1 Department Medische BasisWetenschappen, Limburgs Universitair Centrum, B-3590 Diepenbeek, Belgium; 2 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; 3 Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada J1K 2R1; 4 Institute of Experimental Medicine, H-1450 Budapest, Hungary; and 5 Biochemie, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
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ABSTRACT |
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Membranes of pig kidney cortex tissue were solubilized in the presence of Triton X-100. Partial purification of ATP diphosphohydrolase (ATPDase) was achieved by successive chromatography on concanavalin A-Sepharose, Q-Sepharose Fast Flow, and 5'-AMP-Sepharose 4B. Monoclonal antibodies against ATPDase were generated. Further purification of the ATPDase was obtained by immunoaffinity chromatography with these monoclonal antibodies. NH2-terminal amino acid sequencing of the 78-kDa protein showed a sequence very homologous to mammalian CD39. The protein is highly glycosylated, with a nominal molecular mass of ~57 kDa. The purified enzyme hydrolyzed di- and triphosphates of adenosine, guanosine, cytidine, uridine, inosine, and thymidine, but AMP and diadenosine polyphosphates could not serve as substrates. All enzyme activities were dependent on divalent cations and were partially inhibited by 10 mM sodium azide. The distribution of the enzyme in pig kidney cortex was examined immunohistochemically. The enzyme was found to be present in blood vessel walls of glomerular and peritubular capillaries.
CD39; apyrase; nucleoside triphosphate diphosphohydrolase; Triton X-100
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INTRODUCTION |
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EXTRACELLULAR NUCLEOSIDES and nucleotides influence a
number of cellular processes, mostly by binding to a specific
purinoceptor. Nucleotides, mainly ATP and ADP, act on P2 receptors.
Dephosphorylation of these molecules leads to adenosine, which acts on
P1 receptors and frequently triggers an opposite effect. Coexistence of
P1/P2 receptors has been determined in many cells and will result in reciprocal effects, as was already shown in pancreatic -cells (7),
PC12 cells (22), and vascular smooth muscle (25).
The breakdown of extracellular nucleotides involves several ectoenzymes, called ectonucleotidases. Some of these enzymes are intrinsic proteins of the plasma membrane, with their catalytic site oriented toward the extracellular space. Other ectoenzymes, such as 5'-nucleotidase, are linked to the membrane via a glycosyl phosphatidylinositol anchor (43).
Our study focuses on one of these ectonucleotidases, the ATP
diphosphohydrolase (ATPDase) or apyrase (EC 3.6.1.5) of pig kidney. All
E-type ATPases hydrolyze ATP, thus producing ADP. Although ecto-ATPases
do not significantly break down ADP, ATPDases hydrolyze both the -
and
-phosphate of all naturally occurring nucleoside tri- and
diphosphates. So far, the enzyme has been described in most tissues and
was characterized in a number of them (23), but its physiological
function remains a matter of speculation.
In recent years, it has become clear that the membrane-associated ATPDases are identical to CD39, a lymphoid cell activation antigen (10, 18, 40). Human CD39 is an integral membrane protein, with two membrane-spanning regions, short intracellular NH2- and COOH-terminal ends, and a large extracellular loop containing a more central hydrophobic domain. Its cDNA codes for a 510-amino acid protein of 57 kDa, with 6 potential N-linked glycosylation sites and 11 cysteine residues that may be involved in the formation of oligomers (16, 18). So far, all E-type ATPases share five apyrase-conserved regions (ACR1-5) (6, 39).
Historically, two types of ATPDases have been characterized: type I with a molecular mass of 54 kDa, and type II with a molecular mass of 78 kDa. Amino acid sequencing of the bovine aorta type II ATPDase showed its homology to CD39 (10, 32), but the molecular mass of this last protein is only 57 kDa. The increase in molecular mass from 57 kDa for CD39 to 78 kDa for type II ATPDase is due to N-linked glycosylation (33). Type I ATPDase from porcine pancreatic tissue is a protein with a molecular mass of 35 kDa for the core protein. This type I ATPDase is a truncated part of CD39, probably due to proteolytic cleavage of the NH2 terminus just before ACR4 (10, 31, 32).
In the present study, we identified and localized an ATPDase in porcine kidney and established its identity to CD39. We also showed the presence of 54- and 27-kDa proteins, which are probably cleavage products of the intact 78-kDa enzyme.
