Light modulation and in vitro effects of adenine nucleotides on leaf nitrate reductase activity in cucumber (Cucumis sativus)

Nitrate reductase (NR, EC 1.6.6.1) activity in attached cucumber (Cucumis s ativus L, cv,Ashley) leaves changed rapidly and reversibly during light/dar k transitions, especially when assayed in the presence of free Mg2+, Light decreased and darkness increased the sensitivity of the enzyme to inhibitio n by Mg2+. The SR activation state, i.e. activity in the presence of Mg2+ r elative to activity. in the absence of Mg2+, increased with Light intensity up to 400 mu mol m(-2) s(-1) PAR (photosynthetically active radiation), Wh en a desalted crude extract from illuminated leaves was preincubated with A TP. SR was gradually inactivated, Inactivation nas only observed when activ ity. nas assayed in the presence of Mg2+. The ATP-inactivated SR remained i nactive after removing the excess of ATP by gel filtration and it did not o ccur in partially purified SR preparations. KR extracted front darkened att ached leaves was markedly activated when preincubated with 5-AMP. These res ults support the view that inactivation/activation of cucumber-leaf NR in r esponse to light/dark; signals most likely involves phosphorylation/dephosp horylation of the enzyme catalysed by endogenous proteins. As substantial a ctivation of NR by preincubation with 5-AMP nas also observed when activity nas assayed in the absence of Mg2+, thus indicating that 5-AMP can directl y activate SR, Irradiation of an extract from darkened leaves containing FA D promoted a partial activation of NR. This effect tvas observed both in th e +Mg2+ and in the -Mg2+ assay, indicating that activation nas caused by ph otoexcited flavin and did not involve dephosphorylation of the enzyme.