabolizing damaged proteins. The infrequent and spontaneous occurrence of D-aspartyl residues in proteins adequately explains the broad substrate specificity and limited stoichiometries of protein carboxyl methylation reactions.     

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     

Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney

We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage. It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-linked or O-linked oligosaccharide chains. Reduction of disulfide linkages led to altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loss of catalytic activity. CA IV resembles CA II in being a "high activity" isozyme, relatively resistant to inhibition by halide ions and sensitive to inhibition by sulfonamides. Application of this purification to human kidney membranes produced homogeneous enzyme with nearly identical properties. Amino acid compositions of both lung and kidney CA IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC). Amino-terminal sequences of native enzyme from lung and kidney were identical, as were amino-terminal sequences of the three major tryptic peptides resolved on reverse phase HPLC. Isoelectric focusing revealed microheterogeneity in enzyme from both sources. Antibody raised to human lung CA IV reacted equally strongly with CA IV from kidney, but very weakly or not at all with other CAs. Treatment of lung membranes and kidney membranes with phosphatidylinositol-specific phospholipase C released over half of the membrane-bound CA IV, suggesting that at least half of the CA IV in both organs is anchored to membranes by phosphatidylinositol-glycan linkages.     

Carbonic anhydrase activity of rabbit lungs

Pulmonary carbonic anhydrase (CA) activity was studied in rabbit lungs perfused with solutions containing no CA. Measurements were made of the amount of 14CO2 appearing in the expired gas following injections of H14CO3(-), 14CO2, or a 20:1 mixture of each into the pulmonary artery. The fraction of the injected label in the expired gas was only 17% greater for 14CO2 than for the mixture, suggesting that equilibration between H14CO3(-) and 14CO2 was nearly complete during the capillary transit time. Inhibition of pulmonary CA decreased excretion of H14CO3(-) and the mixture by 40 and 49% and increased the excretion of 14CO2 by 96%. Addition of CA to the perfusate had no effect. Thus, CO2 exchange is not significantly limited by pulmonary CA if inhibitors are absent. Tissue binding of [3H]acetazolamide injected into the pulmonary artery was diminished by 50% when acetazolamide concentrations reached 0.13 x 10(-6) M. Each liter of extravascular lung water contained 1.25 x 10(-6) mol of receptors for acetazolamide that were accessible to plasma during a single circulation. Binding of [3H]acetazolamide was also observed in lungs of anesthetized rabbits, suggesting that pulmonary CA is accessible to plasma in vivo as well as in situ.     

The amino acid substitution and some chemical properties of a variant human erythrocyte carbonic anhydrase: Carbonic anhydrase IdMichigan

Carbonic anhydrase Id_Michigan, an electrophoretic variant of human red cell carbonic anhydrase I, was purified from erythrocytes obtained from an individual heterozygous for the trait. Primary structural analysis indicates that a lysine residue has exchanged for a threonine residue in the variant enzyme. After isolation, there was approximately 1.8 times as much normal as variant enzyme. Thermostability studies demonstrated that carbonic anhydrase Id was more thermolabile than the normal enzyme. The normal and variant enzymes showed no differences in specific carboxylesterase activity or CO₂ hydratase activity. Utilizing the carboxylesterase activity toward -naphthyl acetate, the normal and variant enzymes had similar Michaelis constants, pH profiles, and rates of inhibition by acetazolamide. Immunochemical studies did not demonstrate an antigenic difference for the variant enzyme.Supported in part by Research Grants 2 T1 GM-76, 5 TO1 GM 00071-09, and GM 09252 from U.S. Public Health Service.This report is a portion of a dissertation submitted to the University of Michigan in partial fulfillment of the requirements for the doctor of philosophy degree.     

Carbonic anhydrase activity in intact red blood cells measured with 18O exchange.

