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Location of a membrane-bound carbonic anhydrase isoenzyme (CA IV) in the human male reproductive tract. S Parkkila, A K Parkkila, K Kaunisto, A Waheed, W S Sly and H Rajaniemi J Histochem Cytochem 1993 41: 751 DOI: 10.1177/41.5.8468457 The online version of this article can be found at: http://jhc.sagepub.com/content/41/5/751

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The Journal of Histochemistry and Cytochemistry Copyright 0 1993 by The Histcchcmical Society, Inc

Vol. 41, No. 5, pp. 711-717, 1993 Printed in U5.A.

Original Article

I

Location of a Membrane-bound Carbonic Anhydrase Isoenzyme (CA IV) in the Human Male Reproductive Tract’ SEPPO P A R K K I L A , 2 ANNA-KAISA P A R K K I L A , KARI WILLJAM S. SLY, and HANNU RAJANIEMI

KAUNISTO,

ABDUL

WAHEED,

Department of Anatomy, University of Oulu, Oulu, Finland (S&4KFKK,HR), and Edward A. Dorjy Department of Biochemistry and Molecular Biology, St. Louis University, School of Medicine, St. Louis, Missouri (AKWSS). Received for publication August 13, 1992 and in revised form November 25, 1992; accepted November 28, 1992 (2A2787).

ting, which revealed 35 KD and 33 KD polypeptides. The We studied the location of a membrane-bound carbonic anresults show that the m i d and the apical plasma memhydrase (CA IV)in the human male reproductivetract using brane ofthe lining epithelium of the epididymal duct, ductus a specific antiserum to human CA IV in conjunction with immunoblotting, i m m ~ n o p e d d a s eand , i m m ~ ~ f l ~ ~ t edeferens, sand ampulla of the ductus deferens contain the membrane-bound carbonic anhydrase isoenzyme IV. The cence techniques. The microvilli and apical plasma mempresence of the enzyme in the epithelium of the epididymis brane of the epithelial cells and the subepithelial smooth muscle layer of the epididymis, ductus deferens, and am- and ductus deferens is probably linked to the acidificaton of the epididymal fluid that prevents premature sperm actipulla of the ductus deferens showed specificstaining for CA vation. Its physiological role in the smooth muscle cells reIV. The epithelial cells of the prostate and seminal vesicle mains to be elucidated. (JHistochem Cpochem 41:751-757, failed to stain for CA IV,however, whereas the subepithelial smooth muscle layer showed positive staining. No specific 1993) KEY mm:carbonic anhydrasq Membrane proteins; Human; Male; staining for CA II was seen in the epithelium of the epidiEpididymis; Ductus deferens; Seminal vesicles; Prostate; Sperm modymal duct or the proximal ductus deferens. The presence tility; Immunohistochemistry. of CA IV in the epididymiswas confirmed by immunoblot-

Introduction Carbonic anhydrase (EC 4.2.1.1: CA) catalyzes the reversible hydration of carbon dioxide and is involved in regulation of the ion, fluid, and acid-base balance in various tissues (Carter, 1972; Maren, 1967). Six isoenzymes (CA I-CA VI) have been purified, the best characterized of which are the cytoplasmic enzymes (CA I, 11, and 111) ( k h i a n et al., 1983). The mitochondrial and the secreted salivary carbonic anhydrases are termed CA V and CA VI, respectively (Dodgson et al., 1980; Femley et al., 1979). The membrane-bound isoenzyme is termed CA IV and has been purified from human kidney (Wisuand and Knuuttila, 1989; Wistrand, 1979), bovine lung microsomes (Whltney and Briggle, 1982), and in a catalytically active form from human lung and kidney (Zhu and Sly, 1990) and rat lung (Waheed et al., 1992a). CA IV has re-

Supported by grants from the Medical Research Foundation of Oulu, OulunYliopiston tuk&itio (SP) and by National Institutes of Health grants

DK40163 and GM34182 (WSS). Correspondence ta Seppo F’arkkila. MD, Univ. of Oulu, Dept. of Anatomy. K a j d n t i e 52A. SF90220 Oulu, Finland.