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MATERIALS AND METHODS |
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Materials. Salts, buffers, nucleotides, diadenosine polyphosphates, pristane, Freund's adjuvant, and 3,3'-diaminobenzidine were obtained from Sigma Chemical (Bornem, Belgium). Oligomycin, levamisole, EGTA, EDTA, and ouabain (octahydrate) were purchased from Janssen Chimica (Beerse, Belgium). Immobilon-P blotting membranes were from Millipore (Bedford, MA), and Problott polyvinylidene difluoride (PVDF) membranes were from Perkin Elmer-Applied Biosystems (Nieuwerkerk a/d Ijssel, The Netherlands). All cell culture media and consumables were purchased from Life Technologies (Merelbeke, Belgium). Endoglycosidase F and Triton X-100 (especially purified for membrane research) were purchased from Boehringer Mannheim (Mannheim, Germany). Reagents for SDS-PAGE and immunoblotting were obtained from Bio-Rad Laboratories (Nazareth, Belgium). Glutaraldehyde and durcupan were from Fluka Chemie (Buchs, Switzerland). Commercially available antibodies were obtained from Prosan (Ghent, Belgium) or Pierce (Rockford, IL). Renaissance Chemiluminesence Reagent Plus was from NEN (Boston, MA).
Preparation of crude microsomal pellets. Pig kidneys were obtained at a local slaughterhouse and placed immediately in ice-cold 2 mM Tris · HCl buffer, pH 7.4, containing 10 mM mannitol. Cortex tissue was passed through a meat grinder, and 75 g of the tissue were suspended in 225 ml homogenizing buffer (25 mM Tris · HCl, pH 7.6, containing 250 mM sucrose). After homogenization in a Sorvall Omni-Mixer (DuPont Instruments), the homogenate was centrifuged for 10 min at 1,000 g. The supernatant was centrifuged for 20 min at 15,000 g for removal of most cell organelles, and the resulting supernatant was centrifuged again at 100,000 g for 1 h in a Beckman Optima LE-80K preparative ultracentrifuge.
Solubilization. Each microsomal pellet was resuspended in 20 ml 25 mM Tris · HCl (pH 7.6) to ~10 mg protein/ml and sonicated with a probe sonicator (MSE Instruments) at 75% of the maximum energy setting. An equal volume of the same buffer, containing 0.6% Triton X-100 was added, and the obtained solution was centrifuged for 1 h at 100,000 g. The pellet was discarded, and the supernatant containing the solubilized membranes was used for further purification.
Chromatography. A sample of ~350 mg of solubilized membrane proteins in 160 ml was applied to a 16-mm2 column containing 7.5 ml concanavalin A (Con A)-Sepharose at a flow rate of 0.2 ml/min. Subsequently, the column was washed at 0.5 ml/min with 25 mM Tris · HCl (pH 7.6), 4 mM CaCl2, and 0.01% Triton X-100 and eluted at 1 ml/min with a 100-ml gradient of 0-300 mM methylmannoside in 25 mM Tris · HCl (pH 7.6), 4 mM CaCl2, and 0.3% Triton X-100. The fractions containing the highest specific ATPDase activities were pooled and dialyzed overnight against dialysis buffer (25 mM Tris · HCl, pH 7.6).
The dialyzed fractions were applied to a 7.5-ml, 9-mm2 Q-Sepharose Fast Flow column at 1 ml/min. After the column was washed with 25 mM Tris · HCl (pH 8.2) in 0.01% Triton X-100, the activity was eluted with 40 ml of a gradient of 0-500 mM NaCl in 25 mM Tris · HCl (pH 8.2) in 0.3% Triton X-100 at the same flow rate. After overnight dialysis, the ATPDase peak fractions were applied to 15-ml 5'-AMP-Sepharose 4B in a 16-mm2 column at 0.25 ml/min. The activity was eluted with a 30-ml gradient of 0-10 mM AMP in 25 mM Tris · HCl (pH 7.6), 0.3% Triton X-100 at 1 ml/min. The fractions containing the highest specific ATPDase activity were pooled and dialyzed, and the enzyme preparations were stored atPreparation of ATPDase antibodies and immunoaffinity purification. Polyclonal antibodies were generated by immunizing rabbits with a high-density, multiple antigenic peptide system, synthesized with a peptide corresponding to the NH2-terminal 16 amino acids of porcine pancreas ATPDase. The specificity of these antibodies has been shown previously (33).
