VDAC1 Is a Transplasma Membrane NADH-Ferricyanide Reductase
2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês
10.1074/jbc.m311020200
ISSN1083-351X
AutoresMark A. Baker, Darius J.R. Lane, Jennifer Ly, Vito De Pinto, Alfons Lawen,
Tópico(s)Metabolism and Genetic Disorders
ResumoPorin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1-GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis. Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1-GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis. A number of electron transport systems have been shown to exist within cell membranes. The mitochondrial and endoplasmic reticulum electron transport chains are perhaps two of the best characterized systems in mammalian cells. However, little interest has been shown for the characterization of the members of the plasma membrane redox system (1Ly J.D. Lawen A. Redox. Rep. 2003; 8: 3-21Crossref PubMed Scopus (69) Google Scholar). Evidence to support the existence of a plasma membrane NADH-oxidoreductase (PMOR) 1The abbreviations used are: PMORplasma membrane NADH-oxidoreductaseVDACvoltage-dependent anion-selective channelGFPgreen fluorescent proteinMOPS4-morpholinepropanesulfonic acidPBSphosphate-buffered salineHTPhydroxylapatiteFACSfluorescence-activated cell sortingTBSTris-buffered salineFITCfluorescein isothiocyanateMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight.1The abbreviations used are: PMORplasma membrane NADH-oxidoreductaseVDACvoltage-dependent anion-selective channelGFPgreen fluorescent proteinMOPS4-morpholinepropanesulfonic acidPBSphosphate-buffered salineHTPhydroxylapatiteFACSfluorescence-activated cell sortingTBSTris-buffered salineFITCfluorescein isothiocyanateMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight. or redox system arises from reduction of two artificial impermeable electron acceptors (potassium ferricyanide and dichlorophenolindophenol) by whole cells (2Larm J.A. Vaillant F. Linnane A.W. Lawen A. J. Biol. Chem. 1994; 269: 30097-30100Abstract Full Text PDF PubMed Google Scholar). Reduction of ferricyanide causes concomitant oxidation of cytosolic NADH (3Navas P. Sun I.L. Morré D.J. Crane F.L. Biochem. Biophys. Res. Commun. 1986; 135: 110-115Crossref PubMed Scopus (72) Google Scholar), suggesting the presence of an NADH-ferricyanide reductase in the plasma membrane. The importance of this plasma membrane redox system is exemplified by the fact that, during generation of human Namalwa ρ0 cells (which lack a functional mitochondrial respiratory chain), the rate of whole cell ferricyanide reduction increases ∼4-fold (2Larm J.A. Vaillant F. Linnane A.W. Lawen A. J. Biol. Chem. 1994; 269: 30097-30100Abstract Full Text PDF PubMed Google Scholar). Importantly, these cells remain viable when ferricyanide is added to the media (4Martinus R.D. Linnane A.W. Nagley P. Biochem. Mol. Biol. Int. 1993; 31: 997-1005PubMed Google Scholar), suggesting a major role for the plasma membrane NADH-ferricyanide reductase in cell signaling and survival (5Baker M.A. Lawen A. Antioxid. Redox. Signal. 2000; 2: 197-212Crossref PubMed Scopus (52) Google Scholar). However, little headway has been made in the identification and characterization of the proteins involved in plasma membrane redox function. Two redox enzymes have been suggested to be involved in plasma membrane electron transport from NADH to ferricyanide; however, both NADH-cytochrome b5 reductase flavoprotein (6Enoch H.G. Catala A. Strittmatter P. J. Biol. Chem. 1976; 251: 5095-5103Abstract Full Text PDF PubMed Google Scholar) and glyceraldehyde-3-phosphate dehydrogenase isozyme (7Bulliard C. Zurbriggen R. Tornare J. Faty M. Dastoor Z. Dreyer J.L. Biochem. J. 1997; 324: 555-563Crossref PubMed Scopus (50) Google Scholar) cannot account for transmembraneous electron transfer, because both enzymes are only associated with the inner side of the plasma membrane (8Grebing C. Crane F.L. Löw H. Hall K. J. Bioenerg. Biomembr. 