RICHARD. STEPHENS^, DAFYDD WALTER@, AND NICK CARTER^. 'Medical Genetics Unit and zdepartment of Child Health, St. George's Hospital Medical ...
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Biochemical Scciety Transactions (1995) 23
Characterization of membrane bound carbonic anhydrase :IV (CA IV) 1
- p ISABELLE M A H I E U ~ ,AUDRA BEN JAM IN^. RICHARD STEPHENS^, DAFYDD WALTER@, AND NICK CARTER^
'Medical Genetics Unit and zdepartment of Child Health, St George's Hospital Medical School. London SW 17 ORE. Much of the carbon dioxide produced by the tissues of the body is delivered to the lung in the form of plasma HCO3- ( I ) . The subsequent conversion of H C O y to C 0 2 prior to its release in the expired air. is greatly accelerated by red cell C A I and I1 (2). Reports indicate that although these C A promote rapid formation of C 0 2 from bicarbonate entering red cells, the absence of the enzyme in the plasma could be responsible for a slow decline in plasma hydrogen concentration (3). It appears that extracellular C A is present in the capillary endothelial cells and participates directly in plasma CO&-HCO3--H+ reactions (4,5,6) in a manner which may diminish the suggested disequilibrium (7,8). The aim of the present work was to detect C A IV in sheep lung and to give evidence for its localisation on the endothelid surface. One adult ewe was anaesthetised with 5% thiopentone sodium (0.2-0.3 ml.kg body weight-I i.v.)(INTRAVAL), maintained with 1%' alpha-chloralose (3-5 ml.kg body weight-1 i.v.) and then ventilated via tracheotomy with 100% 0 2 (peak airway pressure: 18-25 cmH20). The chest was opened by midline thoracotomy, and cannulae were inserted in the left atrial appendage and the proximal pulmonary artery. The inferior and superior Vena c u v m and the Aortu were ligated: the animal was killed by circulatory arrest. To ensure the absence of red cells, the pulmonary vessels were washed with normal saline (0.9%)at a pressure of 20-25 mmHg and a temperature of 37°C. Ventilation was then discontinued and the lungs were harvested. SDS-PAGE gel electrophoresis were then performed on lung homogenates. Blots were incubated with 3 independently prepared antisera to highly purified synthetic peptide fragments from the C terminal (fig. I A, lane I), the N terminal (fig. I A, lane 2) or active site (fig. 1A. lane 3 ) amino-acid sequence of the human CA IV. C A IV was visualized as a single immunoreactive band with a molecular mass of 55 kD in membrane containing samples. No staining was found in membrane free homogenates. When 1% Triton-X 100 was incubated overnight with the membrane preparations. a second well defined band appemd, with an apparent molecular weight of about 45 kD. The coexistence of the 2 bands was clearly demonstrated independently of the antibody used. These results suggest that C A IV is present in sheep lung and that the hydrophobic nature of the enzyme causes it to be associated with a lipid or a glycolipid fraction of pulmonary cell membranes. In order to confirm C A IV localization, immunocytochemistry was performed in lung sections. Lung samples were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and then kept in the same fixative for 2-4 h at 4°C. Twenty-micrometer-thick Vibratome sections were cut and washed thoroughly in PBS before immunostaining. Tissue sections were incubated for I h at room temperature with a 1: IOOO dilution of a rabbit antiserum to bovine C A IV. Aftter several washes in PBS, sections were incubated for 1 h with peroxidase-conjugated sheep anti-rabbit IgG (Sigma) at 1:100 dilution. The peroxidase activity was developed with 3.3'diaminobenzidine tetra-hydrochloride and hydrogen peroxide. Some control tissue sections were incubated with rabbit non immune serum and processed for immunoperoxidase a s mentioned above. Staining was found only at the structures which separate capillaries and alveoli, i.e. the alveolar-capillary barrier. However, even at high magnification, light microscopy yielded few clues as to more specific localisations. In order to confirm that C A IV was on the endothelial surface, we decided to perfuse sheep lungs with C A IV antibody and then to purify the antigen-antibody complex. In that purpose, lungs from one adult ewe and two 12 week-old lambs were prepared as previously described b u t , after washing, an antibovine CA IV solution (1/100) was introduced into the pulmonary artery and allowed to dwell for 60 minutes.
The solution was then washed out and collected at the left atrium, before being reintroduced and allowed to dwell for a further 15 minutes. The vessels were then thoroughly washed through with saline for 15 minutes and lungs were harvested. Cell membrane pellets of lung homogenates were incubated overnight in TBS- I B Triton X-100in order to extract the antigen-antibody complex from the membrane. After centrifugation, the supernatants were incubated for 5h with 100 pl of purified proteine A (Sigma). Thc precipitate was then prepared for immunoblotting. After SDS-. PAGE gel electrophoresis and Western blotting, the blots were incubated with the 3 antibodies previously described. C A IV was visualized as a single immunoreactive band with a molecular mass of 45 kD. W e did not routinely observe a single 55 k D band because we incorporated Triton X-100 in our method to extract the antigen-antibody complex from the cells. In this study, we demonstrated the existence of C A IV in lung homogenates. This pulmonary C A IV is readily accessible LO perfused C A IV antibodies demonstrating that it is located on the luminal surface of the capillary endothelium. The presencc o f carbonic anhydrase at such a site should minimize o r eliminiit disequilibrium of hydrogen ions between plasma and red cells traversing the lung. 1. Effros, R.M., Chang. R.S.Y.. and Silverman, P. (1978) Science.
199,427-429. 2. Roughton. F.J.W.( 1935) Physiol. Rev., 15. 241-296. 3. Bidani, A,. and Heming, T.A. ( 199 I ) J. Appl. Physiol.. 7 I , 1460-1468. 4. Lonnerholm. G . . and Wistrdnd, P. (1982) Pediatr. Res.. 16,40741 I . 5. Lonnerholm, G.(1980) Acta Physiol. Scand.. 108. 197- 199. 6.Hanson.M.A.,NYE.P.C.G..andTorrance,R.W. (l981)319. 93- 109. 7. Crandall, E.D., and O'Brasky. J.E. (1978) J. Clin. Invest., 62. 61 8-622. 8. Klocke, R.A. (1978) J. Appl. Physiol., 44,882-888. We gratefully acknowledge financial support from the Association for Spina Bifida and Hydrocephalus.
A. B.C. C A IV characterization by imrnunoblotting using antisera to C terminal (lanes IA, IB, IC). N terminal (lanes 2A, 2B. 2C) and active site (lanes 3A, 3B. 3C) synthetic peptides. SDS-PAGE analysis were performed on lung homogenates (A) and on lung homogenates incubated overnight in Triton X-I(x) (B): note the single 55 k D band in non-treated samples (A) and the appearance of a second band with a monomer molecular weight of 45 kD in Triton X-100 treated samples (B). C. SDS-PAGE analysis performed on C A IV antigen-antibody complex purified from lung perfused with anti-bovine C A IV rabbit antibody. Note the clear 45 k D band.
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55kD-
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55kD45kD-
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