MODULATION OF NITRATE REDUCTASE IN-VIVO AND IN-VITRO - EFFECTS OF PHOSPHOPROTEIN PHOSPHATASE INHIBITORS, FREE MG2-AMP( AND 5')

Nitrate reductase in spinach (Spinacia oleracea L.) leaves was rapidly inactivated in the dark and reactivated by light, whereas in pea (Pis um sativum L., roots, hyperoxic conditions caused inactivation, and an oxia caused reactivation. Reactivation in vivo, both in leaves and roo ts, was prohibited by high concentrations (1030 muM) of the serine/thr eonine-protein phosphatase inhibitors okadaic acid or calyculin, consi stent with the notion that protein dephosphorylation catalyzed by type -1 or type-2A phosphatases was the mechanism for the reactivation of N ADH-nitrate reductase (NR). Following inactivation of leaf NR in vivo, spontaneous reactivation in vitro (in desalted extracts) was slow, bu t was drastically accelerated by removal of Mg2+ with excess ethylened iaminetetraacetic acid (EDTA), or by desalting in a buffer devoid of M g2+. Subsequent addition of either Mg2+, Mn2+ or Ca2+ inhibited the ac tivation of NR in vitro. Reactivation of NR (at pH 7.5) in vitro in th e presence of Mg2+ was also accelerated by millimolar concentrations o f AMP or other nucleoside monophosphates. The EDTA-mediated reactivati on in desalted crude extracts was completely prevented by protein-phos phatase inhibitors whereas the AMP-mediated reaction was largely unaff ected by these toxins. The Mg2+-response profile of the AMP-accelerate d reactivation suggested that okadaic acid, calyculin and microcystin- LR were rather ineffective inhibitors in the presence of divalent cati ons. However, with partially purified enzyme preparations (5-15% polye thyleneglycol fraction) the AMP-mediated reactivation was also inhibit ed (65-80%) by microcystin-LR. Thus, the dephosphorylation (activation ) of NR in vitro is inhibited by divalent cations, and protein phospha tases of the PPI or PP2A type are involved in both the EDTA and AMP-st imulated reactions. Evidence was also obtained that divalent cations m ay regulate NR-protein phosphatase activity in vivo. When spinach leaf slices were incubated in Mg2+-and Ca2+-free buffer solutions in the d ark, extracted NR was inactive. After addition of the Ca2+/Mg2+-ionoph ore A 23187 plus EDTA to the leaf slices, NR was activated in the dark . It was again inactivated upon addition of divalent cations (Mg2+ or Ca2+). It is tentatively suggested that Mg2+ fulfills several roles in the regulatory system of NR: it is required for active NR-protein kin ase, it inactivates the protein phosphatase and is, at the same time, necessary to keep phospho-NR in the inactive state. The EDTA- and AMP- mediated reactivation of NR in vitro had different pH optima, suggesti ng that two different protein phosphatases may be involved At pH 6.5, the activation of NR was relatively slow and the addition or removal o f Mg2+ had no effect. However, 5'-AMP was a potent activator of the re action with an apparent K(m) of 0.5 mM. There was also considerable sp ecificity for 5'-AMP relative to 3'- or 2'-AMP or other nucleoside mon ophoposphates. We conclude that, depending upon conditions, the signal s triggering NR modulation in vivo could be either metabolic (e.g. 5'- AMP) or physical (e.g. cytosolic [Mg2+]) in nature.