Affinity chromatography purification of mitochondrial inner membrane proteins with calcium transport activity.
Immobilized calcium affinity chromatography was used to obtain a preparation enriched in calcium transporters from Triton X-100 extracts of rat liver mitochondria inner membranes (PPCT). The PPCT were reconstituted into preformed asolectin liposomes which contained 120 mM KCl as internal high K+ medium. 45Ca2+ uptake into proteoliposomes was studied under conditions favoring electrophoretic uptake, and H+i/45Ca2+o or Na+i/45Ca2+o exchange, to test for the presence of the three calcium transport modes present in mitochondria. 45Ca2+ uptake in liposomes was studied in parallel. Na+i/45Ca2+o exchange activity was not detectable. H+i/45Ca2+o exchange activity measured in the presence of a pH gradient (acid inside) obtained after suspension in low K medium in the presence of nigericin, was 100-200 nmoles 45Ca2+ per mg protein in 30 s. 45Ca2+ uptake in voltage-dependent assays (a K+ diffusion membrane potential induced by valinomycin in the presence of methylamine) was not electrophoretic since it was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and probably due to secondary Ca2+/H+ countertransport. H+i/45Ca2+o uptake showed a saturable component at around 80 microM Ca and was coupled to an increase in internal pH in pyranine-loaded PPCT proteoliposomes. 45Ca2+ uptake in PPCT proteoliposomes could also be driven by a pH gradient obtained by raising external pH in high K+ medium. The results are consistent with the presence of a functional nH+/Ca2+ antiporter. Polyclonal antibodies raised against the PPCT were able to immunoprecipitate the H+/45Ca2+ uptake activity and recognized two major bands in the PPCT with molecular masses of about 66 kDa and 55 kDa. This is the first report of a partial purified protein(s) which may represent the H+/Ca2+ exchanger of the inner mitochondrial membrane, and represents an important step towards its identification.
Villa
A
A
Departamento de Biología Molecular, Centro de Biología Molecular 'Severo Ochoa', C.S.I.C.-Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049-Madrid, Spain.
García-Simón
M I
MI
Blanco
P
P
Sesé
B
B
Bogónez
E
E
Satrustegui
J
J
D000818:Animals; D017920:Antiporters / Q000737:chemistry*; D001190:Arylsulfonates / Q000378:metabolism; D002118:Calcium / Q000493:pharmacokinetics*; D002132:Calcium Radioisotopes / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry*; D027682:Cation Transport Proteins; D002846:Chromatography, Affinity / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D014634:Valinomycin / Q000494:pharmacology
D017920:Antiporters; D001190:Arylsulfonates; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D008081:Liposomes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids; C027614:calcium-hydrogen antiporters; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C024990:asolectin; D017830:Octoxynol; C005047:pyranine; D002118:Calcium