1984; 16: 517-533Crossref PubMed Scopus (47) Google Scholar). plasma membrane NADH-oxidoreductase voltage-dependent anion-selective channel green fluorescent protein 4-morpholinepropanesulfonic acid phosphate-buffered saline hydroxylapatite fluorescence-activated cell sorting Tris-buffered saline fluorescein isothiocyanate matrix-assisted laser desorption ionization time-of-flight. plasma membrane NADH-oxidoreductase voltage-dependent anion-selective channel green fluorescent protein 4-morpholinepropanesulfonic acid phosphate-buffered saline hydroxylapatite fluorescence-activated cell sorting Tris-buffered saline fluorescein isothiocyanate matrix-assisted laser desorption ionization time-of-flight. VDAC1 is a small, 30-35-kDa protein, predominantly found in the outer membrane of mitochondria (9Sorgato M.C. Moran O. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 127-171Crossref PubMed Scopus (115) Google Scholar), where it constitutes the major protein (10Krimmer T. Rapaport D. Ryan M.T. Meisinger C. Kassenbrock C.K. Blachly-Dyson E. Forte M. Douglas M.G. Neupert W. Nargang F.E. Pfanner N. J. Cell Biol. 2001; 152: 289-300Crossref PubMed Scopus (136) Google Scholar). Predicted structural studies of human VDAC1 suggest the protein is a β-barrel, consisting of either 13 (11Song J. Colombini M. J. Bioenerg. Biomembr. 1996; 28: 153-161Crossref PubMed Scopus (46) Google Scholar) or 16 (12Casadio R. Jacoboni I. Messina A. De Pinto V. FEBS Lett. 2002; 520: 1-7Crossref PubMed Scopus (84) Google Scholar) membrane-spanning strands. VDAC1 has been shown to co-immunoprecipitate with the anti-apoptotic protein Bcl-2 and has been suggested to be involved in forming the pore that releases cytochrome c during apoptosis (13Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1910) Google Scholar). However, a recent report showed Bax-induced pore formation in yeast lacking VDAC (14Pavlov E.V. Priault M. Pietkiewicz D. Cheng E.H.-Y. Antonsson B. Manon S. Korsmeyer S.J. Mannella C.A. Kinnally K.W. J. Cell Biol. 2001; 155: 725-732Crossref PubMed Scopus (237) Google Scholar). Although VDAC1 is predominantly expressed in the outer mitochondrial membrane, recently, it has been demonstrated by several groups that VDAC1 can also be expressed in the plasma membrane (15Thinnes F.P. J. Bioenerg. Biomembr. 1992; 24: 71-75Crossref PubMed Scopus (72) Google Scholar, 16Dermietzel R. Hwang T.K. Buettner R. Hofer A. Dotzler E. Kremer M. Deutzmann R. Thinnes F.P. Fishman G.I. Spray D.C. Siemen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 499-503Crossref PubMed Scopus (142) Google Scholar, 17Thinnes F.P. Hellmann K.P. Hellmann T. Merker R. Schwarzer C. Walter G. Götz H. Hilschmann N. Mol. Genet. Metab. 2000; 69: 240-251Crossref PubMed Scopus (19) Google Scholar, 18Báthori G. Parolini I. Tombola F. Szabó I. Messina A. Oliva M. De Pinto V. Lisanti M. Sargiacomo M. Zoratti M. J. Biol. Chem. 1999; 274: 29607-29612Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 19Buettner R. Papoutsoglou G. Scemes E. Spray D.C. Dermietzel R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3201-3206Crossref PubMed Scopus (122) Google Scholar, 20Gonzalez-Gronow M. Kalfa T. Johnson C.E. Gawdi G. Pizzo S.V. J. Biol. Chem. 2003; 278: 27312-27318Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 21Bahamonde M.I. Fernandez-Fernandez J.M. Guix F.X. Vazquez E. Valverde M.A. J. Biol. Chem. 2003; 278: 33284-33289Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Consistent with these observations, several patch clamp techniques have documented the presence of plasma membrane channels with physiological properties similar to VDAC1 (22Blatz A.L. Magleby K.L. Biophys. J. 1983; 43: 237-241Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 23Jalonen T. Johansson S. Holopainen I. Oja S.S. Arhem P. Acta Physiol. Scand. 