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 II in new world monkeys

Carbonic anhydrase II electrophoretic patterns were investigated in 3113 animals belonging to 12 genera and 24 species of New World primates. Polymorphism was detected in 13 species. A total of 24 different alleles was postulated to explain the variability found; the genusAotus showed the highest (eight) number of such alleles. Three genera of the family Callitrichidae (Callithrix, Saguinus, andCebuella) presented five alleles that were not found among the Cebidae. Important markers at the generic level were observed inCallicebus (CA2 ^*6 andCA2 ^*12),Cebus (CA2 ^*10, CA2^*16, andCA2 ^*21), andAotus (CA2^*3, CA2^*4, CA2^*5, CA2^*9, CA2^*15, CA2^*17, CA2^*22, CA2^*23). CA2^*13 seems to be the most common allele among the Cebidae; six genera of this family showed frequencies higher than 70% of it.     

Carbonic anhydrase isozyme VI in rat lacrimal gland

Using monoclonal antibody specific to rat carbonic anhydrase isozyme VI (CA VI), the isozyme was localized in the lacrimal gland. A minority of acini (less than 10% of the total) contained a few immunoreactive acinar cells. Enzyme histochemistry indicated that the CA VI-positive cells were the only cells possessing CA in the lacrimal acini. In the acinar cells, the reaction product for CA VI was distributed in the secretory granules and cytosol between secretory granules. Except for mitochondrial enzyme (CA V) activity, the intracellular distribution of enzyme activity was similar to that of CA VI immunoreactivity, suggesting that rat lacrimal acinar cells contain only CA VI and CA V. CA VI in the secretory granules was discharged into the acinar lumen and is considered to carry out its function on the surface of the conjunctiva and cornea. The cytosolic CA VI may function in situ and be involved in electrolyte and water secretion by the acinar cells. Polyclonal antibody to rat erythrocyte CA (CA I and CA II) stained only the interlobular ducts. In contrast, all the ductal elements exhibited CA enzyme activity. This discrepancy between immunohistochemistry and enzyme histochemistry suggests the presence of CA isozyme(s) other than CA I, CA II and CA VI in the lacrimal duct.     

Carbonic anhydrase is present in olfactory receptor cells

Summary A modification of Hansson's histochemical technique was used to reveal carbonic anhydrase activity in mounted cryostat sections of rat olfactory mucosa, after glutaraldehyde fixation. A positive reaction that could be inhibited by acetazolamide was found in a population of olfactory receptor cells, whereas the supporting cells were negative. Axons of receptor cells were also positive and could be traced through the cribriform plate to the olfactory bulb.     

Carbonic anhydrase in human platelets.

The carbonic anhydrase activity of human platelets was investigated by measuring the kinetics of CO2 hydration in supernatants of platelet lysates by using a pH stopped-flow apparatus. An average carbonic anhydrase concentration of 2.1 microM was determined for pellets of human platelets. Analysis of the kinetic properties of this carbonic anhydrase yielded a Km value of 1.0 mM, a catalytic-centre activity kcat. of 130000 s-1 and an inhibition constant Ki towards ethoxzolamide of 0.3 nM. From these values, CO2 hydration inside platelets is estimated to be accelerated by a factor of 2500. When platelet lysates were subjected to affinity chromatography, only the high-activity carbonic anhydrase II could be eluted from the affinity column, whereas the carbonic anhydrase isoenzyme I, which is known to occur in high concentrations in human erythrocytes, appeared to be absent.     

Carbonic anhydrase is present in olfactory receptor cells

A modification of Hansson 's histochemical technique was used to reveal carbonic anhydrase activity in mounted cryostat sections of rat olfactory mucosa, after glutaraldehyde fixation. A positive reaction that could be inhibited by acetazolamide was found in a population of olfactory receptor cells, whereas the supporting cells were negative. Axons of receptor cells were also positive and could be traced through the cribriform plate to the olfactory bulb.     

Carbonic anhydrase: an ultrastructural study in rat cerebellum

Summary The cellular and intracellular distribution of carbonic anhydrase isozyme II in rat cerebellum has been investigated with the electron microscope by the indirect antibody immunohistochemical technique. Unequivocal evidence is presented supporting the view that this enzyme is exclusively localized in oligodendrocytes. Myelin does not appear to contain detectable amounts of carbonic anhydrase though it is present in oligodendrocyte processes and in the layer of oligodendrocyte cytoplasm frequently seen to coat the external surface of myelinated fibres. The immune precipitate is found to be confined to the cytosol and the cytosolic surfaces of intracellular membranes. The data are discussed in relation to the possible function of the enzyme and the role of oligodendrocytes in the central nervous system.     

Androgen-linked control of rat liver carbonic anhydrase III

The concentration of carbonic anhydrase III (CAIII) in male rat liver was found to be 30 times greater than that in the female. Castration of male rats led to marked reduction in liver CAIII concentrations which could be partially restored to control levels by testosterone replacement. Marked developmental and senescence changes in liver CAIII were also observed in male rats.     

