Carbonic Anhydrase IV from Human Lung PURIFICATION, CHARACTERIZATION, AND COMPARISON WITH MEMBRANE CARBONIC ANHYDRASE FROM HUMAN KIDNEY*

We have purified carbonic anhydrase (CA) from to apparent homogeneity in a form which is catalytically active and stable to storage. It has an molecular of 35 kDa, to endoglycosidases, seems to no N- O-linked oligosaccharide

bers of a gene family which seems to have arisen from a common precursor by gene duplication (1). They differ from each other in physical and kinetic properties, in susceptibility to inhibitors, in subcellular localization, and in tissue-specific expression (for reviews, see Refs. 2 and 3). The 29-kDa cytoplasmic isozymes CA I, CA II, and CA III have been studied most intensively. A 42-kDa secretory enzyme in saliva, called CA VI (4-6), and a 29-kDa mitochondrial enzyme, called CA V (7), have also been characterized.
Recently, a membrane form of CA was purified from human kidney (8,9). For some time, it had been clear that a membrane-associated CA was present in particulate fractions of tissue homogenates from kidney and in isolated preparations of microvilli and basal infoldings of renal tubular cells (10, 11). McKinley and Whitney (12) achieved a partial purification of the human kidney membrane CA using 3-5% sodium dodecyl sulfate (SDS) to solubilize the membrane proteins. Wistrand (8) subsequently reported purification of the human renal membrane CA to homogeneity by differential centrifugation and affinity chromatography.
Although initially reported to have a molecular mass of 68 kDa (8), it was found on further study (9) to have a molecular mass of 34.4 kDa on SDS gels. A molecular mass of 36.7 kDa was calculated from its amino acid composition.
This purification produced relatively small amounts of catalytically active enzyme. Amino acid composition data were reported, but attempts to determine the amino-terminal amino acid sequence were unsuccessful.
A membrane CA had even earlier been purified to homogeneity from bovine lung microsomes by Whitney and Briggle (13). It was a cystine-containing glycoprotein with a molecular mass of 52 kDa on SDS-polyacrylamide gel electrophoresis. Furthermore, it was inactivated by treatment with reducing agents, suggesting that it was stabilized by one or more disulfide bonds. This and other differences clearly distinguished the bovine membrane-associated enzyme from CA I, CA II, and CA III, the then-known 29-kDa cytoplasmic CAs, and the bovine lung isozyme was provisionally called CA IV. Although the relationship between CA IV from bovine lung and the less well characterized membrane enzyme from human kidney (13) was unclear, both had the remarkable property of being stable to solubilization in SDS. It was this property which enabled Whitney and Briggle (13) to use affinity chromatography to isolate bovine lung CA IV free of other CAs, since solubilization in 5% SDS completely destroyed the activity of the cytoplasmic CAs. However, once purified, the bovine lung enzyme proved too unstable in SDScontaining buffers to permit extensive characterization. We report here a purification which provides relatively large amounts of human lung CA IV in a form which is catalytically active and stable to storage. This purification allowed us to characterize human lung CA IV, to produce specific antibodies to it, and to prepare peptides from it for microsequencing. Furthermore, we applied the purification to human kidney microsomes and plasma membranes, which allowed us to compare the physical and kinetic properties, the amino acid compositions, and the amino-terminal amino acid sequence of the membrane CA from human lung with that prepared from human kidney by the same procedure. EXPERIMENTAL PROCEDURES AND RESULTS'

