Structural Mapping of Anion Inhibitors to ß-Carbonic Anhydrase psCA3 from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative facultative anaerobe belonging to the Pseudomonadaceae family. It is a multidrug-resistant opportunistic human pathogen, a common cause of life-threatening nosocomial infections, and a key bacterial agent in cystic fibrosis and endocarditis. The bacterium exhibits intrinsic resistance to most antibacterial agents, including aminoglycosides and quinolones. Hence, the identification of new drug targets for P. aeruginosa is ongoing. PsCA3 is a ß-class carbonic anhydrase (ß-CA) that catalyzes the reversible hydration of carbon dioxide to bicarbonate and represents a new class of antimicrobial target. Previously, inhibitor screening studies of psCA3 have shown that a series of small anions including sulfamide (SFN), imidazole (IMD), and 4-methylimidazole (4MI), and thiocyanate (SCN) inhibit the enzyme with efficiencies in the micro- to millimolar range. Herein the X-ray crystal structures of these inhibitors in complex with psCA3 are presented and compared with human CA II. This structural survey into the binding modes of small anions forms the foundation for the development of inhibitors against ß-CAs and more selective inhibitors against P. aeruginosa.

Purification of swine carbonic anhydrase isoenzyme III and measurement of its levels in tissues and plasma.

The changes in the levels of carbonic anhydrase isozyme III (CA-III) in swine plasma and urine have not been previously determined or reported. CA-III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA-III from swine muscle tissues and determined CA-III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA-III in the tissues of female swine (age, 3 months) and plasma of young swine (age, 1-5 months) and adult female pigs (age, 2-3 years) were determined using the ELISA system for swine CA-III. The mean (± SD) levels of CA-III in the skeletal muscles were 3.8 ± 3.2 mg/g (wet tissue), and in the plasma, 230 ± 193 ng/ml at 1 month, 189 ± 208 ng/ml at 2 months, 141 ± 148 ng/ml at 3 months, 78 ± 142 ng/ml at 4 months and 53 ± 99 ng/ml at 5 months. The mean level of CA-III in the plasma samples from 2- to 3-year-old pigs was 18 ± 60 ng/ml. CA-III in the plasma samples was found to decrease from 1 month until 3 years of age (p < 0.01). We performed far-western blotting to clarify the cause of the observed decrease in CA-III in plasma. Our results demonstrated that CA-III is bound to the transferrin and albumin. In addition, we determined that the levels of CA-III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.

Carbonic anhydrase inhibitors: cloning, characterization, and inhibition studies of the cytosolic isozyme III with sulfonamides.

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(-1) and k(cat)/K(M) of 2.5 x 10(5) M(-1) s(-1), being a slower catalyst for the physiological reaction as compared to the genetically related cytosolic isoforms hCA I and II. An inhibition study with a library of sulfonamides and one sulfamate, some which are clinically used compounds, is reported. hCA III is less prone to be inhibited by these compounds as compared to hCA I and II for which many low nanomolar inhibitors were detected earlier. The best hCA III inhibitors were prontosil, sulpiride, indisulam, benzolamide, aminobenzolamide, and 4-amino-6-chloro-benzene-1,3-disulfonamide which showed K(I)s in the range of 2.3-18.1 microM. Clinically used compounds such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, topiramate, zonisamide, celecoxib, and valdecoxib were less effective hCA III inhibitors, with affinities in the range of 154-2200 microM. This is the first study in which low micromolar hCA III inhibitors are reported.

Immunohistolocalization of carbonic anhydrase isoenzymes (CA-I, -II and -III ) in canine epididymis.

The immunolocalization of the efferent duct and the epididymis in canine was firstly examined using an the immunohistochemical method with the canine carbonic anhydrase (CA) -I, CA-II and CA-III antisera. The efferent duct was immunonegative for all present canine CA antisera. However, some slender shaped epithelial cells in the head and body segments of the epididymal duct were intensely reacted to the CA-II antiserum. These results suggested that the CA-II might be controlled in the luminal environment in the head and body segments of the canine epididymis by the proton and bicarbonate balance for the maintenance of the spermatozoal stability and movement.

Isolation and measurement of carbonic anhydrase isoenzyme III in plasma, sera, and tissues of dogs.

OBJECTIVE: To purify canine carbonic anhydrase isoenzyme III (CA-III) and determine plasma, serum, and tissue concentrations of CA-III in healthy dogs and dogs with experimentally induced muscle damage. ANIMALS: 121 healthy Beagles. PROCEDURE: Muscle was obtained from 2 Beagles after euthanasia, and CA-III was purified and characterized by use of column chromatography and electrophoresis, respectively. A CA-III-specific ELISA was developed to determine concentrations of CA-III in plasma of 116 dogs and tissues of 1 dog. Serum creatine kinase (CK) activity and CA-III concentration were also determined before and after induction of muscle damage by IM injection of 2 ml of 10% lidocaine to 2 dogs. RESULTS: Canine CA-III had a molecular weight of 28 kd and an isoelectric point of 8.2. Mean (+/- SD) concentration of CA-III in plasma of healthy dogs was 16.91 +/- 9.55 ng/ml. The highest tissue concentration of CA-III was detected in skeletal muscle. Serum concentration of CA-III increased and peaked within the first 2 to 3 hours after induction of muscle damage. The increase in CA-III concentration was more rapid than that of CK activity, and concentration reached its maximum and returned to baseline sooner than did CK activity. CONCLUSIONS AND CLINICAL RELEVANCE: The CA-III ELISA we developed was a sensitive method for determining CA-III concentrations in plasma, serum samples, and tissue specimens of dogs. Use of this ELISA requires only a small volume of serum and may enable the study of changes in CA isoenzyme concentrations associated with muscle disorders in dogs.

Carbonic anhydrase III in liver and muscle of male rats purification and properties.

Cytosolic carbonic anhydrases CAI, CAII, and CAIII from liver, and CAII, and CAIII from muscle of adult male Sprague-Dawley rats were purified to homogeneity. CAIII from liver and muscle had the same amino acid composition and were immunochemically similar. Their kinetic properties at 0 degrees C were also similar. Km(CO2) was 4 mM and kcat 3x105 s(-1). Ki was 0.4 and 0.2 M for acetazolamide and NaCl, respectively. Both CAIIIs ran as single bands on SDS-electrophoresis and high-speed centrifugation, with a mol wt of 29.3 kDa. Their hydrodynamic properties suggest that CAIII is a compact, nearly spherical molecule. It contained 0.9 M zinc per M protein. In both tissues isoelectric focusing identified neutral and acidic isoforms with pIs near 7.0 and 6.3, respectively. These forms were immunologically identical and had the same amino acid composition and mol wts. The acidic forms probably represent subspecies of CAIII in different states of oxidation. CAIII is the major soluble protein in rat liver and muscle. Its function is probably to protect proteins of these tissues from oxidation catalyzed by iron-containing degradation products of haemoglobin and myoglobin. Liver CAI and CAII and muscle CAII were identical to CAI and CAII of rat erythrocytes.