Reference "Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase"

Reference ID

7257

Title

Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

Source

The Journal of biological chemistry, 1988, vol. 263, pp. 11780-11785

Authors (5)

Decottignies-P	Schmitter-J-M
Miginiac-Maslow-M	Jacquot-J-P
Gadal-P

Abstract

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.