Expression in Escherichia coli, Functional Characterization, and Tissue Distribution of Isoforms A and B of the Phosphate Carrier from Bovine Mitochondria
1998; Elsevier BV; Volume: 273; Issue: 35 Linguagem: Inglês
10.1074/jbc.273.35.22782
ISSN1083-351X
AutoresGiuseppe Fiermonte, Vincenza Dolce, Ferdinando Palmieri,
Tópico(s)ATP Synthase and ATPases Research
ResumoThe two isoforms of the mammalian mitochondrial phosphate carrier (PiC), A and B, differing in the sequence near the N terminus, arise from alternative splicing of a primary transcript of the PiC gene (Dolce, V., Iacobazzi, V., Palmieri, F., and Walker, J. E. (1994) J. Biol. Chem. 269, 10451–10460). To date, the PiC isoforms A and B have not been studied at the protein level. To explore the tissue-distribution and the potential functional differences between the two isoforms, polyclonal site-directed antibodies specific for PiC-A and PiC-B were raised, and the two bovine isoforms were obtained by expression in Escherichia coliand reconstituted into phospholipid vesicles. Western blot analysis demonstrated that isoform A is present in high amounts in heart, skeletal muscle, and diaphragm mitochondria, whereas isoform B is present in the mitochondria of all tissues examined. Heart and liver bovine mitochondria contained 69 and 0 pmol of PiC-A/mg of protein, and 10 and 8 pmol of PiC-B/mg of protein, respectively. In the reconstituted system the pure recombinant isoforms A and B both catalyzed the two known modes of transport (Pi/Pi antiport and Pi/H+ symport) and exhibited similar properties of substrate specificity and inhibitor sensitivity. However, they strongly differed in their kinetic parameters. The transport affinities of isoform B for phosphate and arsenate were found to be 3-fold lower than those of isoform A. Furthermore, the maximum transport rate of isoform B is about 3-fold higher than that of isoform A. These results support the hypothesis that the sequence divergence between PiC-A and PiC-B may have functional significance in determining the affinity and the translocation rate of the substrate through the PiC molecule. The two isoforms of the mammalian mitochondrial phosphate carrier (PiC), A and B, differing in the sequence near the N terminus, arise from alternative splicing of a primary transcript of the PiC gene (Dolce, V., Iacobazzi, V., Palmieri, F., and Walker, J. E. (1994) J. Biol. Chem. 269, 10451–10460). To date, the PiC isoforms A and B have not been studied at the protein level. To explore the tissue-distribution and the potential functional differences between the two isoforms, polyclonal site-directed antibodies specific for PiC-A and PiC-B were raised, and the two bovine isoforms were obtained by expression in Escherichia coliand reconstituted into phospholipid vesicles. Western blot analysis demonstrated that isoform A is present in high amounts in heart, skeletal muscle, and diaphragm mitochondria, whereas isoform B is present in the mitochondria of all tissues examined. Heart and liver bovine mitochondria contained 69 and 0 pmol of PiC-A/mg of protein, and 10 and 8 pmol of PiC-B/mg of protein, respectively. In the reconstituted system the pure recombinant isoforms A and B both catalyzed the two known modes of transport (Pi/Pi antiport and Pi/H+ symport) and exhibited similar properties of substrate specificity and inhibitor sensitivity. However, they strongly differed in their kinetic parameters. The transport affinities of isoform B for phosphate and arsenate were found to be 3-fold lower than those of isoform A. Furthermore, the maximum transport rate of isoform B is about 3-fold higher than that of isoform A. These results support the hypothesis that the sequence divergence between PiC-A and PiC-B may have functional significance in determining the affinity and the translocation rate of the substrate through the PiC molecule. The transport of inorganic phosphate across the inner mitochondrial membrane into the matrix compartment is essential for the oxidative phosphorylation of ADP to ATP. This transport is catalyzed by the phosphate carrier (PiC) 1The abbreviations used are: PiCphosphate carrierPiC-Aisoform A of the phosphate carrierPiC-Bisoform B of the phosphate carrierPAGEpolyacrylamide gel electrophoresisPipes1,4-piperazinediethanesulfonic acidPBSphosphate-buffered saline.which has been purified from different sources and reconstituted into liposomes in an active form (1Bisaccia F. Palmieri F. Biochim. Biophys. Acta. 1984; 766: 386-394Crossref PubMed Scopus (68) Google Scholar, 2Kolbe H.V.J. Costello D. Wong A. Lu R.C. Wohlrab H. J. Biol. Chem. 1984; 259: 9115-9120Abstract Full Text PDF PubMed Google Scholar, 3Kaplan R.S. Pratt R.D. Pedersen P.L. J. Biol. Chem. 1986; 261: 12767-12773Abstract Full Text PDF PubMed Google Scholar, 4Guerin B. Bukusoglu C. Rakotomanana F. Wohlrab H. J. Biol. Chem. 1990; 265: 19736-19741Abstract Full Text PDF PubMed Google Scholar). In the reconstituted system the native bovine PiC protein catalyzes the Pi/H+symport as well as the Pi/Pi exchange, which functions by a sequential mechanism (5Stappen R. Krämer R. J. Biol. Chem. 1994; 269: 11240-11246Abstract Full Text PDF PubMed Google Scholar). The primary structure of the mature PiC is made up of three tandemly related domains about 100 amino acids in length (6Runswick M.J. Powell S.J. Nyren P. Walker J.E. EMBO J. 1987; 6: 1367-1373Crossref PubMed Scopus (182) Google Scholar). These repetitive elements are related to those found in the other well characterized members of the mitochondrial carrier family (see Refs. 7Walker J.E. Runswick M.J. J. Bioenerg. Biomembr. 1993; 25: 435-446Crossref PubMed Scopus (196) Google Scholar, 8Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar, 9Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 10Palmieri F. van Ommen B. Papa S. Tager J.M. Guerrieri F. Frontiers of Cellular Bioenergetics: Molecular Biology, Biochemistry and Physiopathology. Plenum, New York London1998Google Scholar for reviews) (11Palmieri L. De Marco V. Iacobazzi V. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1997; 410: 447-451Crossref PubMed Scopus (80) Google Scholar, 12Palmieri L. Lasorsa F.M. De Palma A. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1997; 417: 114-118Crossref PubMed Scopus (121) Google Scholar). They are also found in a number of other proteins of known sequence but of unknown function, which therefore belong to the same protein superfamily (7Walker J.E. Runswick M.J. J. Bioenerg. Biomembr. 1993; 25: 435-446Crossref PubMed Scopus (196) Google Scholar, 8Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar, 9Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 10Palmieri F. van Ommen B. Papa S. Tager J.M. Guerrieri F. Frontiers of Cellular Bioenergetics: Molecular Biology, Biochemistry and Physiopathology. Plenum, New York London1998Google Scholar). By examination of the transmembrane topography of the PiC in the inner mitochondrial membrane, it has been proposed that both the N- and C-terminal regions of the PiC protrude toward the cytosol and that the polypeptide chain spans the membrane six times (13Palmieri F. Bisaccia F. Capobianco L. Dolce V. Fiermonte G. Iacobazzi V. Zara V. J. Bioenerg. Biomembr. 1993; 25: 493-501Crossref PubMed Scopus (43) Google Scholar). Only one gene for the PiC has been detected in man and cow (14Dolce V. Iacobazzi V. Palmieri F. Walker J.E. J. Biol. Chem. 1994; 269: 10451-10460Abstract Full Text PDF PubMed Google Scholar), of which the human one has been localized to chromosome 12q23 (15Marsh S. Carter N.P. Dolce V. Iacobazzi V. Palmieri F. Genomics. 1995; 29: 814-815Crossref PubMed Scopus (7) Google Scholar). In both man and cow two closely related exons named IIIA and IIIB appear to be alternatively spliced (14Dolce V. Iacobazzi V. Palmieri F. Walker J.E. J. Biol. Chem. 1994; 269: 10451-10460Abstract Full Text PDF PubMed Google Scholar). The alternative splicing mechanism affects amino acids 4–45 of the mature PiC. More recently, by Northern blot analysis, isoform A has been found to be limited to heart and skeletal muscle, whereas isoform B is expressed in all tissues examined (16Dolce V. Fiermonte G. Palmieri F. FEBS Lett. 1996; 399: 95-98Crossref PubMed Scopus (33) Google