title: Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts.
abstract: Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.
authors.affiliation: Division of Molecular Plant Biology, University of California, Berkeley 94720.
authors.forename: N A
authors.initials: NA
authors.lastname: Crawford
authors.forename: M
authors.initials: M
authors.lastname: Droux
authors.forename: N S
authors.initials: NS
authors.lastname: Kosower
authors.forename: B B
authors.initials: BB
authors.lastname: Buchanan
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis*; D001643:Bridged Bicyclo Compounds; D002734:Chlorophyll / Q000032:analysis*; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000201:enzymology*; D002846:Chromatography, Affinity; D003624:Darkness; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005288:Ferredoxins / Q000032:analysis*; D005456:Fluorescent Dyes; D007506:Iron-Sulfur Proteins; D008027:Light; D045342:Light-Harvesting Protein Complexes; D010084:Oxidation-Reduction / Q000187:drug effects; D010088:Oxidoreductases / Q000032:analysis; D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins / Q000032:analysis*; D011233:Precipitin Tests; D013438:Sulfhydryl Compounds / Q000032:analysis; D013879:Thioredoxins / Q000032:analysis* / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D001643:Bridged Bicyclo Compounds; D054479:Chloroplast Thioredoxins; D005288:Ferredoxins; D005456:Fluorescent Dyes; D007506:Iron-Sulfur Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins; D013438:Sulfhydryl Compounds; D002734:Chlorophyll; D013879:Thioredoxins; D010088:Oxidoreductases; C021588:ferredoxin-thioredoxin reductase; C028381:monobromobimane