DISCUSSION
A 52-kDa "membrane form" of carbonic anhydrase (CA IV) had been purified from bovine lung (13) and a 34.4-kDa membrane-bound CA from human kidney (8,9). Neither purification provided significant amounts of stable enzyme. Until now, the membrane form of carbonic anhydrase in human lung had not been purified, and the relationship of the membrane-bound carbonic anhydrase activities in lung and kidney has been unclear.
We describe here the purification of CA IV from human lung to homogeneity by affinity chromatography using a procedure which provides milligram amounts of catalytically active enzyme in a form which is stable to storage. Human lung CA IV resembles CA II in its high specific activity, its relative sensitivity to sulfonamide inhibitors, and its resistance to inhibition by halide ions. Unlike the soluble CAs, it is a very hydrophobic protein which is tightly anchored to membranes. We present evidence that CA IV is anchored to membranes through a phosphatidylinositol-glycan linkage, both in lung and kidney.
Like bovine lung CA IV, human lung CA IV contains cysteine and loses catalytic activity on reduction with dithiothreitol. Unlike bovine lung CA IV, human lung CA IV lacks carbohydrate.
Bovine lung CA IV was reduced from 52 to 36 kDa by treatment with endoglycosidases, suggesting that it contains 5-6 N-linked oligosaccharide chains. Purified CA IV from human lung showed no change in M, on SDS-polyacrylamide gel electrophoresis following endoglycosidase treatment, suggesting that CA IV contains no N-linked oligosaccharide chains. In addition, we found no evidence for O-linked carbohydrate chains on CA IV from human lung. The purification reported here was also used successfully to purify the membrane-bound carbonic anhydrase from human renal membranes (8,9), allowing us to compare the membrane-bound enzymes from the two sources. The enzymes purified from human lung and human kidney were nearly identical in physical and kinetic properties, in amino acid composition, and in tryptic peptide patterns, and identical in the amino-terminal amino acid sequence of the native proteins and of the three major tryptic peptides.
Despite the evidence for identity from all of the above criteria, CA IV from human lung and human kidney differed in properties on isoelectric focusing. Although both enzymes showed charge heterogeneity, they differed in the relative prominence of the different charged species. In the lung, the less acidic species predominated.
In the kidney, the more acidic species predominated.
Since the enzyme from both lung and kidney have identical amino-terminal amino acid se-* Portions of this paper (including "Experimental Procedures," "Results," Figs. 1-6, and Tables  I-IV) are presented in miniprint at the end of this paper.
Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
quences (Table IV, Miniprint), there may be differences in posttranslational modifications at the carboxyl terminus. For example, the two enzymes might be degraded to different degrees at the carboxyl terminus, or variably modified by amidation or deamination. In addition, there may be differences in the phosphatidylinositol-glycan anchor which contribute to charge heterogeneity.
In fact, the enzyme from kidney may have more than one kind of carboxyl-terminal membrane anchor corresponding to differences in intracellular localization in different cells. Although 50-70% of the enzyme is found in brush borders, 30-50% of the enzyme can be isolated with basolateral membranes of renal tubular cells (10). Lisanti et al. (41) recently argued that the phosphatidylinositol-glycan linkage was a specific target for localization of membrane proteins to the apical membrane of differentiated cells. If this rule is general, the CA IV in the brush border would presumably contain the phosphatidylinositol-glycan anchor, while the basolateral membrane enzyme, might have a different anchor, possibly a transmembrane-spanning domain. The interesting finding of charge heterogeneity in CA IV from lung and kidney, and the predominance of the more acidic species in the kidney, warrants further study.
The presumed function of CA IV in the lung is to catalyze the dehydration of plasma HCO, to COP, which can readily diffuse across the capillary endothelial surface and pass out of the lungs on expiration. Studies of the rate of CO, hydration and HCO; dehydration in lung capillaries first implicated a carbonic anhydrase on the luminal (plasma) surface of pulmonary capillary endothelial cells (42)(43)(44). This conclusion was also supported by histochemical observations (45,46). Immunolocalization studies with antiserum to CA IV would allow verification that the enzyme identified histochemically is indeed CA IV. Since the antibody described here inhibits CA IV activity, it would also be of interest to test this antibody for inhibition of CO, exchange in perfused lung preparations. Demonstration of such inhibition would verify the proposed role for CA IV in COZ transport across the pulmonary capillary endothelium (44). The membrane CA expressed in the brush border of the proximal tubules of the kidney is thought to play a dominant role in bicarbonate reabsorption from the lumen (47). The antibody described here should be useful for immunolocalization of CA IV in the brush border of the proximal tubules and also in the basolateral membrane in some segments of the renal tubule (10). Since small amounts of brush border enzymes are detectable in normal human urine, one should also be able to detect CA IV immunochemically in membrane fractions isolated from normal human urine. Pharmacologic evidence suggested that the brush border enzyme was not affected by the mutation producing the CA II deficiency syndrome (osteopetrosis and renal tubular acidosis) (48). It will be interesting to test the prediction that these patients have normal levels of immunoreactive CA IV in their kidneys, and therefore, in the membrane fractions from their urine.
Histochemical and biochemical studies have also provided evidence for membrane-associated CAs in other tissues including brain (49), eye (50), and liver (51). The antibody described here should be useful for immunolocalization studies which define the tissue distribution of CA IV. Such studies would disclose whether the membrane-associated CAs in other organs are CA IV and indicate whether CA IV has a physiological role in these organs as well as in kidney and lung.