Membrane-associated Carbonic Anhydrase Purified from Bovine Lung*

We found carbonic anhydrase activity associated with particulate fractions of homogenate8 of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solu- tions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electro-phoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothre- itol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific Cor-hydrating activity was about half that of the erythro- cyte enzyme. Acetazolamide inhibits the membrane-as-sociated enzyme (Ki = 10 n ~ ) nearly as wen as the erythrocyte enzyme (Ki = 3 m). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capil- laries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization. Studies on the rate of CO, hydration and HC03- dehydration in lung capiUaries have pointed

hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 n~) nearly as wen as the erythrocyte enzyme (Ki = 3 m). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.
Studies on the rate of CO, hydration and HC03dehydration in lung capiUaries have pointed to the presence of carbonic anhydrase on the luminal surface of the pulmonary endothelial cells (1-3). Localization of carbonic anhydrase in cultured endothelial cells by activity and by light and electron microscopic techniques (4, 5) have provided further support for this conclusion. The apparent function of the enzyme in this location is to catalyze the dehydration of serum bicarbonate to yield COP that can readily diffuse across the endothelialalveolar barrier and pass out of the lung during expiration.
If carbonic anhydrase is located on the luminal surface of the endothelial ceUs of lung capillaries, then it must be bound very tightly to the membrane so that it will not be washed off into the blood. It did not seem likely to us that one of the well characterized, soluble, carbonic anhydrases would form such a strong association, so we looked for an enzyme similar to the membrane-associated carbonic anhydrase in human kid-* This work was supported by National Institutes of Health Grants GM20815, HL07283, HL20366; and the Veterans Administration Research Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + T o whom correspondence should be addressed, at Pulmonary Division R120, University of Miami School of Medicine, P. 0. BOX 016960, Miami, FL 33101. ney (6-9). We found that lung does contain membrane-associated carbonic anhydrase and proceeded to purify and characterize it.

DISCUSSION
Many properties of the membrane-associated enzyme3 from bovine lung are very similar to those of the er_ythrocyte enzyme. The molecular weights of the polypeptides are probably similar. The specific activity of the membrane-associated enzyme is about half that of the erythrocyte enzyme and its affinity for acetazolamide is about a third of that of the erythrocyte enzyme. Acetazolamide bound to both enzymes at the same rate. It is known that the erythrocyte carbonic anhydrase must have Zn2' in the active site to promote strong binding of acetazolamide (10). Since the membrane-associated enzyme binds acetazolamide strongly, it is likely that it also contains Zn". If it does contain Zn", it is not removed by EDTA at pH 8; the erythrocyte enzyme also does not transfer Zn2' to EDTA at this pH.
On the other hand, there are differences between these enzymes. Antiserum to the erythrocyte enzyme completely inhibited the activity of the erythrocyte enzyme but did not inhibit the membrane-associated enzymes. However, this does not prove that there are no similar antigenic sites on the two proteins. Perhaps the antibody did not bind to the membranebound enzyme due to steric hindrance by neighboring membrane components. Furthermore, it is probable that some antibody molecules could bind to the membrane-associated enzyme without inhibiting its activity, especially with a small substrate like CO,. Thus, the lack of inhibition does not rule out the formation of enzyme-antibody complexes.
There are other notable differences between the two enzymes. The membrane-associated enzyme binds strongly to membranes and is remarkably stable in SDS solutions, properties that the erythrocyte enzyme lacks. The membraneassociated enzyme also has carbohydrate and cysteine residues, components that the erythrocyte enzyme lacks. The disulfide bonds in the membrane-associated enzyme are important. in protecting the enzyme from denaturation with ' Portions of this paper (including "Experimental Procedures," "Results," Figs. 1 and 2, Tables 1-111, and additional references) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. The abbreviation used is: SDS, sodium dodecyl sulfate. Even when the membrane-associated carbonic anhydrase is dissolved in SDS, we continue to designate it as the membrane-associated enzyme to distinguish it from soluble carbonic anhydrase. It is very likely that the membrane associated enzyme is a new isozyme which could be designated as CA IV. SDS, since activity is rapidly lost if dithiothreitol is added with the SDS. The carbohydrate probably has little influence on the stability of the enzyme in SDS but may be important in directing it to the appropriate membrane site.
Since our analyses were not very extensive (because we had so little purified enzyme), the exact nature of the carbohydrate is not yet known. It contains mannose and glucosamine but not galactosamine, so carbohydrate chains would probably be linked to the protein via N-glycosidic linkage from N-acetylglucosamine to asparagine residues. Amino acid analysis showed about 6 residues of glucosamine/100 amino acids, a value which is somewhat low because of partial destruction during acid hydrolysis. If the protein is the same size as the erythrocyte enzyme (with about 270 amino acid residues/ molecule) and if the carbohydrate chains contain an average of 4 residues of glucosamine, then there would be 4-6 carbohydrate chains/molecule. The presence of glucose in the sample is not easily explained, because most known mammalian glycoproteins do not have glucose incorporated into the carbohydrate chains. It is possible that glucose-containing glycolipids are associated with the enzyme and remain attached during purification.
These results permit some speculation about the location of the membrane-associated carbonic anhydrase in lung. The enzyme is F i y bound to membrane and is most concentrated in the pellet (84,OOO x g), results consistent with earlier ideas that it is located on the luminal surface of endothelial cells (1-3). Such localization is supported by studies of acetazolamide binding during perfusion through rabbit lung (11) and by rates of bicarbonate diffusion across the alveolar-capillary barrier (12). It is also supported by histochemical studies of cultured bovine pulmonary endothelial cells (4, 5) and of human, monkey, and rat lungs (13,14). In human and monkey lung, the activity was limited to the capillary walls. In rat lung, carbonic anhydrase activity was found only on that portion of the capillary wall that was in close contact with alveoli. This suggests that not all endothelial cells in rat lung possess significant amounts of the membrane-associated enzyme.
The limitation of carbonic anhydrase to capillary walls in close contact with alveoli might explain earlier findings of the activity of the membrane-associated carbonic anhydrase in developing rat lung (15). The activity was low in newborns, rose slowly for the first 2 weeks, and then rose rapidly to adult levels during the third week. It is during the third week that the capillary-alveolar barrier thins (16-17) to form the structure where carbonic anhydrase was found histochemically.
The level of vascular carbonic anhydrase activity varies markedly from tissue to tissue. It was high in lung and low in liver and hindlimb (18). The distribution of membrane-associated carbonic anhydrase in kidney is different from that found in lung. Histochemical studies of rat kidney showed no activity in the glomeruli and Bowmans' capsule, but the brush-border and basolateral membranes of the convoluted proximal tubules showed intense activity (19). These results agree with biochemical studies that found carbonic anhydrase activity in purified brush-border and basolateral membranes (9). The membrane-associated carbonic anhydrase in human kidney is unusually stable in sodium dodecyl sulfate and is about the same size as the enzyme from bovine lung (6). We think that the membrane-associated carbonic anhydrase of lung is the same as that in the brush-border of the kidney proximal tubules even though the enzyme is apparently associated with endothelial cells in a lung and epithelial cells in kidney. The distribution of the membrane-associated carbonic anhydrase may be similar to that of angiotensin-converting enzyme, an enzyme usually found only on the capillary but which is also found in the brush-border of kidney proximal tubules (20-22). Future studies of membrane-associated carbonic anhydrase may provide important information on the structure and function of membrane proteins and on factors which control their synthesis and distribution.