Purification of multisubunit membrane protein complexes: isolation of chloroplast FoF1-ATP synthase, CFo and CF1 by blue native electrophoresis.
The proton-ATP synthase of thylakoid membranes from chloroplasts (CFoF1) is composed of two parts with different structural and functional properties: the membrane-integral, proton-conducting complex CFo and the hydrophilic part, CF1 which catalyze the formation of adenosine-5'-triphosphate (ATP). To date it is difficult to isolate functional CFoF1 from thylakoids in high purity and yield. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was therefore successfully employed to isolate CFoF1 in a one-step procedure from thylakoid membranes. Using a cathode buffer with low Coomassie Blue G-250 (CBG) concentration (0.002%), CFoF1 remains intact and can be obtained in high purity from solubilized, prepurified ATP synthase. Using BN-PAGE and a cathode buffer with 0.02% CBG, the ATP synthase bifurcates, and we were able to isolate both parts, CFo and CF1, separately. CFoF1, CFo, and CF1, respectively, were electroeluted nearly quantitatively electroeluted from the gel. BN-PAGE is a generally applicable method for the isolation and characterization of multisubunit membrane protein complexes in their native structure. However, the combination of neutral detergents and the negatively charged dye CBG seems to mimic properties of mild ionic detergents. This effect can lead to dissociation of labile subunits and subcomplexes, especially when delipidated membrane protein complexes are applied to BN-PAGE. By variation of the initial electrophoresis conditions, i.e., dye concentration in the cathode buffer, amount of lipid and detergent, BN-PAGE can be used for the isolation of either intact complexes or of subcomplexes.
Neff
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Institut für Biochemie, Abt. Physikalische Biochemie, Technische Universität Darmstadt, Petersenstrasse 22, Darmstadt, D-64287, Germany. dencher@pop.tu-darmstadt.de
Dencher
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NA
D002736:Chloroplasts / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000502:physiology*; D018724:Spinacia oleracea / Q000737:chemistry
D006180:Proton-Translocating ATPases