cently been located by monospecific antibodies and immunohistochemisuy in the plasma membranes ofthe proximal convoluted tubule and a thick ascending limb of Henle in the rat kidney (Brown et al., 1990), in the endothelial cells of the choriocapillariesof the human eye (Hageman et al., 1991), and in the endothelial cells of microcapillaries of rat brain (Ghandour et al., 1992).The cDNA for human CA IV has been cloned (Okuyama et al., 1992). The predicted amino acid sequence reveals similarity to other carbonic anhydrases. Of the 17 “active site” residues that are highly conserved in other human CAS, 16 are also present in CA IV. However, CA IV contains a 27-aminO acid C-terminal extension which probably serves as a recognition signal for cleavage and transfer to a glycosyl phosphatidylinositollinkage through which CA IVs are anchored to plasma membrane (Ghandour et al., 1992;Waheed et al., 1992a; Zhu and Sly. 1990). Carbonic anhydrases play an important role in reproductive physiology, since bicarbonate has been shown to be implicated in the regulation of sperm motility via a bicarbonate-sensitiveadenylate cyclase present in the sperm plasma membrane (Okamura et al., 1985). We have demonstrated in previous studies the presence of CA I1 in human and rat spermatozoa (Parkkila et al., 1991b) and in the human seminal vesicle, ampulla of the ductus deferens, and 75 1

752

distal ductus deferens (Kaunisto et al., 1990). The physiological role of cytoplasmic CA I1 in the distal parts of the reproductive tract is probably linked to equipping of the seminal plasma with bicarbonate required for maintaining sperm motility after ejaculation. However, the spermatozoa are subjected to an acidic environment during passage through the epididymis, which probably accounts for their quiescence there (Caflisch and DuBose, 1990; Carr et al., 1985). It has been suggested that carbonic anhydrases play a central role in the acidification of epididymal fluid (Au and Wong, 1980; Cohen et al., 1976), but it has remained uncertain which one of these isoenzymes is involved. Since CA IV mediates bicarbonate reabsorption in the renal proximal tubule and contributes to acidification of urine in the kidney (Dobyan and Bulger, 1982). it was considered a candidate for acidification of the epididymal fluid. This work was undertaken to determine the distribution of CA IV in the human male reproductive tract in order to contribute to an understanding of the functional significance of the CA isoenzymes in reproductive physiology.

Materials and Methods Antisera. A polyclonal rabbit antiserum to human CA IV was produced and characterized by Zhu and Sly (WO), and antisera to human CA VI and CA I1 by Parkkila et al. (1991a) and Parkkila et al. (1992).respectively. All tissues were stained with these antisera. Crossreactivity of anti-CA 11, anti-CA IV, and anti-CA VI sera with the purified CA I, CA 11, and CA VI was tested in Western blots. Aliquots (1 M )of CA I, CA 11, and CA VI were subjected to SDS-PAGE according to Laemmli (1970) under nonreducing conditions in a Mini-Proteanelectrophoresis unit (Bio-Rad Laboratories; Richmond, CA). The polypeptides were transferred electrophoretically from the gel to a nitrocellulose sheet using a Bio-Rad Mini Trans-Blot apparatus. The sample lanes were incubated first with swine serum for 18 hr at 4’C and then with antisera to CA 11, CA IV, and CA VI diluted 1:lOOO inTAG (10 mM Tris-HCl,l% BSA, 10% glycerol), pH 7.4, for 2 hr at 20%. The sheet was washed three times for 10 min with 1% Triton X-100 and three times for 10 min with 0.3% Triton X-100,and was treated again with swine serum for 1 hr at 20’C before incubation for 30 min at 20’C with peroxidase-conjugatedgoat anti-rabbit IgG (Bio-Rad Laboratories)diluted 1:3000 in TAG. After washing four times for 15 min in PBS, the polypeptides were visualizedwith 3,3’-diaminobenzidinetetrahydrochloride(DAB) (Fluka; Buchs, Switzerland) (9 mg DAB in 30 ml PBS) and hydrogen peroxide.

