Dye-ligand chromatographic purification of intact multisubunit membrane protein complexes: application to the chloroplast H+-FoF1-ATP synthase.
n-Dodecyl-beta-D-maltoside was used as a detergent to solubilize the ammonium sulphate precipitate of chloroplast F(O)F(1)-ATP synthase, which was purified further by dye-ligand chromatography. Upon reconstitution of the purified protein complex into phosphatidylcholine/phosphatidic acid liposomes, ATP synthesis, driven by an artificial DeltapH/Deltapsi, was observed. The highest activity was achieved with ATP synthase solubilized in n-dodecyl-beta-D-maltoside followed by chromatography with Red 120 dye. The optimal dye for purification with CHAPS was Green 5. All known subunits were present in the monodisperse proton-translocating ATP synthase preparation obtained from chloroplasts.
Seelert
H
H
Institut für Biochemie, Abt. Physikalische Biochemie, Technische Universität Darmstadt, Petersenstr. 22, D-64287 Darmstadt, Germany.
Poetsch
A
A
Rohlfs
M
M
Dencher
N A
NA
D000255:Adenosine Triphosphate / Q000378:metabolism; D002501:Centrifugation, Zonal; D002736:Chloroplasts / Q000201:enzymology*; D002793:Cholic Acids / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004396:Coloring Agents / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D008024:Ligands; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D015203:Reproducibility of Results; D012995:Solubility; D018724:Spinacia oleracea
D002793:Cholic Acids; D004396:Coloring Agents; D005960:Glucosides; D008024:Ligands; D008081:Liposomes; D008565:Membrane Proteins; C040358:dodecyl maltoside; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate