Carbonic anhydrase.

1. The G + C content of ribosomal RNA of animals seems correlated with the length of periods required for maturation of those organisms. 2. In Protostomes of the animal kingdom, the size of the 28S rRNA molecule does not seem to correlate with the evolutionary stage of the organism. 3. Aphids and water-fleas as well as some protozoa have the 18S rRNA with mol. wt of 0.9 x 10(6) against an overwhelming pressure of evolution to conserve the rRNA molecule of 0.7 x 10(6) daltons. 4. All the Deuterostomes examined were distinguished from Protostomes by having the 28S rRNA's void of the hidden break at the central point. 5. Aphids and nematodes are exceptional Protostomes in that they have the 28S rRNA's without the hidden break. This was discussed in the light of the evolutionary stage of these organisms. 6. Molecular properties of chloroplast rRNA seem to evidence for endosymbiotic origin of this organelle. Mitochondrial rRNA differs considerably from prokaryotic rRNA with respect to molecular size and base composition.     

Carbonic anhydrase activity of intact carbonic anhydrase II-deficient human erythrocytes

Intact erythrocytes from subjects with deficiency of blood carbonic anhydrase (CA) II and from normal subjects were assayed for enzyme activity by use of an 18O exchange technique in a solution containing 25 mM (CO2 + NaHCO3) plus 125 mM NaCl. At 25 degrees C and pH 7.4, the catalyzed reaction velocity was 0.32 +/- 0.04 M/s for the CA II-deficient and 1.60 +/- 0.12 M/s for the normal cells, a ratio of 1:5. Under the same conditions at 37 degrees C the relative difference between the CA II-deficient and normal cells was much less: the velocity for the CA II-deficient cells was 0.84 +/- 0.07 M/s and for the normal cells 1.60 +/- 0.32 M/s, a ratio of 1:1.9. Results were comparable for the hemolysates with the NaHCO3 reduced to 85 mM (the corresponding intracellular concentration): at 25 degrees C CA II-deficient cells had a velocity of 0.36 +/- 0.01 M/s compared with 1.12 +/- 0.04 M/s for the normal cells, a ratio of 1:3.1. At 37 degrees C again the relative difference between hemolysates from CA II normal and deficient cells was much less: the CA II-deficient cells had a reaction velocity of 1.17 +/- 0.22 M/s vs. 2.60 +/- 0.36 M/s for the normal cells, a ratio of 1:2.2. The greater fractional reduction of enzyme velocity of CA II-deficient cells at 25 degrees C compared with 37 degrees C appears to be explained by a greater chloride inhibition of the presumed CA I at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbonic anhydrase inhibitors

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread enzymes in all organisms, catalyzing CO₂ hydration to bicarbonate and protons. Their inhibition is exploited clinically for decades for various classes of diuretics and systemically acting antiglaucoma agents. In the last years novel applications of CA inhibitors (CAIs) emerged, such as topically acting antiglaucoma, anticonvulsants, antiobesity, antipain, and antitumor agents/diagnostic tools. Such CAIs target diverse isozymes of the 13 catalytically active α-CA isoforms present in mammals. CAs belonging to the α-, β-, γ-, δ-, and ζ-families are found in many organisms all over the phylogenetic tree, and their inhibition was studied ultimately for some pathogenic protozoa (Plasmodium falciparum), fungi (Cryptococcus neoformans, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae), and bacteria (Helicobacter pylori, Mycobacterium tuberculosis, and Brucella suis). Novel interesting chemotypes, in addition to the sulfonamide and sulfamate CAIs, such as coumarins, phenols, and fullerenes, were also reported recently, together with their mechanism of inhibition. This class of enzyme inhibitors shows promise for designing interesting pharmacological agents and understanding in detail protein–drug interactions at molecular level.     