We have used a stirred, temperature-regulated, reaction vessel separated by a Teflon membrane from the ion source of a mass spectrometer to monitor continuously the time course of disappearance of C18O16O, mass 46, at chemical equilibrium as the 18O exchanges with 16O in water. This instrument is sensitive to less than 0.01 mm Hg of partial pressure of C18O16O with a response time of less than 3 s. The equation of Mills and Urey was used to calculate the hydration velocity constant for uncatalyzed or catalyzed homogenous solutions from the exponential disappearance of mass 46. Addition of red blood cells to the reaction mixture produces biphasic (double exponential) disappearance curve for mass 46. A theory of this process has been developed which describes the time course of [C18O16O] as a function of the catalytic factor for intracellular carbonic anhydrase (A) and the permeability of the cell membrane to HCO3- (P) in addition to the known values; water volume of the cells in the suspension, extracellular pH, the extracellular hydration reaction velocity constant, ku, and dehydration reaction velocity constant, ku. Using this theory, A and P were estimated from the disappearance curve for mass 46 at different values of hematocrit in the reaction mixture, both by a trial and error curve fitting procedure and by a more convenient graphical linearization method. The values of A and P obtained were very sensitive to small amounts of lysis (less than 1%), but the graphical method of analysis minimized this effect. For the blood cells of five normal subjects suspended in 24 mM bicarbonate in 145 mM NaCl at pH 7.4 and 37 degrees, using the graphical method we obtained an average value of 9,906 for A as compared to 19,900 for a comparable concentration of hemolysate. Correcting for a lower pH and chloride concentration inside the cell the latter figure would reduce to 17,500, still 80% higher than the intracellular value. The reason for this discrepancy is not clear. The average permeability of the red cell to bicarbonate ion was 3 X 10(-4) cm/s.     

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.     

Purification and properties of carbonic anhydrase from salmon erythrocytes

Carbonic anhydrase (CA) from erythrocytes of the pink salmon, Onchorhyncus gorbushka, was purified using chloroform-ethanol extraction and Sephadex G-75 gel filtration. A single, high specific-activity CA isozyme having a molecular weight of 29,000 was found. The enzyme sedimented as a single boundary at a sedimentation velocity of 2.9S. Amino acid analysis revealed a composition similar to other submammalian CAs with the exception that the cysteine content was low (1 mol cysteine/mol enzyme). Like other submammalian CAs, the presence of a sulfhydryl reducing agent was required to maintain full activity and to prevent structural changes in the enzyme.     

Characterization of a high activity carbonic anhydrase isozyme purified from erythrocytes of Salmo gairdneri

1. A single, high specific activity carbonic anhydrase (CA) isozyme was present in erythrocytes of the teleostean species Salmo gairdneri (rainbow trout). 2. Purification of trout CA to homogeneity was accomplished using chloroform ethanol extraction, Sephadex G-75 gel filtration, and DEAE Bio-Gel anion exchange chromatography. 3. Trout CA was a zinc metalloenzyme of mol. wt 28,300 and pI9.3. 4. Amino acid analysis indicated the presence of 6 half-cystine residues per enzyme molecule, and the presence of a sulfhydryl reducing agent was required to maintain full activity in vitro. 5. Sulfhydryl modification with both N-ethylmaleimide and acrylonitrile indicated the presence of 3 reactive sulfhydryl groups per CA molecule. Modification of those groups had no direct effect on enzyme activity, but modified CA was no longer subject to inactivation by oxidizing conditions.     

Effects of melatonin on carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo

The in vitro effects of melatonin (N-acetyl-5-methoxy-tryptamine) on human carbonic anhydrase isozymes (HCA-I and HCA-II) from human erythrocytes and in vivo effects on rat erythrocytes carbonic anhydrase (CA) were determined. Human erythrocyte carbonic anhydrase isozymes were purified by haemolysate preparation and Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The HCA-I enzyme, having a specific activity of 7337.5 EU/mg protein, was purified 843-fold with a yield of 60% and the HCA-II enzyme, having a specific activity of 17067EU/mg protein, was purified 1962-fold with a yield of 22.7%. For in vitro experiments, the enzyme activity was minimal at 2 x 10(-4) M melatonin concentration and increased above this concentration. Ten mgkg(-1) melatonin was administered intraperitoneally and showed a stimulatory effect on the enzyme. Time-dependent in vivo studies were conducted for melatonin in Sprague-Dawley type rats. It was found that CA activity in the rat erythrocytes was decreased by the melatonin after 1 and 3 hours to 2500 +/- 500.0 and 1875 +/- 239.4 respectively which were statistically significant (p < 0.05) differences to the control (2660 +/- 235.8). However, CA activity was restored to its normal level after 6h (2666 +/- 235.7) (p > 0.05) probably due to metabolism of the melatonin. The findings indicate that melatonin may be pharmacologically useful in some diseases.