Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase.
The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by [14C]iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two [14C]carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated [14C]carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.
Decottignies
P
P
Laboratoire de Physiologie Végétale Moléculaire, Université Paris-Sud, Orsay, France.
Schmitter
J M
JM
Miginiac-Maslow
M
M
Le Maréchal
P
P
Jacquot
J P
JP
Gadal
P
P
D000595:Amino Acid Sequence; D002233:Carbocysteine; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008027:Light; D008291:Malate Dehydrogenase; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D010944:Plants / Q000201:enzymology*; D013879:Thioredoxins / Q000494:pharmacology; D014357:Trypsin; D003313:Zea mays
D004220:Disulfides; D007461:Iodoacetates; D010446:Peptide Fragments; D013879:Thioredoxins; D002233:Carbocysteine; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D014357:Trypsin; D003545:Cysteine; D004229:Dithiothreitol; D019807:Iodoacetic Acid