Prepvation of Tissues. Human prostate gland, seminal vesicle, ampulla of the ductus deferens, ductus deferens, epididymis, and testis specimens were obtained alongside routine histopathologicalspecimens taken during urological operations carried out at Oulu University Central Hospital for bladdcrtarcinoma (n = 2), prostate carcinoma(n = 1). or testis lymphoma (n = 1). The tissue samples were fixed in 4% paraformaldehyde in PBS for 18 hr at 4’C, followed by washing in 20% sucrose (in PBS) for 24 hr at 4°C. The sucrose prevents the formation of ice crystah in the tissue, resulting in a better preservation of the tissue morphology. The specimens were then frozen in liquid nitrogen and stored at -80°C. Frozen sections(CryoCut Microtome, American Optical Corporation) of 8 pm were used for immunohistochemical staining. wunohistochemistry. The sections were stained using immunoperoxidase and immunofluorescencetechniques. The steps in the hmunoperoxidasc staining were as follows: 1. Pre-treatment of the sectionswith swine serum for 40 min and rins-

ing in PBS.

2. Incubation for 1hr with the primary rabbit antiserum diluted 1:lOO

in 1% bovine serum albumin (BSA) in PBS (BSA-PBS). 3. Treatment with swine serum for 40 min and rinsing in PBS. 4. Incubation for 1 hr in biotinylated affinity-isolatedswine immunoglobulins to rabbit immunoglobulins(Dakopatts;Copenhagen,Denmark) diluted 1:300 in BSA-PBS. 5. Treatment with swine serum for 5 min and rinsing in PBS. 6. Incubation for 30 min in a 1:600 dilution of peroxidase-conjugated streptavidin (Dakopatts) in PBS. 7. Incubation for 3 min in DAB (9 mg DAB in 15 ml PBS plus 10 ~1 30% H202). The sections were washed in PBS three times for 10 min after incubation Steps 2,4, and 6. Treatment of the sections with undiluted swine serum before Steps 2, 4, and 6 effectively prevented nonspecific staining. All the incubations and washings were carried out at room temperature and the sectionswere finally mounted in Permount (Fisher Scientific; Fair Lawn, NJ). Steps 1 and 2 for fluorescence microscopy were identical to those of the peroxidase method. After washing three times for 10 min in PBS, the sectionswere then incubated with rhodamine-conjugatedswine anti-rabbit immunoglobulins (Dakopatts) in a 1:50 dilution of BSA-PBS for 1 hr and were then washed three times for 5 min with PBS before being immediately mounted in Mowiol (Hoechst; Frankfurt am Main, Germany). The sectionswere viewed with an Aristoplan microscope (Leitz; Oberkochen, Germany) and photographed on black-and-white negative film (Agfapan 25 I S 0 and Kodak Tri-X pan 400 ISO).

Western Blotting of Tissue Specimens. Fresh tissue samples from the human kidney, testis, proximal ductus deferens, caput epididymis, corpus epididymis, and cauda epididymiswere frozen in liquid nitrogen and stored at -80°C before further processing. They were later homogenized in two volumes of 50 mM %-so4 and 1 mM benzamidine (pH 7.5) with a Brinkman Polytron homogenizer, and total homogenate was used for SDS-PAGE that was performed under reducing conditionsaccording to Laemmli (1970). Protein concentrationswere determined using Peterson’sprocedure (Peterson, 1979) and 100 pg total protein were applied to each lane. The low molecular weight protein standards were from Bio-Rad Laboratories.After the electrophoresis, the polypeptides were transferred to an Immobilon-P PVDF membrane (Millipore; Bedford, MA) using the Polyplot apparatus (American Bionetics; Hayward, CA). After transblotting, the sheets were subjected to immunostaining with anti-CA IV serum diluted 1:1000, followed by treatment with goat anti-rabbit IgG peroxidase antibodies diluted 1:500 (Sigma; St Louis. MO). The polypeptideswere visualized with 4-chloronaphthol and hydrogen peroxide.

Location of CA IV in the Human Reproductive Tract The human epididymis, ductus deferens, ampulla of the ductus deferens, vesicula seminalis, and prostate showed positive staining for CA IV. In the caput (Figure la), corpus (Figure IC),and cauda (Figure Id) epididymis, the enzyme was principally located in the microvilli and apical plasma membrane of the epithelial cells. Particularly in the corpus and cauda epididymis, a distinct staining was also detected in the smooth muscle layer beneath the epithelial lining, but neither normal rabbit serum (data not shown) nor anti-CA VI serum (Figure 1b) showed any positive staining. The pigment particles present in the epithelial cells gave a bright autofluorescence (Figure 1b). The epithelium of the epididymal duct

CARBONIC ANHYDRASE IV IN THE REPRODUCTIVE TRACT

753

Figure 1. Immunohistochemical stainings of CA IV in (a) human caput, (c) corpus, and (d) cauda epididymis. The immunofluorescence stainings were positive in the microvilli region and apical plasma membrane. Note also a distinct immunostainingin the subepithelialsmooth muscle layer. Control staining with anti-CA VI serum in the caput epididymis(b) revealed only nonspecificfluorescence in the pigment granules of the epithelial cells. Antiserum dilutions were 1:lOO.Original magnifications: a,b x 200; c x 160;d x 80.Bars = 100 pm.

failed to stain for CA I1 (data not shown), as reported earlier (Kaunisto et al., 1990). The CA IV-positive staining in the proximal ductus deferens was located in the apical plasma membrane, and a clear staining could also be detected in the smooth muscle layer adjacent to the epithelium (Figures 2a and 2b). Anti-CA I1 serum revealed positive staining only in the erythrocytes (Figure 2c). In the distal ductus deferens (Figure 2d) and the ampulla of the ductus deferens (Figure 2f), the enzyme was located in the apical plasma membrane of the epithelial cells and the subepithelial smooth muscle layer. No positive staining was detected in the control sections (Figures 2e and 2g). Many pigment granules present in the epithelial cells showed bright autofluorescence.

In the prostate (Figure 3a) and seminal vesicle (Figure 3c), the enzyme was located only in the subepithelial smooth muscle layer. Again, no positive staining was seen in the controls (Figures 3b and 3d).

Western Blots The presence of CA IV in the human epididymis was confirmed by Western blotting (Figure 4). The separated parts of the epididymis (caput, corpus, and cauda) and the proximal ductus deferens were all seen to contain a 35 KD polypeptide. An additional band of 33 KD was present in the caput and cauda epididymis blots. Polypeptide bands of the same molecular weights were detected

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Figure 2. Immunohistochemical stainings of CA IV in the human ductus deferens. The enzyme is present in the apical plasma membrane of (a,b) the proximal ductus deferens, (d) distal ductus deferens, and (1)ampulla of the ductus deferens. The subepithelial circular smooth muscle cell layer also showed positive immunostaining with both (a) immunoperoxidase and (b,d,f)immunofluorescence. (c) Anti-CA IIserum showed specific immunostaining in the proximal ductus deferens only in the erythrocytes. No specific immunostaining was detected in (e) the distal ductus deferens and (9) ampulla of the ductus deferens with anti-CA VI serum. Antiserum dilutions were 1:lOO. Original magnifications: e,c,e-g x 200; b x 500;d x 180. Bars: a,c-g = 100 pm; b = 10 pm.

754

C Figure 3. Immunohistochemicaldemonstration of CA IV in (a) the human prostate and (c) seminal vesicle. (a) Positive immunofluorescenceand (c) immunopemxidase staining is present in a narrow subepithelial layer. Control sections of (b)the prostate and (d) seminal vesicle showed no specific immunostaining when anti-CA VI serum (b) or normal rabbit serum (d) was used. Antiserum and normal rabbit serum dilutions 1:lOO. Original magnifications: a,b x 200; c,d x 250. Bars = 100 Nm.

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Figure 4. Western blots of human testis, epididymis, proximal ductus deferens (PDD),and kidney samples. The antiCA IV serum (1:1000) recognizes two major polypeptide bands of 35 and 33 KD in the caput and cauda epididymis, proximal ductus deferens, and kidney blots, whereas only a major band of 35 KD was present in the corpus epididymis blot. An additional 17-18 KD polypeptideband was stained in the protein blots from the corpus and cauda epididymis. Note also that the 35 KD polypeptide was stained most intensely in the corpus epididymis.

31 21.5 14.4 -

in the human kidney protein extract used as a positive control tissue, and minor polypeptide bands of 17-18 KD were present in the corpus and cauda epididymis blots. No immunoreactive determinants were present in the testis blot. As anti-CA IV serum was not found to crossreact with CA I, CA 11, or CA VI, the positive immunohistochemical stainings and Westem blots can be regarded as CA IV specific with respect to these isoenzymes (Figure 5 ) .

