IN-VIVO AND IN-VITRO STUDIES OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE FROM BARLEY ROOT PLASTIDS IN RELATION TO REDUCTANT SUPPLY FOR NO2- ASSIMILATION

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation a nd compared with the in vitro effect of the NADPH/ NADP ratio on the a ctivity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1. 1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio incre ased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to in tact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid de cline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barle y root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ra tios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibit ion by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower K -m for NADP, and was less inhibited by DTT than the enzyme from N-suff icient root plastids, indicating that there was some effect of N starv ation on the G6PDH activity in barley root plastids.