Scholar). phosphate carrier isoform A of the phosphate carrier isoform B of the phosphate carrier polyacrylamide gel electrophoresis 1,4-piperazinediethanesulfonic acid phosphate-buffered saline. The biochemical characterization of potential functional differences between isoforms A and B of the mitochondrial PiC has so far been precluded by the lack of a purification procedure for separating the two isoforms. Similarly, tissue distribution studies of PiC-A and PiC-B at the protein level have been prevented by the lack of specific antibodies. We therefore raised antibodies against PiC isoforms A and B and produced two recombinant proteins that have either the PiC-A or the PiC-B sequence. In this study we demonstrate that the two PiC isoforms are tissue-specific, and that the two recombinant isoforms exhibit different transport affinities (Km) and specific activities (Vmax) after purification and reconstitution into liposomes. To our knowledge, this is the first time that a functional comparison of mitochondrial carrier isoforms has been performed. The coding regions for the mature bovine PiC isoforms A and B were amplified from 10 ng of bovine heart cDNA (isoform A) and bovine liver cDNA (isoform B), respectively, by 30 cycles of polymerase chain reactions. The forward and reverse oligonucleotide primers employed in these reactions corresponded to nucleotides 444–453 linked to 1770–1781 (forward primer A), 444–453 linked to 2051–2062 (forward primer B), and 5955–5978 (reverse primers A and B) of the bovine PiC gene sequence (14Dolce V. Iacobazzi V. Palmieri F. Walker J.E. J. Biol. Chem. 1994; 269: 10451-10460Abstract Full Text PDF PubMed Google Scholar). The forward and reverse primers carried NdeI and XhoI restriction sites as linkers, respectively. The absence of the stop codon in the reverse primer sequences led to the expression of the two PiC isoforms with an extra leucine and glutamic acid, encoded by the XhoI restriction site sequence, plus a tail of six histidines at their C terminus. The reaction products were cloned in the expression vector pET21b, and their sequences were verified by the modified dideoxy chain termination method (17Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52668) Google Scholar). The expression of PiC-A and PiC-B as inclusion bodies in the bacterial cytosol was accomplished in Escherichia coliBL21(DE3), as described first for the bovine oxoglutarate carrier (18Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar) and then for several other mitochondrial carriers (11Palmieri L. De Marco V. Iacobazzi V. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1997; 410: 447-451Crossref PubMed Scopus (80) Google Scholar, 12Palmieri L. Lasorsa F.M. De Palma A. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1997; 417: 114-118Crossref PubMed Scopus (121) Google Scholar, 19Palmieri L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (106) Google Scholar, 20Wohlrab H. Briggs C. Biochemistry. 1994; 33: 9371-9375Crossref PubMed Scopus (53) Google Scholar, 21Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Control cultures containing the empty pET21b vector were processed in parallel. Purified inclusion bodies (18Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar), suspended in TE buffer (10 mm Tris-HCl and 0.1 mm EDTA, pH 8.0), were solubilized in 1.67% (w/v) N-dodecanoylsarcosine (Sarkosyl) for 5 min at 0 °C. The solution was diluted 20 times with SSP buffer consisting of 0.1% Sarkosyl, 0.5 m NaCl, and 20 mm Pi, pH 8.0, and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was chromatographed on Ni+-nitrilotriacetic acid-agarose affinity column (Quiagen). Unspecifically bound proteins were washed with the SSP buffer supplemented with increasing concentrations of histidine. Pure PiC isoforms A and B were recovered when histidine reached 10 mm. The purified proteins were desalted by a Sephadex G-25 column (PD-10 Pharmacia) and stored at −70 °C. All chromatographic steps were performed at 4 °C. Purified PiC-A and PiC-B isoforms were reconstituted by cyclic removal of the detergent with a hydrophobic column (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar, 23Stappen R. Krämer R. Biochim. Biophys. Acta. 1993; 1149: 40-48Crossref PubMed Scopus (50) Google Scholar). The composition of the initial mixture used for reconstitution was 200 μl of purified PiC isoform (about 400 ng of protein), 100 μl of 10% Triton X-114, 90 μl of 10% phospholipids in the form of sonicated liposomes (1Bisaccia F. Palmieri F. Biochim. Biophys. Acta. 1984; 766: 386-394Crossref PubMed Scopus (68) Google Scholar), 30 mm Pi (except where otherwise indicated), 20 mm Pipes (pH 6.5), 0.63 mg of cardiolipin (Sigma), and water to a final volume of 700 μl. After vortexing, this mixture was recycled 13 times through an Amberlite column (Fluka) (3.2 × 0.5 cm) preequilibrated with a buffer containing 20 mm Pipes (pH 6.5) and the substrate at the same concentration as in the starting mixture. All operations were performed at 4 °C, except the passages through Amberlite, which were carried out at room temperature. The reconstituted Pi/Pi antiport activity was determined by measuring the uptake (forward exchange) or the efflux (backward exchange) of [33P]phosphate in exchange for unlabeled substrate (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). For backward exchange measurements, as well as for measuring the efflux of phosphate as a Pi/H+symport, the proteoliposomes containing internal Pi were prelabeled, immediately after reconstitution, by carrier-mediated exchange equilibration, i.e. by adding carrier-free [33P]phosphate of high specific radioactivity for 20 min. The external substrate was removed from proteoliposomes on a Sephadex G-75 column (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar) in the presence of a reversible inhibitor, 5 μm p-chloromercuribenzenesulfonate (23Stappen R. Krämer R. Biochim. Biophys. Acta. 1993; 1149: 40-48Crossref PubMed Scopus (50) Google Scholar), in order to avoid the efflux of internal substrate by the Pi/H+ symport. Transport was started by adding 5 mm dithioerythritol and labeled Pi at the indicated concentrations (forward exchange), 5 mmdithioerythritol and 30 mm cold Pi (backward exchange), or 5 mm dithioerythritol alone (Pi/H+ symport). In all cases the carrier-mediated transport was terminated by addition of 25 mm pyridoxal 5′-phosphate (23Stappen R. Krämer R. Biochim. Biophys. Acta. 1993; 1149: 40-48Crossref PubMed Scopus (50) Google Scholar). In control samples the inhibitor was added at time 0 according to the inhibitor stop method (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). The assay temperature was 25 °C. All transport measurements were carried out at the same internal and external pH value of 6.5. Finally, the external substrate was removed, and the radioactivity in the liposomes was measured (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). In forward exchange kinetic measurements, the initial transport rate was calculated in millimoles/min/g of protein from the time course of isotope equilibration, as has been published previously (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). In the case of backward exchange and Pi/H+ symport, the decrease in radioactivity inside the liposomes was fitted to the equation α = 100(1 − e −kt ) (where α is the percentage of isotopic equilibration) (24Schroers A. Krämer R. Wohlrab H. J. Biol. Chem. 1997; 272: 10558-10564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The rates were expressed as apparent velocities, i.e. the product ofk and the substrate concentration inside the liposomes, and they are directly proportional to the actual transport rate (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar,24Schroers A. Krämer R. Wohlrab H. J. Biol. Chem. 1997; 272: 10558-10564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The antisera anti-PiC-A and anti-PiC-B were raised against the amino acid sequences 1–10 of the mature bovine PiC-A and 7–16 of the mature bovine PiC-B, respectively. The synthesis of peptides, the coupling to ovalbumin of the first peptide through Tyr-6 or Tyr-10, and of the second one through Cys-7, as well as the generation of the antibodies were carried out as described previously (25Bisaccia F. Capobianco L. Brandolin G. Palmieri F. Biochemistry. 