Possible target proteins of chloroplast thioredoxin (Trx) have been investigated in the stroma lysate of spinach chloroplasts. For that purpose, we immobilized a mutant of m-type Trx in which an internal cysteine at the active site was substituted with serine, on cyanogen bromide-activated resin. By using this resin, the target proteins in chloroplast were efficiently acquired when they formed the mixed-disulfide intermediates with the immobilized Trxs. We could acquire Rubisco activase (45 kDa) and 2-Cys-type peroxiredoxin (Prx), which were recently identified as targets of chloroplast Trxs. Glyceraldehyde-3-phosphate dehydrogenase and sedoheputulose 1,7-bisphosphatase, well-known thiol enzymes in the Calvin cycle, also were recognized among the collected proteins, suggesting the method is applicable for our purpose. Furthermore, four proteins were identified from a homology search of the NH 2-terminal sequence of the acquired proteins: glutamine synthetase, a protein homologous to chloroplast cyclophilin, a homolog of Prx-Q, and the Rubisco small subunit. The Trx susceptibilities of the recombinant cyclophilin and Prx-Q of Arabidopsis thaliana were then examined. The method developed in the present study is thus applicable to investigate the various redox networks via Trxs and the related enzymes in the cell.
Rotation of the Q Q subunit in chloroplast F 1 -ATPase (CF 1 ) was investigated by using a single molecule observation technique, which is developed by Noji et al. to observe the rotation of a central Q Q subunit portion in the K K 3 L L 3 Q Q sub-complex of F 1 -ATPase from thermophilic Bacillus PS3 (TF 1 ) during ATP hydrolysis [Noji, H. et al. (1997) Nature 386, 299^302]. We used two cysteines of the Q Q subunit (Cys-199 and Cys-205) of CF 1 -ATPase, which are involved in the regulation of this enzyme, to fix the fluorochrome-labeled actin filament. Then we successfully observed a unidirectional, counter-clockwise rotation of the actin filament with the fluorescent microscope indicating the rotation of the Q Q subunit in CF 1 -ATPase. We conclude that the rotation of the Q Q subunit in the F 1 -motor is a ubiquitous phenomenon in all F 1 -ATPases in prokaryotes as well as in eukaryotes.z 1999 Federation of European Biochemical Societies.
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