Discussion The present results demonstrate for the first time the location of the membrane-boundcarbonic anhydrase CA IV in the human male reproductive tract. Isoenzyme CA IV was found in the microvilli and apical plasma membrane of the epithelial cells in the epididymal

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duct, ductus deferens, and ampulla of the ductus deferens but was absent from the seminal vesicle and prostate epithelia. On the other hand, the subepithelial smooth muscle layer throughout the reproductive tract seemed to contain the enzyme. The membraneassociated CA in skeletal muscle was recently shown to be CA IV (Waheed et al.. 1992b). Western blotting confirmed the presence of CA IV in the human epididymisand ductus deferens. The caput and cauda epididymis and proximal ductus deferens showed evidence of 35 and 33 KD polypeptides, which were also present in the human kidney blot, whereas only the 35 KD polypeptide band was present in the corpus epididymis. The 3 3 KD and 17-18 KD polypeptides are probably proteolytic fragments of the 35 KD native CA IV (Sato et al., 1990). The fluid leaving the testis is acidified during its passage through

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anti-CA IV

anti-CA VI

Figure 5. Crossreactivity of antiCA II, antiCA IV, and antiCA VI sera with CA I, CA II, and CA VI. Note that the antiCA IV serum (1:lOOO) did not crossreact with the CA isoenzymes I, II. and VI.

CARBONIC ANHYDRASE IV IN THE REPRODUCTNE TRACT

the epididymis. Carr et al. (1985) have proposed that the sperm quiescence noted in the cauda epididymis results from a lowering of the intracellular pH of the spermatozoa by weak acids or other factors constituting an acidic environment. Correspondingly, an increase in intraspermatozoalpH has been shown to result in activation of the bovine cauda epididymal spermatozoa (Babcock et al., 1983). Although the mechanism for the acidification of the epididymal fluid is not well understood, earlier research has suggested that carbonic anhydrases may be implicated in it (Goyal et al., 1980; Cohen et al., 1976), as the investigators demonstrated by a histochemical method that carbonic anhydrase activity is located in the cytoplasm of the epithelial cells of the rat caput and cauda epididymis and in the apical plasma membrane of the epithelial cells of the corpus epididymis. This technique does not distinguish individual isoenzymes, however. Our results here demonstrate that the isoenzyme present in the epithelium of the epididymis, ductus deferens, and ampulla of the ductus deferens is CA IV. This isoenzyme has also been located in the plasma membranes of the proximal convoluted tubules and the thick ascending limb of Henle in the rat kidney, where it is thought to account for luminal acidification by reabsorbing bicarbonate (Brown et al., 1990). Therefore, its presence in the epithelium of the epididymis, ductus deferens, and ampulla of the ductus deferens may also be linked to the acidification of the epididymal fluid via bicarbonate reabsorption. This hypothesis is compatible with an earlier report suggesting that bicarbonate reabsorption rather than H’ secretion is the mechanism of epididymal fluid acidification in the rat (Caflisch and DuBose, 1990). The lack of CA I1 in the epithelia of the human epididymis and the proximal ductus deferens also supports the concept that bicarbonate reabsorption by CA IV plays a pivotal role in the acidification of the epididymal fluid. The acidic environment in the epididymisand proximal ductus deferens probably prevents premature sperm activation, while their activation during ejaculation is accounted for by bicarbonate secreted by the epithelial cells of the distal ductus deferens and seminal vesicle, which contain CA I1 (Kaunisto et al., 1990).

Acknowledgments We thank Ms Aino Kuha and MY Eero Oja for their skdgultechnical assistance.

Literature Cited Au CL,Wong PYD (1980): Luminal acidification by the perfused rat cauda epididymis. J Physiol 309:419 Babcock DF, Rufo GA Jr, Lardy HA (1983): Potassium-dependentincreases in cytosolic pH stimulate metabolism and motility of mammalian sperm. Proc Natl Acad Sci USA 80:1327 Brown D, Zhu XL, Sly WS (1990): Localization of membrane-associated carbonic anhydrase type IV in kidney epithelial cells. Proc Natl Acad Sci USA 87:7457 Caflisch CR, DuBose TD Jr (1990): Direct evaluation of acidification by rat testis and epididymis: role of carbonic anhydrase. Am J Physiol258:E143 Carr DW, Usselman MC, Acott TS (1985): Effects of pH, lactate, and viscoelastic drag on sperm motility: a speciescomparison. Biol Reprod 33:588 Carter MJ (1972): Carbonic anhydrase: isoenzymes, properties, distribution, and functional significance. Biol Rev 47:465 Cohen JP, Hoffer AP, Rosen S (1976): Carbonic anhydrase localization in the epididymis and testis of the rat: histochemical and biochemical analysis. Biol Reprod 14339