1994; 33: 3705-3713Crossref PubMed Scopus (38) Google Scholar). Samples, solubilized in SDS sample buffer and boiled for 5 min, were separated by SDS-PAGE (as described in Capobianco et al. (26Capobianco L. Bisaccia F. Mazzeo M. Palmieri F. Biochemistry. 1996; 35: 8974-8980Crossref PubMed Scopus (36) Google Scholar)) and electrotransferred to nitrocellulose membranes. The membranes were incubated with the antiserum anti-PiC-A or the antiserum anti-PiC-B (each diluted 1:40,000 in PBS-TM (PBS containing 0.5% (w/v) non-fat dried milk and 0.05% (v/v) Tween-20)) for 2 h, washed three times in PBS-TM for 10 min each, and then incubated for 2 h with the secondary antibody, horseradish peroxidase conjugated anti-rabbit IgG (Pierce) diluted 1:2000 in PBS-TM. The membranes were washed and developed using the ECL system (Amersham Pharmacia Biotech). The films were scanned with an LKB 2202 Ultroscan laser densitometer. To determine the amount of PiC-A and PiC-B in mitochondria, standard calibration curves were constructed using 5–30 ng pure recombinant PiC-A or PiC-B as the standard. Nitrocellulose membranes containing the standards and the mitochondrial samples were simultaneously immunodecorated as described above. Once it was checked that mitochondrial sample loading was within the linear range of the calibration curves, the densitometric signal intensity was used to measure the amount of PiC-A and PiC-B (27Pardridge W.M. Boado R.J. Farrell C.R. J. Biol. Chem. 1990; 265: 18035-18040Abstract Full Text PDF PubMed Google Scholar, 28Dyer J. Barker P.J. Shirazi-Beechey S.P. Biochem. Biophys. Res. Commun. 1997; 230: 624-629Crossref PubMed Scopus (59) Google Scholar). After SDS-PAGE, proteins were either stained with Coomassie Blue dye or transferred to polyvinylidene difluoride membranes, stained with Coomassie Blue dye, and their N-terminal sequences determined with a pulsed liquid protein sequencer (Applied Biosystems 477A). The pure recombinant PiC isoforms were estimated from Coomassie Blue-stained SDS-PAGE gels with an LKB 2202 Ultroscan laser densitometer, using carbonic anhydrase as protein standard. To assay the protein incorporated into liposomes, the vesicles were passed through a Sephadex G-75 column, centrifuged at 300,000 ×g for 30 min and delipidated with organic solvents as described in Capobianco et al. (26Capobianco L. Bisaccia F. Mazzeo M. Palmieri F. Biochemistry. 1996; 35: 8974-8980Crossref PubMed Scopus (36) Google Scholar). Then, the SDS-solubilized protein was determined by comparison with carbonic anhydrase in SDS gels and/or by quantitative immunodecoration (as described above) with the antiserum anti-PiC-A and the antiserum anti-PiC-B. The sequence divergence between isoforms A and B of PiC is confined to the N-terminal region of the PiC protein and is precisely localized between amino acids 4–45 of the mature protein corresponding to exons III A and III B of the PiC gene (Fig.1). The isolation of the cDNA clones encoding PiC isoforms A and B (see “Experimental Procedures”) created the possibility to produce, in bacteria, homogeneous A and B isoforms of PiC, which could be used to explore the functional differences between the two isoforms. To this end, PiC-A and PiC-B isoforms were expressed in E. coli,purified, and refolded into the active form using a pET expression system and the Sarkosyl refolding procedure described under “Experimental Procedures.” After induction with isopropyl-β-d-thiogalactopyranoside, PiC-A and PiC-B accumulated in the bacterial cytosol as inclusion bodies (Fig. 2,A and B, lane 1). The presence of the histidine tail at the C-terminal end of the expressed PiC isoforms allowed their purification by a Ni+-agarose affinity column (Fig. 2, A andB, lane 5). About 2 mg of each isoform were obtained per liter of bacterial culture. The identity of PiC-A and PiC-B was confirmed by the determination of their N-terminal sequences, and by their reaction with specific antisera, as shown in Fig.3.Figure 3Tissue specificity of PiC isoforms A and B explored by Western blot analysis. 