Jean-Pierre Jacquot

Evidence for the existence of several enzyme-specific thioredoxins in plants

Thioredoxin, a small molecular weight protein, functioning as hydrogen carrier in DNA synthesis has recently been identified to be an indispensable factor in the light activation of certain regulatory enzymes of plant chloroplasts [l] . In the presence of thioredoxin and ferredoxin-thioredoxin reductase, photochemically-reduced ferredoxin is able to activate enzymeglike NADP malate dehydrogenase [2] and fructose 1,6-bisphosphatase (FBPase) [3] , both enzymes restricted to the chloroplasts [3,4]. In vitro reduced ferredoxin and ferredoxin-thioredoxin reductase can be replaced by the non-physiological sulfhydryl reagent dithiothreitol (DTT), thus eliminating the need for light, but not the need for thioredoxin [3,5,6] . During our efforts to isolate and purify thioredoxin from different plant species (spinach, sorghum and French beans) and plant materials (leaves and roots) we have realized that there are several forms of thioredoxin which are specific for the activation of either NADP malate dehydrogenase or FBPase. Similar findings have recently been reported by [7] . We describe here a simple method for the separation of different forms of thioredoxin which is applicable to various plant materials. In addition our results indicate that the different forms of thioredoxin are enzyme specific. However a given form of thioredoxin isolated from one plant species can activate the respective enzyme isolated from another plant species and vice versa.

Separation by high-performance liquid chromatography of the ferredoxin-thioredoxin system proteins

Abstract In order to separate the four proteins of the ferredoxin—thioredoxin system, their behaviour was studied on different high-performance liquid chromatographic columns. When the proteins were not activated, the mixture could be totally resolved by gel filtration chromatography on a TSK 3000 SW column, using a phosphate buffer containing 0.3 M sodium chloride, or by anion-exchange chromatography. Hydrophobic interaction chromatography did not allow a one-step separation. When the proteins were light activated and their cysteinyl residues derivatized with iodoacetate or iodoacetamide, filtration on the TSK 3000 SW column was found to be the only efficient method for separating the four proteins in a one-step process.

Further evidence for a role of sulfhydryls in the thioredoxin dependent activation of corn NADP-malate dehydrogenase

The kinetics of activation and reduction of the corn leaf NADP‐malate dehydrogenase reveal that plant thioredoxins play a dual role in the activation of this enzyme in the presence of dithiothreitol (e.g. reductive and conformational). A mutant thioredoxin of E. coli in which Cys32 and Cys35 are replaced by an Ala and a Ser residue, respectively, was tested in the activation of corn NADP‐malate dehydrogenase either in the presence of DTT or in a light reconstituted system. While native E. coli or plant thioredoxinm strongly activated the NADP‐malate dehydrogenase, the mutant thioredoxin was unable to promote any activation of the enzyme. At the same time the mutant thioredoxin stimulated the activity of T7 DNA polymerase. These results clearly confirm that the thioredoxin requirement observed in the activation of NADP‐malate dehydrogenase is primarily needed for redox reactions and not for structural/conformational effects as is the case for the T7 DNA polymerase assay.