Expression, Purification, and Characterization of Isoform 1 of the Plasma Membrane Ca2+ Pump
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m302400200
ISSN1083-351X
AutoresDanilo Guerini, Bin Pan, Ernesto Carafoli,
Tópico(s)Pancreatic function and diabetes
ResumoThe plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms. The plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms. The plasma membrane Ca2+ ATPase (PMCA) 1The abbreviations used are: PMCA, plasma membrane Ca2+ ATPase; MOPS, 4-morpholinepropanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid.1The abbreviations used are: PMCA, plasma membrane Ca2+ ATPase; MOPS, 4-morpholinepropanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid. pumps Ca2+ out of the cell, reducing its concentration in the cytosol to the level compatible with the messenger function (1Carafoli E. FASEB J. 1994; 8: 993-1002Crossref PubMed Scopus (360) Google Scholar). In excitable tissues, e.g. heart, it does so in concert with the Na+/Ca2+ exchanger (2Philipson K.D. Nicoll D.A. Curr. Opin. Cell Biol. 1992; 4: 678-683Crossref PubMed Scopus (45) Google Scholar). The pump has been detected in all mammalian cells studied so far (1Carafoli E. FASEB J. 1994; 8: 993-1002Crossref PubMed Scopus (360) Google Scholar, 3Carafoli E. Guerini D. Trends Cardiovasc. Med. 1993; 3: 177-184Crossref PubMed Scopus (36) Google Scholar), although differences in the level of its expression have been observed (4Stauffer T. Hilfiker H. Carafoli E. Strehler E.E. J. Biol. Chem. 1993; 268: 25993-26003Abstract Full Text PDF PubMed Google Scholar). cDNA cloning has identified four independent PMCA transcripts in human and rat tissues, and the corresponding human genes have been located on four different chromosomes (3Carafoli E. Guerini D. Trends Cardiovasc. Med. 1993; 3: 177-184Crossref PubMed Scopus (36) Google Scholar, 5Brandt P. Neve R.L. Kammesheidt A. Rhoads R.E. Vanaman T.C. J. Biol. Chem. 1992; 267: 4376-4385Abstract Full Text PDF PubMed Google Scholar, 6Olson S. Wang M.G. Carafoli E. Strehler E.E. McBride O.W. Genomics. 1991; 9: 629-641Crossref PubMed Scopus (67) Google Scholar, 7Wang G.M. Huafang Y. Hilfiker H. Carafoli E. Strehler E.E. McBride O.W. Cytogenet. Cell Gen. 1994; 67: 41-45Crossref PubMed Scopus (38) Google Scholar). The primary transcripts are spliced in a complex way: two major sites of splicing, termed sites C and A, in principle produce about 30 different isoforms (1Carafoli E. FASEB J. 1994; 8: 993-1002Crossref PubMed Scopus (360) Google Scholar). The cloning work has indicated that certain transcripts of the PMCA isoforms and of their splicing products have striking tissue-specific expression (8Shull G.E. Greeb J. J. Biol. Chem. 1988; 263: 8646-8657Abstract Full Text PDF PubMed Google Scholar, 9Strehler E.E. Strehler-Page M.-A. Vogel G. Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6908-6912Crossref PubMed Scopus (90) Google Scholar, 10Greeb J. Shull G.E. J. Biol. Chem. 1989; 264: 18569-18576Abstract Full Text PDF PubMed Google Scholar, 11Stahl W.L. Eakin T.J. Owens J.W.M. Breininger J.F. Filuk P.E. Anderson W.R. Mol. Brain Res. 1992; 16: 223-231Crossref PubMed Scopus (98) Google Scholar, 12Keeton T.P. Burk S.E. Shull G.E. J. Biol. Chem. 1993; 268: 2740-2748Abstract Full Text PDF PubMed Google Scholar, 13Stauffer T. Guerini D. Carafoli E. J. Biol. Chem. 1995; 270: 12184-12190Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 14Genazzani A.A. Carafoli E. Guerini D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5797-5801Crossref PubMed Scopus (154) Google Scholar). For example, although PMCA1 and PMCA4 have been detected in all tissues examined, PMCA2 and PMCA3 have only been found in significant amounts in the brain and a few other tissues. These results on the transcripts have been confirmed at the protein level with isoform-specific antibodies (13Stauffer T. Guerini D. Carafoli E. J. Biol. Chem. 1995; 