1989; 136: 611-612Crossref PubMed Scopus (24) Google Scholar). Furthermore, immunocytochemical studies using antibodies raised against the N terminus of VDAC1 block a high conductance anion channel found in the plasma membrane of bovine astrocytes (16Dermietzel R. Hwang T.K. Buettner R. Hofer A. Dotzler E. Kremer M. Deutzmann R. Thinnes F.P. Fishman G.I. Spray D.C. Siemen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 499-503Crossref PubMed Scopus (142) Google Scholar). These antibodies also appear to specifically label the plasma membrane in human B-lymphocytes. In vivo, VDAC1 harbors two alternative first exons, which leads to the expression of VDAC1 isoforms with different N-terminal sequences. One of these isoforms carries a leader sequence that directs the protein via the Golgi apparatus into the secretory pathway (19Buettner R. Papoutsoglou G. Scemes E. Spray D.C. Dermietzel R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3201-3206Crossref PubMed Scopus (122) Google Scholar). The other VDAC1 isoform, devoid of any cleavable pre-sequence, is targeted to the mitochondria, where it is efficiently inserted into the outer membrane (19Buettner R. Papoutsoglou G. Scemes E. Spray D.C. Dermietzel R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3201-3206Crossref PubMed Scopus (122) Google Scholar). The presence of VDAC1 in the plasma membrane leads to the question of its role there. In this report we establish that VDAC1 at the level of the plasma membrane can function as a redox enzyme, capable of reducing extracellular ferricyanide in the presence of intracellular NADH. Cell Culture—Namalwa cells were cultured as previously described (2Larm J.A. Vaillant F. Linnane A.W. Lawen A. J. Biol. Chem. 1994; 269: 30097-30100Abstract Full Text PDF PubMed Google Scholar). COS7 cells were detached by pretreating with cell dissociation solution (Sigma, Castle Hill, New South Wales, Australia) and incubated for 5 min prior to gentle agitation of the flasks. Cell concentrations were determined as previously described (2Larm J.A. Vaillant F. Linnane A.W. Lawen A. J. Biol. Chem. 1994; 269: 30097-30100Abstract Full Text PDF PubMed Google Scholar). Preparation of Plasma Membrane-enriched Fractions from Namalwa Cells—Unless indicated otherwise, all steps were performed at 4 °C. Approximately 6-8 × 108 cells were pelleted and washed twice with ice-cold PBS. Following the second wash, the supernatant was removed and the cells were adjusted to 2 × 108 cells/ml using ice cold lysis buffer consisting of 210 mm mannitol, 70 mm sucrose, 5 mm Hepes, pH 7.2, and 1 mm EGTA. Digitonin (1.4 mg/109 cells) was added, and the cells were lysed on ice using 72 strokes in a glass homogenizer (Sigma). After lysis, the homogenate was centrifuged at 2000 × g for 5 min. The supernatant was removed and kept at -20 °C for later analysis. The pellet was washed twice with buffer A (50 mm Tris-HCl, pH 8.0) and then resuspended in buffer A adjusted to 4 × 108 cells/ml. The resuspended pellet was then loaded onto a two-phase system. The two-phase system contained 6.4% (w/w) dextran T-500, 6.4% (w/w) poly(ethylene glycol) 3350, and 0.1 m sucrose. The homogenate (2 g) was added to the two-phase system, and the weight of the system was brought to 16 g with distilled water. The tubes were inverted vigorously 40 times in the cold (4 °C). The phases were separated by centrifugation at 150 × g in an IEC Centra-7R refrigerated centrifuge. The upper phase was carefully withdrawn, together with the interface, and transferred into 10-ml centrifuge tubes. The sample was then centrifuged at 5000 × g for 30 min. The fluffy pellet resulting from the spin constituted the plasma membrane-enriched fraction. Purity of plasma membrane preparations was determined by subcellular marker enzyme assays (Refs. 2Larm J.A. Vaillant F. Linnane A.W. Lawen A. J. Biol. Chem. 1994; 269: 30097-30100Abstract Full Text PDF PubMed Google Scholar and 24Vaillant F. Larm J.A. McMullen G.L. Wolvetang E.J. Lawen A. J. Bioenerg. Biomembr. 