For the generation of monoclonal antibodies, 500 µl of the pooled 5'-AMP-Sepharose 4B fractions (±100 µg protein) were used. Immunized mouse spleen cells were fused with Sp2/0-Ag14 cells (ATCC CRL1581, American Type Culture Collection, Rockville, MD), and hybridoma cells were selected in HAT medium and grown in DMEM with 15% fetal calf serum. Anti-ATPDase-producing hybridoma cells were selected by using the ATPDase capture assay (35) with minor modifications (see Determination of ATPDase activity). For the production of ascites, antibody-producing hybridoma cells (4 × 106) were suspended in 1 ml of 10 mM phosphate buffer (pH 7.4), containing 130 mM NaCl and 3 mM KCl (PBS), and injected intraperitoneally in a mouse previously injected (10 days earlier) with 0.7 ml of pristane. IgG was purified on a protein G-Sepharose 4 Fast Flow column, and a mixture of 100 mg purified monoclonal antibodies (4A9 and 4D11) was mixed for 48 h at 4°C with 2 g of swollen cyanogen bromide-activated Sepharose 4B. The gel was stabilized by cross-linking with 0.025% glutaraldehyde in 0.25 M NaHCO3 (12). The final step in the purification of the ATPDase was performed by applying 80 ml of solubilized cell membrane (2 mg protein/ml) to the immunoaffinity column (16 mm2) at 0.25 ml/min. The ATPDase activity was recovered by elution with 25 mM Tris · HCl, 3 M NaSCN, and 0.3% Triton X-100, pH 7.6. The eluted fractions were dialyzed overnight and tested for ATPDase activity.Determination of ATPDase activity. The ATPDase activity was determined by adding 10 µl of an enzyme preparation to 40 µl of a reaction mixture containing 1 mM ouabain, 0.1 mg/ml Con A, 1 mM N-ethylmaleimide, 5 µg/ml oligomycin, 1 mM levamisole, 100 µM sodium vanadate, 100 mM NaCl, 5 mM KCl, and 2 mM substrate (any NTP or NDP) in 30 mM Tris · HCl, pH 7.5. To check for enzymes specifically activated by calcium, 2 mM CaCl2 and 0.1 mM EDTA were added, whereas for magnesium-activated enzymes 2 mM MgCl2 and 0.1 mM EGTA were used (21). After 30-min incubation at 37°C, the reaction was stopped by the addition of 150 µl of 0.25% (wt/vol) CuSO4, 1% (wt/vol) SDS, and 4.6% (wt/vol) sodium acetate (pH 4.0), and the inorganic phosphate was determined according to the method of LeBel et al. (14).
For characterization experiments, an ATPDase capture assay (35) was performed in 96-well, flat-bottomed ELISA plates coated with 1 µg rabbit anti-mouse antibodies/well, in 100 µl coating buffer (40 mM sodium carbonate, pH 9.6). The plates were incubated overnight with hybridoma supernatant at 4°C. After washing, 50 µl of solubilized membranes/well were incubated at room temperature for 1 h. For the characterization of the enzyme, 50 µl of the ATPDase reaction mixture were added. After 1-h incubation at 37°C, 40 µl of the mixture were transferred to an empty 96-well plate, and the phosphate released was determined as described above.Electrophoresis and Western blotting. Proteins were separated by SDS-PAGE according to Laemmli (13), with or without 1% (vol/vol) 2-mercaptoethanol depending on the use of reducing or nonreducing conditions, respectively. The proteins were transferred to PVDF membranes (immobilon P) by electroblotting (36), and the bands were visualized by using horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG, both at a dilution of 1:4,000, and Renaissance Chemiluminesence Reagent Plus according to the manufacturer's instructions.
Enzymatic deglycosylation. N-linked carbohydrates were removed by using endoglycosidase F. Approximately 20 µg (in 20 µl) of immunoaffinity-purified ATPDase were boiled for 2 min in the presence of 1% SDS. Two hundred and twenty-five microliters of 20 mM phosphate buffer (pH 7.5) containing 0.2% NaN3, 50 mM EDTA, and 0.5% Nonidet P-40 were added, and the sample was boiled again for 2 min. Finally, 8 µl of endoglycosidase F (0.4 U) were added, and the mixture was rotated overnight at 37°C.
