Fig. 1.

Pea chloroplast FBPase oxidation under atmospheric conditions. A, western blot analysis of the in vitro oxidation of the recombinant FBPase. Recombinant cFBP1 was incubated in a solution with 100 mM Tris-HCl (pH 8.0) and 50 mM DTT. After a 30 min incubation at room temperature free cysteines were blocked by adding four volumes of an alkylating solution containing 100 mM Tris-HCl (pH 8.0), 2.7% CHAPS, and 40 mM IAM (lane 1) or after removing excess DTT by gel filtration before adding the alkylating solution (lane 2). 100 ng of the recombinant protein were loaded per lane. B, western blot analysis of the in vitro oxidation of the chloroplast FBPase present in pea extracts. Leaves soluble proteins (detailed extraction protocol in the Section 2) were incubated with DTT and free cysteines alkylated as described in panel A. 10 μg of total soluble protein were loaded per lane. 1, non-reduced protein extract; 2, DTT-incubated protein extract; 3, DTT-incubated protein extract after gel filtration. C, schematic model representing the pea FBPase oxidation exposed to atmospheric air. The green colour represents reduced subunits and the red colour represents oxidized subunits. The structures were prepared using PyMOL Molecular Graphics software (www.pymol.org). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)