1996; 28: 531-540Crossref PubMed Scopus (46) Google Scholar; see following section). Alternatively, after the second wash the cells were resuspended in ice-cold 1 mm NaHCO3, 0.2 mm EDTA (dipotassium salt) at an approximate ratio of 1 ml/108 cells to osmotically swell the cells (25Morré D.M. Morré D.J. J. Chromatogr. B Biomed. Sci. Appl. 2000; 743: 377-387Crossref PubMed Scopus (47) Google Scholar). The resulting cell suspension was then incubated at 4 °C with gentle shaking for 40 min in a 10-ml disposable centrifuge tube. Following incubation, cells were mechanically disrupted using 110 strokes in a 7-ml all-glass Kontes Dounce homogenizer (Sigma). The extent of cell rupture was monitored by light microscopy and trypan blue exclusion, and was found to be consistently ≥85%. The crude homogenate was centrifuged at 500 × g for 10 min in Sorvall Evolution centrifuge (SS34 rotor) for 10 min, 4 °C, to remove nuclei and unruptured cells. The resulting supernatant was then centrifuged at 25,000 × g for 30 min as above at 4 °C. The supernatant was extracted with a plastic transfer pipette and stored at 4 °C for later analysis, whereas the pellet was thoroughly resuspended in 50 mm Tris-HCl, pH 8.0 (buffer A), to a net mass of 8 g. This concentrated suspension was divided into four 2-g aliquots, each of which was then individually loaded onto one of four 16-g aqueous two-phase systems constructed on a weight basis. Each system had a final composition of 6.6% poly(ethylene glycol) 3350 (Sigma) + 6.6% Dextran-500 (Sigma) + 1.9 g of 1 m sucrose solution + 0.4 g of 200 mm K2HPO4 (pH 7.4) + 2 g of resuspended membranes in buffer A. In each case the total mass of the system was brought to 16 g with distilled Milli-Q H2O. Each two-phase system was constructed in 15-ml glass Corex centrifuge tubes, sealed with Parafilm, and mixed vigorously with at least 70 inversions at 4 °C. Following the separation of the phases by centrifugation at 1000 × g for 5 min as above at 4 °C, the upper phase of each system was extracted (taking care not to disturb the material collected at the interface) and repartitioned against a fresh lower phase of a second set of two-phase systems constructed in parallel, except containing 2 g of buffer A in place of the crude membrane suspension. The material from the second set of upper phases was collected, diluted 1:5 in buffer A, pelleted at 25,000 × g for 30 min (as above) at 4 °C, and then resuspended in fresh buffer A. All preparations used were highly enriched in plasma membranes and showed minimal mitochondrial contaminations (Table I).Table IEnrichment of plasma membranes by two-phase separationEnzyme (organelle)Crude extractSpecific activityAfter two-phase separationSpecific activityEnrichment factorμkatnkat/mgμkatnkat/mgSuccinate dehydrogenase (mitochondria)44.61557.6 ± 46.11.21121.2 ± 18.20.22Alkaline phosphatase (plasma membrane)11.64145.5 ± 26.54.16415.8 ± 36.42.86Succinate dehydrogenase: alkaline phosphatase ratio1.000.08 Open table in a new tab Enzyme Assays—All enzyme assays and subcellular enzyme markers have been used as described elsewhere (24Vaillant F. Larm J.A. McMullen G.L. Wolvetang E.J. Lawen A. J. Bioenerg. Biomembr. 1996; 28: 531-540Crossref PubMed Scopus (46) Google Scholar), with a slight modification. Alkaline phosphatase was assayed for as the plasma membrane marker, using an adaptation of a previously described method (26Pekarthy J.M. Short J. Lansing A.I. Lieberman I. J. Biol. Chem. 1972; 247: 1767-1774Abstract Full Text PDF PubMed Google Scholar). Briefly, 50 μg of protein were added to 1 ml of 50 mm Tris, pH 10.5, 2 mm MgCl2. The reaction was started with the addition of 4 mmp-nitrophenyl phosphate. The increase in absorbance at 420 nm was measured at 37 °C for 1 h. Rates of activity are expressed in nanokatals/mg of protein using the extinction