Immunolocalization. Freshly dissected pig kidney tissue samples were fixed overnight in a solution containing 2% paraformaldehyde, 0.17% glutaraldehyde, and 4% sucrose in 0.1 M sodium cacodylate buffer (pH 7.4). Tissues were dehydrated in graded ethanol solutions and embedded in paraffin. Sections were cut at a thickness of 4 µm and mounted on polyionic slides (Superfrost Plus, Fisher, Montréal, Canada). After removal of the paraffin with xylene, the tissue sections were rehydrated with graded ethanol-water solutions and washed with 150 mM NaCl in 0.1 M Tris, pH 7.5, (TBS). The sections were incubated for 10 min in TBS-containing 0.1 M glycine and subjected to the pressure cooker "heat-induced epitope retrieval" procedure by incubating in 1 mM EDTA, 10 mM Tris, pH 8.0, for 9 min (19). After a wash of 10 min in TBS, at room temperature, nonspecific binding sites were blocked with 1% BSA and 1% fat-free skimmed milk in TBS for 30 min at room temperature. The sections were then incubated overnight at 4°C with type I ATPDase antiserum or preimmune serum (1:100), washed several times with TBS, and incubated with alkaline phosphatase conjugated to rabbit IgG (Sigma Chemical, St. Louis, MO) at a dilution of 1:100 for 2 h at room temperature. After several washings, the alkaline phosphatase reaction was visualized with nitro blue tetrazolium and 5-bromo-1-chloro-3-indolyl phosphate as the substrates. Sections were mounted in 5% gelatin, 27% glycerin, and 0.1% sodium azide, preheated at 45°C. Photographs were taken under bright-field illumination by using a Zeiss photomicroscope and Kodak T-Max-100 print film.
Enzyme histochemistry. Small blocks were cut out of the inner part of the pig kidney cortex and fixed with 3% paraformaldehyde, 1% glutaraldehyde, 0.25 M sucrose, and 2 mM CaCl2 in a 0.05 M cacodylate buffer, pH 7.4, for 1 h at 4°C. After being washed with the cacodylate buffer, 50-µm Vibratom sections for electron microscopic investigation were cut. Other blocks for light microscopic purposes were put into 10% sucrose for 30 min at room temperature and incubated in 30% sucrose overnight at 4°C. Sections of 10 µm were cut and washed with a 0.05 M Tris-maleate buffer, pH 7.4, containing 0.2 M sucrose. The ATPDase reaction was performed for 45 min at room temperature, in 0.05 M Tris-maleate buffer, containing (in mM) 2 PbNO3 (as a phosphate complexing agent), 5 KCl, 1 levamisole, 1 ouabain, 1.5 CaCl2 , and 1 ATP. The reaction was stopped by rinsing thoroughly with buffer, and the Pb3(PO4)2 was converted to PbS by incubating the sections with 1% (NH4)2S for 1 min.
The cryosections for light microscopy were rinsed with 0.05 M Tris-maleate buffer, water, and subsequently mounted on a glass slide in Aquatex (Merck). For electron microscopy, the Vibratom sections were incubated with 1% osmium tetroxide in the cacodylate buffer for 30 min in the dark. After being washed in 30 and 50% ethanol, the sections were incubated for 30 min with 2% uranyl acetate in 70% ethanol in the dark. After further dehydration with ethanol, the sections were incubated for 30 min at 37°C in a 1:1 (vol/vol) absolute ethanol-durcupan resin, followed by 30 min in durcupan at 60°C and overnight embedding with durcupan at 60°C. Ultrathin sections were cut and examined in a Hitachi 2001 transmission electron microscope.Amino acid sequencing. A sample of ATPDase (20 µg protein), purified by immunoaffinity chromatography, was subjected to electrophoresis on a 10% SDS-polyacrylamide gel under reducing conditions and blotted on a ProBlott PVDF membrane. The ProBlot membrane was subjected to three washing cycles of 50% HPLC-grade methanol and HPLC-grade water, and the band of interest was excised and subjected to NH2-terminal sequencing on a Procise 492 sequenator (Perkin-Elmer Applied Biosystems), operating in pulsed-liquid mode.
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RESULTS |
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Purification. In a previous study, we showed that ATPDase activity was mainly present in the brush-border membranes of porcine kidney (38). However, no clear characterization of the enzyme could be achieved. In this paper we describe a new and more successful approach to the purification of ATPDase from pig kidney cortex.