757

Dobyan DC, Bulger RE (1982): Renal carbonic anhydrase. Am J Physiol 243:F311 Dodgson SJ, Foster RE, Storey BT, Mela L (1980): Mitochondrial carbonic anhydrase. Proc Natl Acad Sci USA 775562 Femley RT, Wright RD, CoghlanJP (1979): A novel carbonic anhydrase from the ovine parotid gland. FEBS Lett 1053299 Ghandour MS, Langley OK, Zhu XL, Waheed A, Sly WS (1992): Carbonic anhydrase IV on brain capillary endothelial cells: a marker associatedwith the blood-brain barrier. Proc Natl Acad Sci USA, in press Goyal HO, Ferguson JG, Hrudka F (1980): Histochemical activity of carbonic anhydrase in testicular and excurrent ducts of immature, mature intact and androgen-deprived bulls. Biol Reprod 22:991 Hageman GS, Zhu XL, Waheed A, Sly WS (1991): Localization of carbonic anhydrase IV in a specific capillary bed of the human eye. Proc Natl Acad Sci USA 88:2716 Kaunisto K, Parkkila S, k m e l a T,Romberg L, Rajaniemi H (1990): Immunohistochemical localization of carbonic anhydrase isoenzymes in the human male reproductive tract. Histochemistry 94381 Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 Maren TH (1967): Carbonic anhydrase: chemistry, physiology, and inhibition. Physiol Rev 47595 Okamura N, Tajima Y, Soejima A, Masuda H. Sugita Y (1985): Sodium bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa through direct activation ofadenylate cyclase.J Biol Chem 260:9699 Okuyama T,Sato S, Zhu XL, Waheed A, Sly WS (1992): Human carbonic anhydrase IV: cDNA cloning, sequence comparison,and expression in COS cell membranes. Proc Natl Acad Sci USA 89:1315 Parkkila S, Kaunisto K, Kellokumpu S,Rajaniemi H (1991b): A high activity carbonic anhydrase isoenzyme (CAII) is present in mammalian spermatozoa. Histochemistry 95:477 Parkkila S,Kaunisto K, Rajaniemi H (1991a): Location of the carbonic anhydrase isoenzymes VI and I1 in human salivary glands by immunohistochemistry. In Botre F, Gros G, Storey BT, eds. Carbonic anhydrase. From biochemistry and genetics to physiology and clinical medicine. Weinheim, VCH, 254 Parkkila A-K. Parkkila S, Juvonen T, Rajaniemi H (1992): Carbonic anhydrase isoenzymes I1 and I are present in the zona glomerulosa cells of the human adrenal gland. Histochemistry, in press Peterson GL (1979): Review of the Folin phenol protein quanutation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem 100:201 Sato S, Zhu XL, Sly WS (1990): Carbonic anhydrase isozymes IV and I1 in urinary membranes from carbonic anhydrase 11-deficientpatients. Proc Natl Acad Sci USA 87:6073 k h a n RE, Hewett-Emmett D, Goodman M (1983): On the evolution and genetics of carbonic anhydrasesI. I1 and 111. In Rattazi MC, ScandaliosJG, Whin GS, eds. Isozymes: current topics in biological and medical research. Vol 7. New York, AR Liss, 79 Waheed A, Zhu XL, Sly WS (1992a): Membrane-associatedcarbonic anhydrase from rat lung. J Biol Chem 267:3308 Waheed A, Zhu XL, Sly WS, Wetzel P, Gros G (1992b): Rat skeletalmuscle membrane associated carbonic anhydrase is 39-kDa, glycosylated. GPIanchored CA IV. Arch Biochem Biophys 294550 Whitney PL, Briggle TV (1982): Membrane-associatedcarbonic anhydrase purified from bovine lung. J Biol Chem 257:12056 Wistrand PJ (1979): Renal membrane-bound carbonic anhydrase.Purification and properties. Upsala J Med Sci (suppl) 26:75 Wistrand PJ, Knuuttila K-G (1989): Renal membrane-bound carbonic anhydrase. Purification and properties. Kidney Int 35:851 Zhu XL. Sly WS (1990): Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney. J Biol Chem 265:8795