coefficient of p-nitrophenol in alkaline solution of 9620 m-1 cm-1. Succinate dehydrogenase activity was determined via the spectrophotometric measurement of the initial linear rates of the reduction of the artificial electron acceptor, dichlorophenolindophenol, at 600 nm (decrease in absorbance) in the presence of succinate. Each 1 ml of reaction medium contained 50 mm dipotassium phosphate, pH 7.4, 40 μm dichlorophenolindophenol, and with the reaction being started by the addition of 20 mm sodium succinate. Rates of activity are expressed in nanokatals/mg of protein using the extinction coefficient of dichlorophenolindophenol at physiological pH of 5500 m-1 cm-1. NADH-ferricyanide reductase activity was measured in a Beckman DU 7500 spectrophotometer. Reduction of ferricyanide to ferrocyanide was ascertained in 1-ml reactions containing 50 mm Tris-HCl, pH 8.0, 3% (v/v) Triton X-100, 750 μl of fraction, 250 μm β-NADH. The reaction was initiated by the addition of 250 μm potassium ferricyanide, following a 5-min pre-incubation period at 37 °C. Rates were measured at either 420 or 340 nm at 37 °C for 500 s, with the linear rate of decrease in absorbance being determined from the second 250-s duration. Actual rates of enzymic activity were determined using an extinction coefficient for ferricyanide (420 nm) of 1000 m-1 cm-1 or 6220 m-1 cm-1 for NADH (340 nm). Determination of Protein Concentrations—Protein concentrations were determined by using the bicinchoninic acid assay (27Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18501) Google Scholar). Purification of the NADH-Ferricyanide Reductase—The chromatographic steps of DEAE-Sephacel and Blue Sepharose affinity column were carried out using stepwise gradient elution, respectively. Solubilized proteins from the enriched plasma membrane pellet were applied to the DEAE-Sephacel column (5-ml bed volume) pre-equilibrated with buffer B (buffer A + 0.05% (v/v) Triton X-100). The column was washed three times with 20 ml of buffer B. The material bound onto the column was eluted by 10 ml of buffer B containing 50 mm NaCl. Active fractions eluting from the column were pooled and applied to the Blue Sepharose affinity column (5-ml bed volume), which had been pre-equilibrated with buffer B. The column was washed twice with 10 ml of buffer B, then eluted with a linear 10-ml gradient (0-10 μm) of NADH in buffer B. One-ml fractions were collected and assayed for NADH-ferricyanide reductase activity. Solubilization of Plasma Membrane Proteins—The plasma membrane-enriched fraction was centrifuged at 25,000 × g as above for 30 min at 4 °C. The resulting creamy white pellet was resuspended in 50 mm Tris-HCl, pH 8.0, 3% (v/v) Triton-X-100 to an approximate concentration of 5 mg/ml and stirred for 1 h at 4 °C. The solubilized fraction was obtained by pelleting the insoluble material at 100,000 × g for 40 min in a Beckman TLX optima ultracentrifuge (TLA-100.3 rotor) at 4 °C. Purification of VDAC1—VDAC1 was purified according to Ref. 28De Pinto V. Prezioso G. Palmieri F. Biochim. Biophys. Acta. 1987; 905: 499-502Crossref PubMed Scopus (86) Google Scholar with a slight modification from either Namalwa cell plasma membrane or rat liver mitochondria preparations. For the preparation of mitochondria, Wistar rats were decapitated, and their livers excised and immediately washed twice in lysis buffer. Following the second wash, the livers were diced into 2-mm3 pieces and diluted 1:3 (w/w) with ice-cold lysis buffer (as outlined for Namalwa cells). The diced livers were then homogenized in a 50-ml glass Teflon homogenizer (Sigma) at 1500 rpm, for five strokes. The homogenate was centrifuged at 600 × g for 5 min to remove nuclei and unbroken cells. The supernatant was decanted, and a second centrifugation was performed at 600 × g for 5 min to remove any contaminating nuclei. Following the second spin, the supernatant was carefully