Table 1 represents the ATPase activities with Ca2+ as a divalent cation in a typical balance sheet of the purification of porcine kidney ATPDase. Homogenization of the cortex and differential centrifugation produced a crude microsomal pellet, containing 12% of the original ATPase activities. The specific activity of this preparation was comparable to that of the starting material.
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Identification of the ATPDase with monoclonal antibodies. By immunizing a mouse with the partially purified enzyme preparation obtained after 5'-AMP-Sepharose 4B chromatography, monoclonal antibodies were generated. They were screened for their potential to bind ATPDases by using the ATPDase capture assay. This allowed the detection of seven different ATPDase antibodies, all of the IgG1 subtype. By using an epitope competition assay, we could distinguish at least four distinct epitopes recognized by the monoclonal antibodies. Antibodies 2C3 and 4A9 seem to react with neighboring epitopes as indicated by competition assays. This similarity also applies to antibodies 2C7, 1A4, and 4D11. Antibodies 1H12 and 4D8 apparently react with epitopes unrelated to the others. Antibody 4D8 has the highest affinity for ATPDase in this capture assay (results not shown).
The antibodies were further characterized on Western blot, to identify the molecular mass of the protein with which they react. For this purpose, zymogen granule membranes of the pig pancreas were prepared as previously described (31). As shown in Fig. 2, all the antibodies reacted with a 78-kDa protein under nonreducing conditions. Six antibodies also showed a reaction with a protein of ~54 kDa, and only one (1H12) reacted with a 27-kDa protein. All the antibodies also interreacted with proteins in the high-molecular-weight range. Under reducing conditions, reaction only occurred with antibody 1H12, which now only recognizes the 78-kDa band, as can be seen in Fig. 2 for the kidney particulate fraction. This means that the six other antibodies need the disulfide bridges for immunoreactivity.
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Immunoaffinity chromatography. A mixture of two monoclonal antibodies was employed to prepare an ATPDase-affinity column as described in MATERIALS AND METHODS. After 80 ml of a solubilized microsomal preparation were loaded, one-half of the ATPDase activity bound to the column and could be eluted with 3 M NaSCN. This resulted in an increase in specific activity of ~1,000-fold in one single step.
After separation of the eluted material by denaturing SDS-PAGE, the 78-kDa protein was blotted onto a ProBlot membrane and used for NH2-terminal amino acid sequencing. A sequence of 17 amino acids was obtained (DRRESELKTFCSKNILV) and used to run a Fasta3 Database Search. A search in the SWALL Non-Redundant Protein sequence database (Swissprot, Trembl, and TremblNew) showed 88% identity to bovine CD39 and 70% identity to human CD39 for this 17-amino acid sequence (Table 2).
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Characterization of the ATPDase. The pooled fractions obtained after 5'-AMP-Sepharose were treated with endoglycosidase F and then subjected to reducing SDS-PAGE and Western blotting with antibody 1H12. Figure 2 shows that the deglycosylated 78-kDa protein has a molecular mass of ~57 kDa, concordant with the calculated molecular mass based on the human CD39 cDNA sequence.
With the capture assay, using monoclonal antibody 4D8, a number of biochemical characteristics of the enzyme were determined. Measurement of the ATPase and the ADPase activities showed that their ratio was ~1:1, with Ca2+ as a divalent cation. A number of other naturally occurring NTPs and NDPs were also tested. Figure 3 clearly shows that, despite the physiological prevalence of adenine nucleotides, they are not the best substrates for the pig kidney ATPDase. Both the NTPase and NDPase activities required divalent cations for enzymatic activity. After addition of 0.5 mM EGTA and 0.5 mM EDTA to the reaction mixture, no activity could be detected with any of the 12 substrates tested (data not shown). Interestingly, the presented data show that the enzyme generally has a preference for Ca2+ ions over Mg2+ ions. AMP, p-nitrophenylphosphate, and diadenosine polyphosphates (AP3A, AP4A, and AP5A) were not hydrolyzed by the enzyme (data not shown).
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Immune and enzyme histochemical localization by light and electron
microscopy.
All ATPDase monoclonal antibodies were used for the
immunolocalization of the ATPDase in pig kidney. These antibodies all reacted with the blood vessel walls, the glomerular capillaries and the
peritubular capillaries, as displayed in Fig.
5 for antibody 4D8.