270: 12184-12190Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Quantitative analysis has shown that, in general, the human PMCA1 product is more abundant than the PMCA4, both at the mRNA (4Stauffer T. Hilfiker H. Carafoli E. Strehler E.E. J. Biol. Chem. 1993; 268: 25993-26003Abstract Full Text PDF PubMed Google Scholar) and the protein level (13Stauffer T. Guerini D. Carafoli E. J. Biol. Chem. 1995; 270: 12184-12190Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The overexpression of the PMCA4 and the PMCA2 proteins has permitted the study of their biochemical properties (15Heim R. Iwata T. Zvaritch E. Adamo H.P. Rutishauser B. Strehler E.E. Guerini D. Carafoli E. J. Biol. Chem. 1992; 267: 24476-24484Abstract Full Text PDF PubMed Google Scholar, 16Enyedi A. Verma A.K. Filoteo A.G. Penniston J.T. J. Biol. Chem. 1993; 268: 10621-10626Abstract Full Text PDF PubMed Google Scholar, 17Enyedi A. Verma A.K. Heim R. Adamo H.P. Filoteo A.G. Strehler E.E. Penniston J.P. J. Biol. Chem. 1994; 269: 41-43Abstract Full Text PDF PubMed Google Scholar, 18Hilfiker H. Guerini D. Carafoli E. J. Biol. Chem. 1994; 269: 26178-26183Abstract Full Text PDF PubMed Google Scholar, 19Seiz-Preianò B. Guerini D. Carafoli E. Biochemistry. 1996; 35: 7946-7953Crossref PubMed Scopus (38) Google Scholar). A number of methods (transient transfection, stable cell lines, vaccinia virus as a vector) have been used (20Foletti D. Guerini D. Carafoli E. FASEB J. 1995; 9: 670-680Crossref PubMed Scopus (38) Google Scholar, 21Zvaritch E. Vellani F. Guerini D. Carafoli E. J. Biol. Chem. 1995; 270: 2679-2688Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 22Schwab B.L. Guerini D. Didszun C. Bano D. Ferrando-May E. Fava E. Tam J. Xu D. Xanthoudakis S. Nicholson D.W. Carafoli E. Nicotera P. Cell Death Differ. 2002; 9: 818-831Crossref PubMed Scopus (230) Google Scholar), significantly advancing knowledge on the differential properties of the isoforms. Strikingly, however, very little information is available on PMCA1, which is the most common isoform. Only one successful attempt to express this isoform (a truncated variant C) has so far been reported (23Liu B.-F. Xu X. Fridman R. Muallem S. Kuo T.H. J. Biol. Chem. 1996; 271: 5536-5544Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Difficulties in expressing this isoform have been repeatedly reported (23Liu B.-F. Xu X. Fridman R. Muallem S. Kuo T.H. J. Biol. Chem. 1996; 271: 5536-5544Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24Adamo H.P. Verma A.K. Sanders M.A. Heim R. Salisbury J.L. Wieben E.D. Penniston J.T. Biochem. J. 1992; 285: 791-797Crossref PubMed Scopus (56) Google Scholar). The PMCA pump is a substrate of intracellular proteases. Initial observations had indicated that it was a substrate for the Ca2+-dependent protease calpain (CANP, EC 3.4.22.17) (25Molinari M. Anagli J. Carafoli E. J. Biol. Chem. 1994; 269: 27992-27995Abstract Full Text PDF PubMed Google Scholar), and more recent work has shown that effector caspases (e.g. caspases 1 and 3) also cleave PMCA pump isoforms (22Schwab B.L. Guerini D. Didszun C. Bano D. Ferrando-May E. Fava E. Tam J. Xu D. Xanthoudakis S. Nicholson D.W. Carafoli E. Nicotera P. Cell Death Differ. 2002; 9: 818-831Crossref PubMed Scopus (230) Google Scholar, 26Paszty K. Verma A.K. Padanyi R. Filoteo A.G. Penniston J.T. Enyedi A. J. Biol. Chem. 2002; 277: 6822-6829Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Both activation and inactivation of PMCA2 and PMCA4 by caspases have been reported (22Schwab B.L. Guerini D. Didszun C. Bano D. Ferrando-May E. Fava E. Tam J. Xu D. Xanthoudakis S. Nicholson D.W. Carafoli E. Nicotera P. Cell Death Differ. 