aspirated so as not to disturb the pellet. The supernatant was then centrifuged at 30,000 × g for 30 min to pellet the mitochondria. The supernatant was removed, and the pellet was resuspended in lysis buffer 2 (225 mm mannitol, 25 mm sucrose, 2 mm MOPS, pH 7.4, 1 mm EGTA). A second centrifugation was performed at 30,000 × g for 30 min to wash the mitochondria. Following the second spin, the mitochondrial pellet was taken and diluted to 5 mg/ml in buffer A. After incubating in buffer A for 1 h, the sample was centrifuged at 30,000 × g for 30 min and the pellet, consisting mainly of mitochondrial membranes, was used further. The membranes were then solubilized in buffer A + 5% (v/v) Triton X-100. Following constant stirring for 1 h, the sample was centrifuged at 100,000 × g for 30 min, and the supernatant was taken. The supernatant was then applied onto a DEAE column, which had been pre-equilibrated with buffer B. The flow through was taken and applied to a dry 2:1 (w/w) hydroxylapatite:celite (HTP:celite) column. The flow through from the HTP:celite column was collected, which consisted of purified VDAC1. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)—SDS-PAGE and silver staining were performed as described elsewhere (29Nesterenko M.V. Tilley M. Upton S.J. J. Biochem. Biophys. Methods. 1994; 28: 239-242Crossref PubMed Scopus (309) Google Scholar, 30Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3734) Google Scholar). Western Blotting—Following transfer of proteins to nitrocellulose, the membrane was blocked in 5% (w/v) skim milk powder (Bio-Rad, Regents Park, New South Wales, Australia) in TBS (20 mm Tris-HCl, pH 7.6, 137 mm NaCl) for 1 h at 37 °C. Following three washes with washing buffer (0.05% (v/v) Tween 20 in TBS) for 20 min, the membrane was hybridized overnight at 4 °C with primary antibody at 1/1000 dilution. The membrane was then washed three times with TBS and incubated with a dilution of 1/1250 horseradish peroxidase-labeled secondary antibody (Amersham Biosciences, Castle Hill, New South Wales, Australia), for 1 h at 37 °C. Following three washes, ECL was used as previously described (31Grubb D.R. Ly J.D. Vaillant F. Johnson K.L. Lawen A. Oncogene. 2001; 20: 4085-4094Crossref PubMed Scopus (39) Google Scholar). Immunoprecipitation—Approximately 1-2 μg of rabbit anti-porin IgG antibody was added to the 10-ml purified fraction and allowed to agitate at 4 °C overnight. Following this, 50 μl of a 50% protein G bead slurry was added and the mixture was agitated for another 2 h. The sample was spun at 10,000 × g for 15 s, and the supernatant containing the unbound material was removed and kept for later analyses. The protein G-antibody-antigen complex was further washed three times with buffer A + 0.5 m NaCl and once with buffer B. The immunopellet was then resuspended in 1 ml of buffer B. Precipitation of Protein from Detergents—Proteins were precipitated according to the method of Wessel and Flügge (32Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3141) Google Scholar). FACS Analysis—Approximately 1 × 107 cells were harvested, pelleted (800 × g, 5 min) and washed twice in 1% fetal calf serum in PBS. The cells were suspended in 4 ml of PBS and incubated for 40 min with goat anti-mouse IgG Fc fragment (Bethyl Laboratories, Montgomery, TX). The cells were then washed and divided equally into four tubes. Tubes 2 and 4 were incubated for 40 min at 4 °C with anti-VDAC1 antibody at 1/1000 dilution. The cells were pelleted (800 × g, 5 min) and the supernatant aspirated. The pellet was then washed twice with 1% fetal calf serum in PBS. Following the second wash, tubes 3 and 4 were incubated with FITC-conjugated anti-mouse antibody for 40 min. FITC histograms were obtained by analyzing 15,000 cells with the MODfit program (Becton-Dickinson, Wheelers Hill, Victoria, Australia). The amount of viable cells was simultaneously obtained