Improved in vitro light activation and assay systems for two spinach chloroplast enzymes

Enzyme Regulation in C4 Photosynthesis

“…Partially purified NADP-MDH preparations are known to undergo a light-dependent activation when supplied with chloroplast membranes and the components of the ferredoxin/thioredoxin system, i.e. ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin (in this case thioredoxin m) (19,24,25). shows that a homogeneous preparation of corn leaf NADP-MDH was also activated photochemically by the ferredoxin/thioredoxin system and that, as found previously with less pure preparations, activation was dependent on each of the components of that system.…”

“…Prior to effecting this catalytic reaction, NADP-MDH requires an activation that can be achieved in vivo by light (13) or in vitro by DTT added in the presence of a soluble protein factor (11,14,22,23) that has been identified as a specific chloroplast thioredoxin (thioredoxin m) (12,19,24,25). NADP-MDH can also be activated photochemically in vitro by thioredoxin m reduced via ferredoxin and ferredoxin-thioredoxin reductase (19,24,25) or by a soluble protein factor that appears to be independent of the ferredoxin/thioredoxin system (2).Although NADP-MDH has been partially purified from leaves of several different C3 and C4 plants (13,19,(22)(23)(24), a procedure for obtaining homogeneous preparations has not been reported and consequently the enzyme has not been characterized. We have therefore focused our attention on NADP-MDH and now describe a purification procedure that yields homogeneous preparations of the enzyme from corn leaves.…”

“…Although NADP-MDH has been partially purified from leaves of several different C3 and C4 plants (13,19,(22)(23)(24), a procedure for obtaining homogeneous preparations has not been reported and consequently the enzyme has not been characterized. We have therefore focused our attention on NADP-MDH and now describe a purification procedure that yields homogeneous preparations of the enzyme from corn leaves.…”

“…NADP-MDH is particularly abundant in C4 plants where it functions photosynthetically in the NADP-dependent reduction of oxaloacetate to malate. Prior to effecting this catalytic reaction, NADP-MDH requires an activation that can be achieved in vivo by light (13) or in vitro by DTT added in the presence of a soluble protein factor (11,14,22,23) that has been identified as a specific chloroplast thioredoxin (thioredoxin m) (12,19,24,25). NADP-MDH can also be activated photochemically in vitro by thioredoxin m reduced via ferredoxin and ferredoxin-thioredoxin reductase (19,24,25) or by a soluble protein factor that appears to be independent of the ferredoxin/thioredoxin system (2).…”

Residue Glu‐91 of Chlamydomonas reinhardtii ferredoxin is essential for electron transfer to ferredoxin‐thioredoxin reductase

“…The proteins used in the reconstituted light activation system were spinach thioredoxin m purified as in [16], spinach FTR purified as in [6] and recombinant sorghum NADP malate dehydrogenase (NADP-MDH) obtained as in [17]. The ferredoxin-FNR interaction was followed by the cytochrome c reduction assay [18].…”