2002; 9: 818-831Crossref PubMed Scopus (230) Google Scholar, 26Paszty K. Verma A.K. Padanyi R. Filoteo A.G. Penniston J.T. Enyedi A. J. Biol. Chem. 2002; 277: 6822-6829Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Calpain attacks calmodulin binding enzymes (27Molinari M. Anagli J. Carafoli E. J. Biol. Chem. 1995; 270: 2032-2035Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), removing portions of the calmodulin binding sequence and leading to their "irreversible" activation. The protease contains regions with strong homology to calmodulin (domains IV and VI; Refs. 28Minami Y. Emori Y. Imajoh-Ohmi S. Kawasaki H. Suzuki K. J. Biochem. 1988; 104: 927-933Crossref PubMed Scopus (31) Google Scholar, 29Minami Y. Emori Y. Kawasaki H. Suzuki K. J. Biochem. 1987; 101: 889-895Crossref PubMed Scopus (41) Google Scholar), which may be important in directing it to its target sequences. Support for this concept was provided by the binding of calpain to the calmodulin binding domain of the erythrocyte pump (27Molinari M. Anagli J. Carafoli E. J. Biol. Chem. 1995; 270: 2032-2035Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The proteolysis of the pump by calpain removes most of the C-terminal portion protruding into the cytosol, leaving behind a constitutively active fragment of about 124 kDa. The process has been studied on erythrocyte membranes or on the pump purified from them, i.e. on a mixture of the pumps isoforms present in erythrocytes, PMCA1, and PMCA4 (13Stauffer T. Guerini D. Carafoli E. J. Biol. Chem. 1995; 270: 12184-12190Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Because PMCA4 represents at least 80% of the total erythrocyte pump, it was not surprising that most of the peptides isolated after calpain digestion should have derived from this isoform (30James P. Vorherr T. Krebs J. Morelli A. Castello G. McCormick D.J. Penniston J.T. De Flora A. Carafoli E. J. Biol. Chem. 1989; 264: 8289-8296Abstract Full Text PDF PubMed Google Scholar). Nevertheless, the failure to detect high molecular mass peptides deriving from PMCA1 indicated that the latter isoform had different calpain sensitivity. The work described here was performed to characterize biochemically the human PMCA1 isoform, including its calpain sensitivity. The pump was expressed in Sf9 insect cells with the help of the baculovirus system, but a complex DNA manipulation was required to generate a full-length PMCA1 clone devoid of mutations, which could be transferred to the baculovirus. The protein became expressed at lower levels than the PMCA2 and PMCA4 isoforms but could still be purified on a calmodulin-Sepharose column. The purified PMCA1 was activated by calmodulin, interacting with it with an affinity similar to that of PMCA4. However, it had a distinctly higher affinity for ATP in experiments measuring the formation of the phosphoenzyme intermediate. In agreement with previous observations (31James P. Pruschy M. Vorherr T. Penniston J.T. Carafoli E. Biochemistry. 1989; 28: 4253-4258Crossref PubMed Scopus (104) Google Scholar), the purified PMCA1 was a better substrate for the cAMP-dependent kinase than PMCA4 and PMCA2. The PMCA4, PMCA2, and PMCA1 proteins were tested for calpain sensitivity in vitro. Although the PMCA1 isoform was rapidly and completely degraded, the PMCA4 and PMCA2 proteins were proteolyzed much more slowly and only to large active fragments. The digestion patterns of the PMCA2 and PMCA4 isoforms also differed. Whereas only the calmodulin binding domain and the sequence downstream of it were quantitatively removed in the case of PMCA4, a more complex pattern of degradation prevailed in the PMCA2 protein. Materials—m-CANP was isolated from freshly collected human erythrocytes as described in Ref. 27Molinari M. Anagli J. Carafoli E. J. Biol. Chem. 1995; 270: 2032-2035Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar. The TNM-FH medium was from Sigma. Antibiotics and fetal calf serum were purchased from Invitrogen. The monoclonal antibody 5F10 was obtained from Milan Analytic AG (Hoffmann-La Roche). Nitrocellulose was from Schleicher & Schuell (Dassel, Germany). Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate were from Promega (Madison, WI). All other reagents were of the highest purity grade commercially available. Construction of the Expression Vectors—To construct a full-length cDNA of human PMCA1, two clones (t6, t8.1) were used (32Verma A.K. Filoteo A.G. Standford D.R. Wiebenm E.D. Penniston J.T. Strehler E.E. Fisher R. Heim R. Vogel G. Mathews S. Strehler-Page M.-A. James P. Vorherr T. Krebs J. Carafoli E. J. Biol. Chem. 1988; 263: 14152-14159Abstract Full Text PDF PubMed Google Scholar). The human PMCA1 isoform was used (also known as PMCA1b). The vectors used for the constructions were the pTZ18/19 (United States Biological, Swampscott, MA), the pSG5 (Stratagene, GmbH, Zürich, Switzerland), and the pVL1393 (provided by Dr. M. D. Summers, Texas A&M University, College Station, TX). DNA was purified by CsCl centrifugation (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Vols. 1–3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) or by Qiagen columns (Quiagen, Chatsworth, CA). Different attempts to ligate fragments together to obtain a full-length construct resulted in a number of deletion-containing products. The use of PCR-directed mutagenesis to create suitable restriction sites or cDNA fragments was similarly unsuccessful. In a first step an XbaI, located in vector sequences proximal to the 3′-end of the PMCA1 and BamHI (nt 950, HUMPMPCA, GenBank™ accession number J04027) fragment, was subcloned together with a 900-bp SalI-BamHI (nt 950, J04027) fragment; the SalI was located in the vector proximal to the 5′-end, in the pSG5 SalI-XbaI cut vector. This vector was used because it yielded the highest amount of DNA of all those that were tried. No mutations were observed using this vector. After modifying the XbaItoa KpnI site, the PMCA1 cDNA was cut by SalI, blunt ended, cut by KpnI, and subcloned in pVL1393. The bacteria bearing the pVL1393 PMCA1 construct produced very low amounts of plasmid DNA. Generally, 2–4 μg of DNA could be purified from 250–500 ml of liquid cultures in 2XYT, corresponding to about one plasmid copy per bacterial cell. Culturing of Sf9 Cells and Construction of a Recombinant Baculovirus—Spodoptera frugiperda (Sf9) cells were grown in TNM-FH medium supplemented with 10% fetal calf serum and 50 μg/ml gentamicin (both from Invitrogen) at 30 °C. PMCA1-containing DNA purified by two CsCl gradient centrifugations was cotransfected with Bak-Pak DNA (Invitrogen) to Sf9 cells using the Ca3(PO4)2 precipitation method as described (34Summers, M. D., and Smith, G. E. (1988) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experiment Station Bulletin 1555, Texas A&M University, College Station, TXGoogle Scholar). The recombinant baculoviruses were identified by visual inspection of the viral plaques after plating on Sf9 cells and analyzed by dot blotting with PMCA1-specific probes. Putative recombinant viral plaques were checked for the correct insertion in the presence of 150 μg/ml X-gal (5-bromo-chloro-3-indolyl-b-d-galactoside; Bachem Feichemikalien AG, Dübendorf, Switzerland). The recombinant plaques were amplified and titrated as described (34Summers, M. D., and Smith, G. E. (1988) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experiment Station Bulletin 1555, Texas A&M University, College Station, TXGoogle Scholar). More than 20 different recombinant viruses were isolated and tested for their ability to express the PMCA1 pump. Seven of them (see also Fig. 3A) expressed a protein of about 140 kDa. The band was absent in non-infected Sf9 cells. From some clones, viral DNA was prepared and analyzed by Southern blotting (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Vols. 1–3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Viral DNA was obtained from sucrose gradient purified viruses (34Summers, M. D., and Smith, G. E. (1988) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experiment Station