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 II and H+-ATPase in osteoclasts of four osteopetrotic mutations in the rat

 Osteopetrosis in laboratory animals is a metabolic bone disease characterized by increased skeletal mass. It is inherited as an autosomal recessive and results from a defect in the development and/or function of osteoclasts. We studied two enzymes essential for bone resorption, carbonic anhydrase II isoenzyme (CA II) and H⁺-ATPase, in osteoclasts from four osteopetrotic mutations in the rat; namely incisors-absent (ia), osteopetrosis (op), toothless (tl), and microphthalmia (mib), to test the hypothesis that reduced bone resorption in one or more of these mutations results from defects in the synthesis or activity of one of these enzymes. CA II was present in most osteoclasts from normal, tl, op, and mib littermates and was homogeneously distributed in cytoplasm. CA II staining in ia osteoclasts was more variable and less intense than in the other mutations. H⁺-ATPase was also present in osteoclasts from normal animals and mutants and immunostaining showed clear polarization to the ruffled border region in all normal rats and mutants except ia, which showed diffuse distribution of staining in the cytoplasm. H⁺-ATPase activity (proton transport) in a related tissue, kidney, was normal in tl and ia rats but increased in op and mib rats compared to their normal littermates. These results suggest that the osteoclasts in osteopetrotic rat mutations are not abnormal with respect to the distribution of CA II and H⁺-ATPase and that the function of these enzymes in the skeleton, while likely normal, needs to be tested directly in bone. Accepted: 25 September 1998     

A high activity carbonic anhydrase isoenzyme (CA II) is present in mammalian spermatozoa

Summary Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to contain only CA II, which was located principally in the postacrosomal region of the human spermatozoa and in the acrosomal cap region of the rat spermatozoa. The presence of CA II could be confirmed by immunoblotting, which revealed a 29 K polypeptide in both the human and rat spermatozoa. No CA I or VI-specific fluorescence could be detected in the spermatozoa of either species. The immunoblottings were also negative. The results show mammalian spermatozoa to contain the high activity carbonic anhydrase isoenzyme II. Its presence is probably linked to hydration of CO₂ produced by active energy metabolism and thereby to the maintaining of an adequate intraspermatozoal bicarbonate concentration as required for the maintenance of sperm motility.     

A high activity carbonic anhydrase isoenzyme (CA II) is present in mammalian spermatozoa

Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to contain only CA II, which was located principally in the postacrosomal region of the human spermatozoa and in the acrosomal cap region of the rat spermatozoa. The presence of CA II could be confirmed by immunoblotting, which revealed a 29 K polypeptide in both the human and rat spermatozoa. No CA I or VI-specific fluorescence could be detected in the spermatozoa of either species. The immunoblottings were also negative. The results show mammalian spermatozoa to contain the high activity carbonic anhydrase isoenzyme II. Its presence is probably linked to hydration of CO2 produced by active energy metabolism and thereby to the maintaining of an adequate intraspermatozoal bicarbonate concentration as required for the maintenance of sperm motility.     

Carbonic anhydrase II deficiency: single-base deletion in exon 7 is the predominant mutation in Caribbean Hispanic patients.

To date, three different structural gene mutations have been identified in patients with carbonic anhydrase II deficiency (osteopetrosis with renal tubular acidosis and cerebral calcification). These include a missense mutation (H107Y) in two families, a splice junction mutation in intron 5 in one of these families, and a splice junction mutation in intron 2 for which many Arabic patients are homozygous. We report here a novel mutation for which carbonic anhydrase II-deficient patients from seven unrelated Hispanic families were found to be homozygous. The proband was a 2 1/2-year-old Hispanic girl of Puerto Rican ancestry who was unique clinically, in that she had no evidence of renal tubular acidosis, even though she did have osteopetrosis, developmental delay, and cerebral calcification. She proved to be homozygous for a single-base deletion in the coding region of exon 7 that produces a frameshift that changes the next 12 amino acids before leading to chain termination and that also introduces a new MaeIII restriction site. The 27-kD truncated enzyme produced when the mutant cDNA was expressed in COS cells was enzymatically inactive, present mainly in insoluble aggregates, and detectable immunologically at only 5% the level of the 29-kD normal carbonic anhydrase II expressed from the wild-type cDNA. Metabolic labeling revealed that this 27-kD mutant protein has an accelerated rate of degradation. Six subsequent Hispanic patients of Caribbean ancestry, all of whom had osteopetrosis and renal tubular acidosis but who varied widely in clinical severity, were found to be homozygous for the same mutation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbonic anhydrase III: the phosphatase activity is extrinsic

The carbonic anhydrases reversibly hydrate carbon dioxide to yield bicarbonate and hydrogen ion. They have a variety of physiological functions, although the specific roles of each of the 10 known isozymes are unclear. Carbonic anhydrase isozyme III is particularly rich in skeletal muscle and adipocytes, and it is unique among the isozymes in also exhibiting phosphatase activity. Previously published studies provided evidence that the phosphatase activity was intrinsic to carbonic anhydrase III, that it had specificity for tyrosine phosphate, and that activity was regulated by reversible glutathionylation of cysteine186. To study the mechanism of this phosphatase, we cloned and expressed the rat liver carbonic anhydrase III. The purified recombinant had the same specific activity as the carbonic anhydrase purified from rat liver, but it had virtually no phosphatase activity. We attempted to identify an activator of the phosphatase in rat liver and found a protein of approximately 14 kDa, the amount of which correlated with the phosphatase activity of the carbonic anhydrase III fractions. It was identified as liver fatty acid binding protein, which was then purified to test for activity as an activator of the phosphatase and for protein-protein interaction, but neither binding nor activation could be demonstrated. Immunoprecipitation experiments established that carbonic anhydrase III could be separated from the phosphatase activity. Finally, adding additional purification steps completely separated the phosphatase activity from the carbonic anhydrase activity. We conclude that the phosphatase activity previously considered to be intrinsic to carbonic anhydrase III is actually extrinsic. Thus, this isozyme exhibits only the carbon dioxide hydratase and esterase activities characteristic of the other mammalian isozymes, and the phosphatase previously shown to be activated by glutathionylation is not carbonic anhydrase III.