150 μg of mitochondria isolated from various bovine tissues and 0.2 μg of purified PiC-A and PiC-B were run on SDS-PAGE. After electroblotting, the nitrocellulose sheets were treated with the anti-PiC-A antiserum (panel A) or the anti-PiC-B antiserum (panel B). Lanes A, recombinant PiC-A; lanes B, recombinant PiC-B; lanes H–K, mitochondria from heart (H), diaphragm (D), skeletal muscle (SM), liver (L), lung (Lu), brain (Br), and kidney (K). The autoradiography films were exposed for 15 s (panel A) or 60 min (panel B).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To prepare antisera specific for PiC-A and PiC-B, we first raised antibodies against three synthetic peptides corresponding to residues 1–10 and 8–17 of PiC-A, and residues 7–16 of PiC-B. Only the antibodies generated by using the latter two peptides were found, at the dilution used in this work, to be highly specific for PiC-A and PiC-B, respectively (Fig. 3, A and B, lanes A and B). We then investigated the presence of the two PiC isoforms in the mitochondria of several bovine tissues by Western blotting. Fig. 3 A shows that the antiserum specific for PiC-A immunodecorated a band of 34 kDa in the lysates of heart, diaphragm, and skeletal muscle, but none in the lysates of liver, lung, brain, and kidney. On the contrary, the antiserum specific for PiC-B (Fig. 3 B) reacted with a band of 34 kDa in the mitochondrial lysates of all the tissues investigated. These results indicate that the PiC-A isoform is present only in muscles, whereas the PiC-B isoform is present in all the mitochondria tested. It should be noted that the autoradiography films were exposed for 15 s in the case of the immunoblotting with anti-PiC-A antiserum (Fig. 3 A) and 60 min in the case of the immunoblotting with the anti-PiC-B antiserum (Fig. 3 B). Therefore, the expression levels of PiC-A in mitochondria of heart, skeletal muscle, and diaphragm are much higher than those of PiC-B in the mitochondria of all the tissues. Furthermore, no immunoreaction was observed with the anti-PiC-A antiserum and the lysates of liver, lung, brain, and kidney when the exposure time was prolonged to 60 min. To quantify PiC-A and PiC-B levels in mitochondria, various amounts of mitochondrial samples were loaded onto the gel and immunoblotted simultaneously with the appropriate range of recombinant PiC-A and PiC-B standards (see “Experimental Procedures”). In four determinations, the abundance of PiC isoforms was calculated to be 69 ± 13 pmol/mg of protein of PiC-A in heart mitochondria, and 10 ± 2 and 8 ± 1 pmol/mg of protein of PiC-B in heart and liver mitochondria, respectively. For kinetic analysis of the two PiC isoforms the reconstitution procedure has been optimized by adjusting the parameters that influence the efficiency of carrier incorporation into the liposomes. With both isoforms optimal transport activity was obtained with 0.6 μg/ml and 12.8 mg/ml protein and phospholipid concentration, respectively, with a Triton X-114/phospholipid ratio of 1.1 and with 13 passages through the same Amberlite column (see “Experimental Procedures”). Furthermore, since cardiolipin has been shown to be essential for the reconstitution of purified PiC (1Bisaccia F. Palmieri F. Biochim. Biophys. Acta. 1984; 766: 386-394Crossref PubMed Scopus (68) Google Scholar, 29Kadenbach B. Mende P. Kolbe H.V.J. Stipani I. Palmieri F. FEBS Lett. 1982; 139: 109-112Crossref PubMed Scopus (114) Google Scholar, 30Mende P. Huther F.J. Kadenbach B. FEBS Lett. 1983; 158: 331-334Crossref PubMed Scopus (27) Google Scholar), we tested the effect of this phospholipid on reconstitution of the two recombinant PiC isoforms. Fig.4 shows that the activity of both isoforms was increased 5-fold by optimal concentrations of cardiolipin and that isoform B was more active than isoform A at all cardiolipin concentrations applied (at 1 and 0.33 mm external phosphate for PiC-A and PiC-B, respectively). The different external Pi concentrations used for the two isoforms were chosen according to the Km values of PiC-A and PiC-B (see below) to accomplish comparable conditions in kinetic terms