by staining intracellular DNA with propidium iodide, and the fluorescence was measured using the same program. Growth and Maintenance of Escherichia coli—Transformed E. coli bearing the recombinant plasmids were grown in liquid Luria Broth supplemented with kanamycin. Cells were incubated at 37 °C overnight for growth. DNA Plasmid Amplification—pl-VDAC1-GFP was a kind gift from Prof. Dermietzel (19Buettner R. Papoutsoglou G. Scemes E. Spray D.C. Dermietzel R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3201-3206Crossref PubMed Scopus (122) Google Scholar). Approximately 1 μg of DNA was taken and used to inoculate 107 viable E. coli cells. The cells were grown on solid Luria Broth medium with kanamycin to select for transformed cells. Plasmid DNA was purified according to the instructions from the manufacturer (Clontech, East Meadow Circle, PA). As a control, the commercially available pEGFP vector was used (Clontech). Electroporation of COS7 Cells—Cells to be electroporated were grown logarithmically (70% confluent). Each transfection required 2 × 106 cells to yield a reasonable number of transfectants. One ml of trypsin was added for 5 min per 10-cm culture dish, and the cells were collected and harvested by centrifuging for 5 min at 1200 rpm and then resuspending in 400 ml of ice-cold medium and placed in a 0.8-cm cuvette (Bio-Rad). The cells were then electroporated at 200 V in a Gene-Pulser electroporator (Bio-Rad) with the capacitor set at 960 microfarads. The time constants obtained ranged from 15 to 45 ms. The cells were then transferred to growth medium and allowed to grow for 48 h. Purification of a Plasma Membrane NADH-Ferricyanide Reductase—Human Namalwa cells were harvested and washed, and the plasma membrane extracted to a purity of >90% as described under “Materials and Methods.” Triton X-100 (3% (v/v)) was the best detergent for solubilizing maximal enzyme activity (data not shown). To further purify the enzyme, the solubilized membrane proteins were applied onto a DEAE-Sephacel column. Active enzyme was eluted with 50-100 mm NaCl (data not shown). Those fractions with NADH-ferricyanide reductase activity eluting from the DEAE-Sephacel column were pooled and applied to a Blue Sepharose, a 2′,5′-ADP affinity, or an HTP:celite (1:2) column. Following washing with 5 column volumes, elution of the enzyme from the former two columns was achieved with a 10-ml gradient of 0-50 μm NADH. One-ml fractions were collected and assayed for enzyme activity. Those fractions with NADH-ferricyanide reductase activity were then precipitated and run in an SDS-PAGE. A 35-kDa band correlated with enzyme activity, suggesting it might be the NADH-ferricyanide reductase (data not shown). Both purification protocols yielded more than 200-fold enrichment from the plasma membrane enriched fractions of the enzyme activity (Table II) and the purification of a major protein band of ∼35 kDa (Fig. 1, A and B).Table IIPurification of a plasma membrane NADH-ferricyanide reductaseStepProteinVolumeEnzyme activitySpecific activityPurification factormgmlfkatfkat/mgPlasma membrane252527751111Solubilization10252775277.52.5DEAE0.163364.52278.120.6a. Blue Sepharose0.01125025,000225b. 2′, 5′-ADP0.001127.727,700249.5 Open table in a new tab The 35-kDa band was excised from the gel and subjected to a MALDI-TOF analysis. Four resulting peptides with their predicted amino acid sequences were used to search the BLAST and TREMBL data bases for sequence homology to known proteins. This search revealed the 35-kDa band to be identical to human VDAC1 (Fig. 1C). VDAC1 Is an NADH-Ferricyanide Reductase—Co-elution of VDAC1 with enzyme activity from various columns suggested that VDAC1 is an NADH-ferricyanide reductase. However, because the eluants were not homogeneous, further evidence was sought to confirm the function of VDAC1 as a re
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