Bulletin 1555, Texas A&M University, College Station, TXGoogle Scholar). As expected, digestion with BamHI (a BamHI site is present in the pVL1393 vector just upstream of the PMCA1 sequence) and PstI-BamHI produced one fragment of 1000 bp and two fragments of 500 and 400 bp, respectively, which were recognized by probes spanning the coding sequence of PMCA1. Digestion with HindIII resulted in a PMCA1-specific fragment of 9500 bp, confirming that the PMCA1 was inserted at the right place in the genome of the baculovirus. After transferring the PMCA1 DNA to the latter, problems with the stability of the DNA were no longer encountered. Preparation of RNA, Northern Blotting, and Preparation of Digoxigenin Probes—2–4 × 107 Sf9 cells were infected with recombinant baculovirus for 48 h. The cells were collected, washed twice in 25 mm Tris-HCl, pH 7.4, 150 mm NaCl, and resuspended in 600 μl of 20 mm Tris-HCl, pH 7.4, 10 mm NaCl, 3 mm MgCl2 on ice before the addition of 12.5 mm vanadyl-ribosyl complex (Invitrogen). Cytosolic RNA was then purified as described in Ref. 35Berger S. Birkenmeier C. Biochemistry. 1979; 18: 5143-5149Crossref PubMed Scopus (257) Google Scholar. Northern blotting was performed essentially as described in Ref. 36Guerini D. Garcia-Martin E. Gerber A. Volbracht C. Leist M. Merino C.G. Carafoli E. J. Biol. Chem. 1999; 274: 1667-1676Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar. DNA fragments were labeled with digoxigenin-dCTP (Roche Applied Science), using the protocol suggested by the manufacturer. Preparation of Membranes—Sf9 cells were seeded at a density of 104/cm2 and left to attach for 1 h. They were infected at a multiplicity of infection of 5–10 for 1 h, and then the inoculum was replaced by fresh medium. After 48 h the infected cells were collected, washed twice with TBS (25 mm Tris-HCl, pH 7.5, 150 mm NaCl), and resuspended in 2 ml of 10 mm Tris-HCl, pH 7.5 (1 × 107 cells/ml). After 10 min on ice, the cells were homogenized in the presence of 75 μg/ml phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, 5 μg/ml leupeptin, 5 μg/ml anti-papain, and 5 μg/ml pepstatin. The homogenate was diluted by adding an equal volume of 10 mm Tris-HCl, pH 7.5, 20% sucrose, and 300 mm KCl and centrifuged at low speed (750 × g) for 5 min. The supernatant was centrifuged at 100,000 × g and resuspended in 20 mm HEPES-KOH, pH 7.5, 130 mm NaCl, 500 μm MgCl2, 50 μm CaCl2. In some cases the membranes were prepared by a freeze and thaw procedure: the cells were washed twice with TBS, resuspended in 10 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol, 75 μg/ml phenylmethylsulfonyl fluoride, and 1 mm EDTA and disrupted by three cycles of freezing at –70 °C and thawing at 37 °C. Determination of the Protein Stability by Pulse and Chase Experiments—48 h after infection the cells were washed twice, incubated with methionine-deficient minimal Eagle's medium (Invitrogen), and buffered to pH 6.2 with 25 mm MES-KOH. After 20 min at 27 °C, the same medium containing 200 μCi/ml [35S]Met (Amersham Biosciences) was added and incubated for3hat27 °C. A portion of the cells was collected (time = 0). To the remainder a 10,000 excess of cold Met in TNM-FH, 10% fetal calf serum, 50 μg/ml gentamicin was added, and the cells were transferred to 27 °C. Membranes were prepared from them by the freeze and thaw method described above, solubilized in 10 mm Tris-HCl, pH 8.0, 1 mm EDTA, and 1% SDS and boiled for 5 min. Membrane proteins corresponding to 3 × 106 cpm were diluted in 500 μl of NEM (50 mm Tris-HCl, 7.5, 150 mm NaCl, 1 mm EDTA, 0.2% gelatin, 0.1% NaN3, 0.2% SDS, 0.3% Nonidet P-40) and mixed with 1–2 μl of monoclonal antibody 5F10. After gentle shaking for 1–2 h at 4 °C, 20–30 μl of protein A-Sepharose CL-4B were added, and the incubation was continued for2hor overnight at 4 °C. The pellet was washed twice with NEM, twice with 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.1% NaN3, 0.3% Nonidet P-40, twice with 10 mm Tris-HCl, pH 8.0, and 1 mm EDTA, and resuspended in SDS-PAGE loading buffer. Activity Measurements—The ATPase activity was measured as described in Ref. 37Lanzetta P. Alvarez L. Reinach P. Candia O. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1810) Google Scholar. The assays were performed on 10–30 μg of membrane proteins or, in the case of the purified protein, with 50–200 ng of protein in 500–1000 μl of 120 mm KCl, 30 mm HEPES-KOH, pH 7.2, 1 mm MgCl2, 0.5 mm EGTA, 0.5 mm EDTA, 1 μm thapsigargin, and 5 μg/ml oligomycin. The amount of CaCl2 needed to produce the free calcium concentration desired was calculated as described in Ref. 38Fabiato A. Fabiato F. J. Physiol. 1979; 75: 463-505PubMed Google Scholar. The formation of the phosphoenzyme intermediate from ATP was followed as described before (18Hilfiker H. Guerini D. Carafoli E. J. Biol. Chem. 1994; 269: 26178-26183Abstract Full Text PDF PubMed Google Scholar): additional details are given in the legends for the figures. In brief, total membrane proteins or purified enzymes were diluted in 20 mm MOPS-KOH, pH 6.8, 100 mm KCl in the presence of different concentrations of Ca2+, La3+, or EGTA. The reaction was started by the addition of [γ-32P]ATP (100–3000 Ci/mmol) on ice and stopped after 20 s by the addition of 6% trichloroacetic acid, 1 mm KH2PO4. The pelleted proteins were separated on acidic gels (39Sarkadi B. Enyedi A. Földes-Papp Z. Gardos G. J. Biol. Chem. 1986; 261: 9552-9557Abstract Full Text PDF PubMed Google Scholar). After drying, the gels were exposed at –70 °C for 2–5 days. SDS-PAGE, Western Blotting, and Protein Determination—Proteins were separated on SDS-polyacrylamide gel electrophoresis as indicated in Refs. 40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206615) Google Scholar or 39Sarkadi B. Enyedi A. Földes-Papp Z. Gardos G. J. Biol. Chem. 1986; 261: 9552-9557Abstract Full Text PDF PubMed Google Scholar. Protein concentrations were determined as described in Ref. 37Lanzetta P. Alvarez L. Reinach P. Candia O. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1810) Google Scholar. In some cases the gels were stained with a silver impregnation method (41Merryl C. Dunan M. Goldman D. Anal. Biochem. 1981; 110: 201-207Crossref PubMed Scopus (290) Google Scholar). The proteins were transferred to nitrocellulose or to polyvinylidene difluoride membranes (Schleicher & Schuell) according to Ref. 42Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44843) Google Scholar. The blots were processed essentially as described by Stauffer et al. (13Stauffer T. Guerini D. Carafoli E. J. Biol. Chem. 1995; 270: 12184-12190Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) in the presence of PMCA-specific antibodies. After incubation with 1% bovine serum albumin in TBST (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.05% Tween 20), the nitrocellulose was treated with monoclonal antibody 5F10 (1:2000 in TBST) or polyclonal antibodies 2A, 1N, 2N, and 4N (1:500 in 0.5% bovine serum albumin, TBST) for 1 h at room temperature. After a further 30-min incubation with alkaline phosphatase coupled to goat anti-mouse or goat anti-rabbit immunoglobulins (DAKO, Glostrup, Denmark), the immunocomplexes were detected with 66 μl of nitroblue tetrazolium and 33 μl of 5-bromo-4-chloro-3-indolyl-phosphate solutions (Promega) in 10 ml of alkaline phosphatase buffer (100 mm Tris-HCl, pH 9.0, 100 mm NaCl, 5 mm MgCl2, and 1 mm CaCl2). Calmodulin Overlay—The proteins were transferred to nitrocellulose membrane sheets (42Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44843) Google Scholar). Nonspecific binding was blocked by 1% bovine serum albumin in TBSM (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm MgCl2, 0.2 mm CaCl2, 0.05% Tween 20). For control experiments (unspecific binding), 1 mm EGTA was included in TBSM. The blots were incubated separately with different concentrations of biotinylated calmodulin (10–7 to 10–10m) in TBSM for 1 h, washed twice in TBSM, and incubated for another hour with avidin-coupled alkaline phosphatase in TBSM. The filters were developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate in 10 ml of alkaline phosphatase buffer (100 mm Tris-HCl, pH 9.0, 100 mm NaCl, 5 mm MgCl2, and 1 mm CaCl2). In all experiments the amount of the pump present was carefully calibrated by Western blotting performed in parallel. Only blots in which an identical amount of pump was present were evaluated. Phosphorylation of PMCA by the cAMP-dependent Kinase—Prior to the purification the membranes were treated with alkaline phosphatase (3 units/μg protein) (Roche Applied Science) for 10 min at 37 °C. After addition of DNase (20 μg/ml) and of RNase (10 μg/ml) on ice, the different PMCA isoforms were purified on calmodulin-Sepharose. The proteins were phosphorylated in a final volume of 400 μl of 20 mm Hepes-NaOH, pH 7.4, 130 mm NaCl, 0.05% Triton X-100, 0.5 mg/ml phosphatidylcholine, 10 mm MgCl2, 2 mm EDTA, 1 mm EGTA, 10 μCi of [γ-32P]ATP (3000 Ci/mmol), 1–5 μg of reconstituted protein kinase A (Sigma) for 30–60 min at 37 °C. The phosphorylated proteins were treated with 0.2 m hydroxylamine for 30 min at room temperature and separated by SDS-PAGE. Calpain Digestion of the Ca2 + -ATPases—100 μg of membrane proteins obtained from infected Sf9 cells were incubated with 10 μl of calpain (1.5–2.0 μg) at 25 °C. The medium (0.2 ml final volume) contained 10 mm Tris-HCl, pH 7.5, and 200 μm CaCl2. A zero time point was taken before adding calpain. Aliquots were withdrawn at the times indicated in the legends for the figures, and the reaction was stopped by adding SDS gel loading buffer (17 mm Tris-HCl, pH 8.0, 10% urea, 1.7% SDS, 2% dithiothreitol, 1.7 mm EDTA, and bromphenol blue). Formation of the Phosphorylated Intermediate by the Pumps after Proteolysis with Calpain—25 μg of membrane proteins of Sf9 cells were incubated without or with calpain (0.37–0.5 μg) for 30 min in 50 μl of 20 mm MOPS-KOH, pH 6.8, 100 mm KCl, 100 μm Ca2+, and 100 μm LaCl3. The phosphorylation was started by adding 0.2 μm [γ-32P]ATP (500 Ci/mmol). After 30 s on ice, the reaction was stopped with 600 μl of cold 7% trichloroacetic acid, 10 mm Na2HPO4. The precipitated proteins were divided into two aliquots (20 and 5 μg) and separated by acidic SDS-PAGE (39Sarkadi B. Enyedi A. Földes-Papp Z. Gardos G. J. Biol. Chem. 1986; 261: 9552-9557Abstract Full Text PDF PubMed Google Scholar). The portion of the gel containing the 20-μg aliquots was stained with Coomassie Brilliant Blue, dried, and exposed overnight at –70 °C with an intensifying screen. The other portion of the gel was subjected to Western blot analysis using the 5F10 antibody. Trypsin Digestion of Membrane Protein-expressing PMCA Isoforms— 100 μg of Sf9 cell membrane proteins were resuspended in 200 μl of 20 mm Hepes-KOH, pH 8.0, 100 mm KCl. After taking the zero time aliquot, 2 μg of trypsin (Promega) were added to start the reaction. 40 μl of the digestion mixture were withdrawn at different times, transferred to a tube containing 2 μl of soybean trypsin inhibitor (1 mg/ml), and separated by SDS-PAGE. Characterization of the PMCA1 Pump Overexpressed in Sf9 Cells—Blots of membrane proteins obtained from Sf9 cells infected with recombinant baculoviruses for PMCA1, and with recombinant viruses for PMCA4 (15Heim R. Iwata T. Zvaritch E. Adamo H.P. Rutishauser B. Strehler E.E. Guerini D. Carafoli E. J. Biol. Chem. 1992; 267: 24476-24484Abstract Full Text PDF PubMed Google Scholar) and PMCA2 (18Hilfiker H. Guerini D. Carafoli E. J. Biol. Chem. 1994; 269: 26178-26183Abstract Full Text PDF PubMed Google Scholar) used for comparison, were exposed to polyclonal antibody 2A (13Stauffer T. Guerini D. Car
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