Separation by Blue Native and Colorless Native Polyacrylamide Gel Electrophoresis of the Oxidative Phosphorylation Complexes of Yeast Mitochondria Solubilized by Different Detergents: Specific Staining of the Different Complexes
✍ Scribed by Xavier Grandier-Vazeille; Martine Guérin
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 361 KB
- Volume
- 242
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Blue native polyacrylamide gel electrophoresis ers for BN-PAGE, induces a charge shift on the proteins (BN-PAGE) or colorless native polyacrylamide gel so that the electrophoretic mobility of the proteins is electrophoresis (CN-PAGE) allowed separation of the mainly determined by the negative charges of bound oxidative phosphorylation complexes of yeast mito-Coomassie dye. In CN-PAGE, performed at a deterchondria. These complexes were characterized by spemined pH, the intrinsic charge of the proteins allows cific staining related to their enzymatic activity. Solutheir separation (1). Further analysis of the polypepbilization of mitochondria by different nonionic tide composition of each of the complexes was perdetergents such as Triton X-100, dodecyl maltoside, formed in a second dimension by Tricine-SDS-PAGE Nonidet P-40, Lubrol, octyl glucoside, or Hecameg led (1, 2). This system was essentially used in analyzing to the separation of F 1 -F O ATPase complexes exhibmammalian mitochondria (1, 2) and allowed to screen iting distinct apparent molecular masses related to for OXPHOS defects in human diseases (3-5).
different contaminating proteins and lipids. All these
We attempted to apply this system to yeast mitodifferent forms were active in ATP hydrolysis as rechondria and more particularly to the analysis of the vealed directly on the gel. Analysis of the subunit com-F 1 -F O ATPase complex. This membrane-bound enposition of these complexes was carried out by a twozyme catalyzes the synthesis of ATP from ADP and dimensional Tricine-SDS-PAGE and showed that the P i in a reaction driven by the electrochemical proton purest F 1 -F O ATPase complex was obtained with Lugradient maintained across the membrane by the respibrol, whereas with Hecameg and octyl glucoside, only ratory chain. This enzyme is structurally conserved the F 1 part of ATPase was solubilized. ᭧ 1996 Academic Press, among the different organisms and is composed of two Inc.
structurally and functionally distinct moieties, F 1 and F O . The F 1 part, which is composed of five different subunits termed a, b, g, d, and e, carries the catalytic Separation of enzymatically active heteromultimeric sites. The F O sector is embedded in the membrane and complexes of the inner mitochondrial membrane was forms a specific proton channel. It is composed of at greatly improved by the use of either blue native polyleast six hydrophobic subunits, the subunits 6, 8, and acrylamide gel electrophoresis (BN-PAGE) 2 or colorless 9 which are mtDNA encoded and the subunits 4, d, and native polyacrylamide gel electrophoresis (CN-PAGE). OSCP which are nDNA encoded (for review see Ref. 6). However, according to Cox et al. (7), the F O sector may comprise only the three mitochondrial hydrophobic pro-