for the two isoforms. The clear stimulation of the activity of the two isoforms by low concentrations of cardiolipin (<5%) contrasts with the lack of effect observed with PiC purified from bovine heart (23Stappen R. Krämer R. Biochim. Biophys. Acta. 1993; 1149: 40-48Crossref PubMed Scopus (50) Google Scholar), a difference probably due to the absence of cardiolipin in the recombinant PiC isoforms. Kinetic constants were determined for pure recombinant PiC-A and PiC-B over a wide range of Pi concentrations from a standard double-reciprocal set of experiments (see Table I). The transport affinities (Km) for Pi on the external membrane surface of the reconstituted PiC-A and PiC-B isoforms, measured by the forward exchange method, were determined to be 2.21 ± 0.15 and 0.78 ± 0.02 mm for PiC-A and PiC-B, respectively. The specific activities of recombinant PiC-A and PiC-B (Vmax) were 125 ± 18 and 349 ± 22 mmol/min/g of protein for PiC-A and PiC-B, respectively. These activities were calculated by taking into account the amount of PiC isoforms recovered in the proteoliposomes after reconstitution. Under our experimental conditions the inserted protein varied between 14 and 20% of the protein added to the reconstitution mixture. Furthermore, no difference in the efficiency of reconstitution (i.e. the share of successfully incorporated protein) was observed between PiC-A and PiC-B.Table IKinetic constants of recombinant PiC-A and PiC-B isoformsPiCForward exchange measurementsBackward exchange measurementsPi/Pi antiportVmaxPi/Pi antiport externalKmPi/Pi antiport external KiPi/Pi antiport internal KmPi/H+ symport internal Kmmmol · min−1 · g protein−1mmmmPiC-A125 ± 18 (6)aValues given are means ± S.D. (n).2.21 ± 0.15 (6)2.04 ± 0.12 (3)9.7 ± 0.6 (6)8.1 ± 0.7 (4)PiC-B349 ± 22 (6)0.78 ± 0.02 (6)0.84 ± 0.03 (4)6.3 ± 0.5 (6)7.0 ± 0.5 (4)Lineweaver-Burk plots were obtained from forward exchange measurements of exchange velocity, v, or from backward exchange measurements of rate constant, k, under variation of external or internal Pi as described under “Experimental Procedures.” Substrate concentrations were as follows: Pi/Pi antiport measured by the forward procedure, 30 mm internal Pi, and 0.33–9.0 mmexternal Pi; Pi/Pi antiport measured by the backward procedure, 0.3–40 mm internal Pi and 30 mm external Pi; Pi/H+ symport, 0.3–40 mm internal Pi in the absence of external Pi. For the determination of Ki of arsenate, Dixon plots were obtained from forward exchange measurements of exchange velocity, v, in the presence of 30 mminternal Pi, and three external concentrations of [33P]phosphate (0.8, 1.2, and 2.0 mm for PiC-A or 0.2, 0.33, and 0.8 mm for PiC-B) and external arsenate from 0.5 to 8.0 mM (PiC-A) or from 0.2 to 3.0 mm (PiC-B).a Values given are means ± S.D. (n). Open table in a new tab Lineweaver-Burk plots were obtained from forward exchange measurements of exchange velocity, v, or from backward exchange measurements of rate constant, k, under variation of external or internal Pi as described under “Experimental Procedures.” Substrate concentrations were as follows: Pi/Pi antiport measured by the forward procedure, 30 mm internal Pi, and 0.33–9.0 mmexternal Pi; Pi/Pi antiport measured by the backward procedure, 0.3–40 mm internal Pi and 30 mm external Pi; Pi/H+ symport, 0.3–40 mm internal Pi in the absence of external Pi. For the determination of Ki of arsenate, Dixon plots were obtained from forward exchange measurements of exchange velocity, v, in the presence of 30 mminternal Pi, and three external concentrations of [33P]phosphate (0.8, 1.2, and 2.0 mm for PiC-A or 0.2, 0.33, and 0.8 mm for PiC-B) and external arsenate from 0.5 to 8.0 mM (PiC-A) or from 0.2 to 3.0 mm (PiC-B). The analysis of the inward-facing substrate binding site was carried out by using the backward-exchange method for the methodological reasons that have been described previously (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). From these experiments an internal Km of 9.7 ± 0.6 mm for PiC-A and 6.3 ± 0.5 mm for PiC-B was calculated. The intraliposomal Km was also determined by measu
Referência(s)