Histochemical staining and quantification of plant mitochondrial respiratory chain complexes using blue-native polyacrylamide gel electrophoresis

Journal article

Our knowledge of the respiratory chain and associated defects depends on the study of the multisubunit protein complexes in the inner mitochondrial membrane. Functional analysis of the plant mitochondrial respiratory chain has been successfully achieved by a combination of blue-native polyacrylamide gel electrophoresis (BN-PAGE) for separation of the protein complexes, and in-gel histochemical staining of the enzyme activities. We have optimized this powerful technique by determining linear ranges of amount of protein and enzyme activity for each respiratory complex. Time courses of the in-gel enzyme activities were also performed to determine optimal reaction times. Using the in-gel activity staining method we have previously shown decreased activity of complex V (F₁F₀-ATPase) in male-sterile sunflowers (Sabar et al., 2003). Here we have identified unique supercomplexes comprising complex IV (cytochrome c oxidase) in sunflower mitochondria. This method therefore represents a reliable tool for the diagnosis of respiratory dysfunction. In addition, the wider application of BN-PAGE in combination with enzyme activity staining is discussed.

Authors: Sabar, M; Balk, J; Leaver, C

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Blackwell Publishing Ltd.
• Journal: Plant Journal
• Volume: 44
• Issue: 5
• Pages: 893-901
• Publication date: 2005-12-01
• DOI: 10.1111/j.1365-313X.2005.02577.x
• EISSN: 1365-313X
• ISSN: 0960-7412
• Language: English
• Keywords: histochemical staining; plant electron transfer complexes; ATPase; BN-PAGE; respiratory chain
• Subjects: Plant Sciences