Carbonic anhydrase IX

Cell migration can be principally viewed as a chain of well-orchestrated morphological events that lead to dynamic reshaping of the cell body. However, behind the scene of such a “morphological theater” there are very complex, interrelated molecular and physiological processes that drive the cell movement. Among them, ion transport and pH regulation play a key role, with carbonic anhydrase IX (CA IX) emerging as one of the important “molecular actors.” CA IX is a highly active cell surface enzyme expressed in a broad range of solid tumors in response to hypoxia and explored as a clinically useful biomarker of hypoxia and as a therapeutic target. Its biological role is to protect tumor cells from hypoxia and acidosis in the tumor microenvironment. The study published recently by our group showed that CA IX actively contributes to cell migration and invasion. For the first time, we demonstrated CA IX accumulation in lamellipodia of migrating cells and its direct in situ interaction with bicarbonate transporters. Our findings indicate that tumor cells need CA IX not only as a pro-survival factor in hypoxia and acidosis, but also as a pro-migratory component of the cellular apparatus driving epithelial-mesenchymal transition.     

Carbonic anhydrase C in white-skeletal-muscle tissue.

We investigated the activity of carbonic anhydrase in blood-free perfused white skeletal muscles of the rabbit. Carbonic anhydrase activities were measured in supernatants and in Triton extracts of the particulate fractions of white-skeletal-muscle homogenate by using a rapid-reaction stopped-flow apparatus equipped with a pH electrode. An average carbonic anhydrase concentration of about 0.5 microM was determined for white skeletal muscle. This concentration is about 1% of that inside the erythrocyte. Some 85% of the muscle enzyme was found in the homogenate supernatant, and only 15% appeared to be associated with membranes and organelles. White-skeletal-muscle carbonic anhydrase was characterized in terms of its Michaelis constant and catalytic-centre activity (turnover number) for CO2 and its inhibition constant towards ethoxzolamide. These properties were identical with those of the rabbit erythrocyte carbonic anhydrase C, suggesting that a type-C enzyme is present in white skeletal muscle. Affinity chromatography of muscle supernatant and of lysed erythrocytes showed that, whereas rabbit erythrocytes contain about equal amounts of carbonic anhydrase isoenzymes B and C, the B isoenzyme is practically absent from white skeletal muscle. Similarly, ethoxzolamide-inhibition curves suggested that white skeletal muscle contains no carbonic anhydrase A. It is concluded that white skeletal muscle contains essentially one carbonic anhydrase isoenzyme, the C form, most of which is probably of cytosolic origin.     

Carbonic anhydrase in mammalian vascular smooth muscle.

Carbonic anhydrase (CA) is ubiquitously expressed and plays a pivotal role in acid-base balance, ion transport, and gas exchange. Limited observations by others, derived from functional, pharmacological, and histochemical studies, suggest that CA is present in vascular smooth muscle and is involved in vasoregulation. The present study, using measurements of bioactivity, inhibition characteristics, and immunohistochemical analysis, was undertaken to more fully evaluate CA in vascular smooth muscle. In isolated bovine aortic smooth muscle, which is devoid of erythrocytes, CA is present in low concentrations with a CO(2) hydration activity (at 0C) of 3.5 +/- 2.7 U/g. The I(50) for acetazolamide inhibition is 0.07 +/- 0.01 microM. Results with dorzolamide and bromopyruvate, selective inhibitors of the CA II and I isozymes, respectively, show that roughly 75% of the CA activity is accounted for by CA I, with 20% due to CA II. These results accord qualitatively with immunocytochemical staining with specific CA I and II antibodies, showing that both isozymes are present and that their staining co-localizes with cells positive for smooth muscle alpha-actin. These data establish the activity, inhibition, and isozyme pattern of carbonic anhydrase expression in mammalian vascular smooth muscle.     

Carbonic anhydrase from Vicia canencens leaves.

Carbonic anhydrases (CA: Carbonate hydrolase; E, C,4.2.1.1) from leaves Vicia canencens were purified and were characterized. The purification level of enzyme was 76-fold. The optimum temperature of this enzyme was 70 degrees C. pH optimum was 9.2. Each of the enzyme molecules was a decamer having MW of 262,000 and the subunit MW was 26,400.     

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 VI in the mouse nasal gland.

Western blotting analysis of mouse nasal tissue using a specific anti-mouse secreted carbonic anhydrase (CA VI) antibody has shown that CA VI is present in this tissue. A single immunoreactive band of 42 kD was observed, as has been found previously for salivary tissues. RT-PCR analysis has shown that nasal mucosa expressed CA VI mRNA. By immunohistochemistry (IHC), CA VI was observed in acinar cells, in duct contents of the anterior gland of the nasal septum, and in the lateral nasal gland. The Bowman's gland, the posterior gland of the nasal septum, and the maxillary sinus gland were negative. Immunoreactivity was also observed in the mucus covering the respiratory and olfactory mucosa and in the lumen of the nasolacrimal duct. In contrast, an anti-rat CA II antibody (that crossreacts with the mouse enzyme) stained only known CA II-positive cells and an occasional olfactory receptor neuron. These results indicate that CA VI is produced by the nasal gland and is secreted over the nasal mucosa. By reversible hydration of CO(2), CA VI is presumed to play a role in mucosal functions such as CO(2) sensation and acid-base balance. It may also play a role in olfactory function as a growth factor in maturation of the olfactory epithelial cells.     

Carbonic anhydrase in the monkey kidney

The distribution of carbonic anhydrase in the kidney of the cynomolgus monkey was studied by the histochemical method of Hansson. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed high enzyme activity at the brush border and the basolateral membranes and the cytoplasm. Straight proximal tubules were less intensely stained. In nephrons with long loops of Henle, the descending thin limb contained weak enzyme activity, whereas the ascending thin limb was inactive. The thick limb of Henle's loop displayed most enzyme activity at the luminal cell border. In distal convoluted tubules enzyme activity was restricted to the basal part of the cells. In the late distal tubule, intercalated cells appeared among the "ordinary" distal cells and contained abundant cytoplasmic enzyme. Many intensely stained intercalated cells were also found in the cortical and outer medullary segments of the collecting duct, intermingled with more weakly stained chief cells. In the inner medullary segment of the collecting duct, enzyme activity gradually disappeared. Many capillaries were clearly stained for enzyme activity. The capillary staining apparently varied with that of the kidney tubules; virtually all capillaries in the cortex, but very few in the inner medulla, were stained. The distribution of carbonic anhydrase in the kidney tubules of the monkey is very similar to that in man and in the rat, but the primate kidney differs from the rat kidney by the presence of capillary enzyme activity. The functional importance of this difference is not clear at present.     

Carbonic anhydrase is present in human oesophageal epithelium and submucosal glands

Summary Carbonic anhydrase (EC 4.2.1.1) activity was investigated in normal human oesophageal mucosa using the Hansson and Ridderstråle catalytic cobalt methods. The enzyme was detected in the cell membranes and nuclei and, to a lesser extent, in the cytoplasm of the epithelial cells of the mucosa giving a chicken wire appearance. Activity decreased towards the lumen. Other stratified squamous epithelia - buccal mucosa, ectocervix and skin - gave a similar pattern. Acinar cells of oesophageal submucosal glands also exhibited activity for the enzyme, but the ducts did not. The formation of reaction product was prevented by acetazolamide and ethoxzolamide and by the omission of bicarbonate from the substrate medium. Carbonic anhydrase in oesophageal squamous epithelium may be involved in the control of intra- and extracellular pH, while that in the glands is more likely to be concerned with bicarbonate secretion.     

Carbonic anhydrase from bovine bone

In this research, carbonic anhydrase enzyme, which was taken from the bones of an animal, was purified and characterized for the first time. For this, the bones of a young cow were used. The purification treatment was completed in three steps. Three different isoenzymes, such as peripheral, cystolic, and integral from the bone-cell cytozolic isoenzyme were purified and characterized. In purification of the three isoenzymes, the technique of affinity chromatography, which utilized Sepharose-4B-L-Tyrosine-Sulphanylamide, was used. In measuring the activities of enzymes, two different methods were applied. These are the esterase methods that utilize hydratase and p-nitrophenylacetate as substrate. The measurement of proteins was done with the methods of Bradford and Coomassie Brillant Blue. The optimum pH and temperature of each enzyme were measured and molecular weights were measured by gel-filtration. Its purity was examined by SDS-PAGE (3-10% alternating) electrophoresis and the inferior unit was defined. The inhibition effects of some chemicals were tested for each of the three isoenzymes.