Figure 4.

Effects of Pyruvate and Citrate on the AOX Redox State of Purified Leaf Mitochondria.

Mitochondria from N. sylvestris wild-type and CMSII leaves were isolated as described in Methods. Pyruvate (P) at 5 mM was added (+) or not (−) to all buffers during the mitochondrial isolation process. Purified mitochondria were resuspended in the washing buffer, supplemented (+) or not (−) with 20 mM citrate (C), incubated for 1 h, and then pelleted by centrifugation.

(A) Immunodetection of reduced and oxidized forms of AOX. Mitochondrial proteins (50 μg per lane) were separated by nonreducing SDS-PAGE on 12% acrylamide gels. After electrotransfer to nitrocellulose membranes, proteins were probed with S. guttatum anti-AOX antibody and detected by chemiluminescence.

(B) Coomassie blue–stained control gel of mitochondrial proteins. The quality of the mitochondrial preparations is demonstrated by the presence of the P subunit of glycine decarboxylase (GDC P) and the αβ-subunits of mitochondrial ATPase (∼60 kD; De Paepe et al., 1993).

(C) Quantification of oxidized and reduced forms of AOX. Reduced AOX, black bars; oxidized AOX, gray bars; ratios of oxidized/reduced forms of AOX, white bars.