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DISCUSSION |
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A previous purification of porcine kidney ATPDase has revealed an abundant 94-kDa protein and led at that time to the erroneous conclusion that this protein was a likely candidate for the ATPDase (37). However, the use of ATPDase antibodies in this study demonstrated that the 78-kDa protein was in fact responsible for the enzyme activity. Our results also indicate the presence in porcine kidney of an ATPDase sharing immunological identity with the ATPDases of pig pancreas (type I, 54 kDa) and bovine aorta (type II, 78 kDa).
A polyclonal antibody generated against the NH2-terminal sequence of type I ATPDase (33) was found to react with type I ATPDases of bovine heart (1), bovine blood vessels (33), and bovine lung (34). In all of these tissues, the type I ATPDase antibody also reacted with a 78-kDa type II ATPDase, corresponding to CD39 (10). The NH2-terminal amino acid sequence of type I ATPDase shows a very high level of homology to an internal sequence of human CD39 (10). It was therefore concluded that type I ATPDase (the 54-kDa protein) is the COOH-terminal part of type II ATPDase (the 78-kDa protein). This brings up the possibility that type I ATPDase originates by proteolysis of the 78-kDa ATPDase (29). This conclusion is supported by the presence in our purified fractions of a 27-kDa protein, which could be the remainder of the 78-kDa protein after removal of the 54-kDa moiety. The copurification of the 27- and the 54-kDa protein also strengthens the idea that both were formed by proteolysis of the native 78-kDa ATPDase. However, it remains unclear whether this proteolysis occurs spontaneously during purification rather than as a natural process.
Because monoclonal antibodies only react with a single epitope, it is obvious that they will recognize either the 54- or the 27-kDa protein, together with the 78-kDa ATPDase. Indeed, Western blots showed that both the 78-kDa and either the 54- or the 27-kDa proteins were present simultaneously.
The high-molecular-weight proteins with which the antibodies react under nonreducing conditions are probably multimers. These protein bands disappear under reducing conditions, probably because of cleavage of the disulphide bridges.
We examined the effect of sodium azide on both ATPase and ADPase activities of porcine ATPDase and found that the ADPase activity is more sensitive to sodium azide than the ATPase activity. A small inhibition of the ADPase activity was seen at 1 mM azide, increasing to 62 ± 5% inhibition in the presence of 10 mM sodium azide. In pig pancreas, 10 mM azide reduces the ADP hydrolysis by 51% (15), but in the latter study the inhibition of the ATPase activity is not mentioned. In chicken oviduct, 10 mM sodium azide reduces the hydrolysis of ATP by ATPDase by 35%, and, for ADP, the reduction is even 87% (35). In other studies, the ADPase activity was often inhibited slightly more than the ATPase activity (23).
NH2-terminal amino acid sequence determination of the 78-kDa ATPDase of pig kidney shows that this protein has a very high level of homology with bovine and human CD39. Apparently, porcine ATPDase is lacking a methionine and a glutamic acid at the NH2-terminal end, compared with both bovine and human CD39. It should however, be noted, that the described sequences for bovine and human CD39 were derived from nucleic acid sequences and were not obtained from amino acid analysis of the purified proteins. It is probable that both amino acids are removed by a posttranslational modification and are not detected in the expressed protein. Therefore, we conclude that the pig kidney ATPDase is the porcine counterpart of bovine and human CD39.
Deglycosylation of the 78-kDa ATPDase results in a protein of ~57 kDa, consistent with the calculated molecular mass of the protein coded by human CD39 cDNA (18). Deglycosylation of the pancreatic 54-kDa protein yields a core protein with a molecular mass of 35 kDa (31). This corresponds to the molecular mass of the COOH-terminal fragment of human CD39, calculated from the position of the NH2-terminal sequence of the 54-kDa ATPDase. Again, these data suggest that the 27- and the 54-kDa (type I) proteins are part of the 78-kDa type II ATPDase.
Previous studies showed that the ATPDase in the kidney was only present in the brush borders (20, 38). This was also concluded by immunofluorescence in rat kidney using antibodies against rat liver ATPDase (26). However, after the cloning of the rat liver ATPDase cDNA (17) it became clear that the encoded protein was not ATPDase (11). After reinvestigation, Sabolic et al. (27) concluded that the antibody against the putative liver ATPDase was not specific because the labeling with this antibody in renal brush border did not coincide with the ATPDase activity (27).
Electronmicroscopic histochemistry in this study clearly shows the presence of ATPase and ADPase activity on both basolateral and brush-border membranes, but the activity associated with the brush borders is definitely higher than on the basolateral membranes. This is probably due to the tremendous surface increase as a result of the microvilli in the brush borders. The demonstration in the kidney of ATPDase activity with monoclonal antibodies only shows the presence of this protein in the blood vessels, the glomerular, and the peritubular capillaries. This indicates that another ATPDase may be present on the brush-border membranes of the proximal tubuli, as seen on the light micrograph showing the ADPase activity on these sites. Immunological staining with a polyclonal antibody against the ATPDase, which probably also recognizes other members of the GDA1/CD39 family of nucleoside phosphatases, also shows a positive reaction in the brush borders of the proximal convoluted tubules and in the glomerular capsule. This activity is not due to CD39/ATPDase but must be assigned to another ATPDase. Previously, ADPase activity was demonstrated in rat kidney on the glomerular basement membrane and the surrounding endothelial and epithelial cells (24), the glomerular cells (4), and microvilli and vascular endothelial cells (28). In our study, however, there is only very weak immunological staining of the glomerular capillaries with the anti-CD39/ATPDase antibodies. Even by enzyme histochemistry, only a weak reaction was obtained, indicating that the amount of ATPDase on the glomerular cells is very low compared with the tubular cells (Fig. 10).
We presume that ectonucleotidases in the kidney play a key role in the regulation of the extracellular concentrations of nucleotides and nucleosides. Both substances have important biological functions in the kidney. By binding to P2X receptors, localized almost exclusively in the preglomerular blood vessels, ATP causes vasoconstriction of the renal microvasculature (8). In cell culture, extracellular ATP and UTP stimulate the growth of glomerular mesangial cells and inner medullary collecting duct cells by binding to a P2U receptor (9, 30). Furthermore, ATP increases the intracellular Ca2+concentration in the epithelial cells of the terminal collecting duct via a P2U purinergic receptor (2).
The antidiuretic effect of adenosine is due to the reduction in the glomerular filtration rate via A1 receptor-mediated vasoconstriction of afferent and A2 receptor-mediated vasodilation of efferent arterioles. This effect is slower but more prolonged than the preglomerular vasoconstriction triggered by ATP. Binding of adenosine, released as AMP and converted by 5'-nucleotidase, to the A1 receptor in the thick ascending limbs inhibits the active ion transport (3). Furthermore, adenosine is also known to inhibit the release of renin via interaction with A1 receptors (for review, see Ref. 42). This A1 receptor is mostly expressed in collecting ducts of the papilla and inner medulla and in the cells of the juxtaglomerular apparatus (41). This raises the possibility that adenosine regulates the glomerular filtration, a phenomenon known as the tubuloglomerular feedback. Both mechanisms protect the cells under ischemic conditions. In proximal tubular fluid, the adenosine is mainly formed by the action of phosphodiesterase and 5'-nucleotidase on cAMP, which has a modulatory effect on the renal phosphate transport (5). Furthermore, extracellular produced adenosine is reabsorbed, presenting a salvage pathway for the recovery of nucleotides.
Clearly, extracellular nucleotides and nucleosides play a variety of physiological functions in kidney. The modulation of the concentration of these ligands is essential in the regulation of their functions.
In this study we described an ATPDase, present in all the vasculature of the kidney, as CD39. Furthermore, our histological and immunohistological data suggest the presence of an unidentified but related enzyme in kidney tubuli. This information can be the basis to further characterize the modulation and function of the extracellular nucleotides and nucleosides in renal homeostasis.
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ACKNOWLEDGEMENTS |
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We thank A. Roosen, K. Lengyel, and E. Csizmadia for technical assistance.
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FOOTNOTES |
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This work was supported by grants from the Bilateral Scientific and
Technological Cooperation (BIL97/32) and Fonds pour la Formation de
Chercheurs et l'Aide à la Recherche du Québec (FCAR) and
by the Natural Sciences and Engineering Research Council of Canada. J. Sévigny is a recipient of fellowships from the Heart and Stroke
Foundation of Canada and from FCAR. E. Waelkens is a research associate
of the Fonds voor Wetenschappelijk OnderzoekVlaanderen.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. Vanduffel, Limburgs Universitair Centrum, Dept. MBW, Universitaire Campus; Bldg. D, B-3590 Diepenbeek, Belgium (E-mail: Luc.Vanduffel{at}luc.ac.be).
Received 4 June 1999; accepted in final form 30 December 1999.
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