No abstract

Abstract. Nitrate reductase in spinach (Spinacia oleracea L.) leaves was rapidly inactivated in the dark and reactivated by light, whereas in pea (Pisum sativum L., roots, hyperoxic conditions caused inactivation, and anoxia caused reactivation. Reactivation in vivo, both in leaves and roots, was prohibited by high concentrations (10-30 gM) of the serine/threonine-protein phosphatase inhibitors okadaic acid or calyculin, consistent with the notion that protein dephosphorylation catalyzed by type-1 or type-2A phosphatases was the mechanism for the reactivation of NADH-nitrate reductase (NR). Following inactivation of leaf NR in vivo, spontaneous reactivation in vitro (in desalted extracts) was slow, but was drastically accelerated by removal of Mg 2+ with excess ethylenediaminetetraacetic acid (EDTA), or by desalting in a buffer devoid of Mg 2+. Subsequent addition of either Mg 2+, Mn 2+ or Ca 2+ inhibited the activation of NR in vitro. Reactivation of NR (at pH 7.5) in vitro in the presence of Mg 2+ was also accelerated by millimolar concentrations of AMP or other nucleoside monophosphates. The EDTA-mediated reactivation in desalted crude extracts was completely prevented by protein-phosphatase inhibitors whereas the AMP-mediated reaction was largely unaffected by these toxins. The Mg2+-response profile of the AMP-accelerated reactivation suggested that okadaic acid, calyculin and microcystin-LR were rather ineffective inhibitors in the presence of divalent cations. However, with partially purified enzyme preparations (5 15% polyethyleneglycol fraction) the AMPmediated reactivation was also inhibited (65-80%) by microcystin-LR. Thus, the dephosphorylation (activation) of NR in vitro is inhibited by divalent cations, and protein phosphatases of the PP1 or PP2A type are involved in both the EDTA and AMP-stimulated reacThis paper is dedicated to Prof. O.H. Volk on the occasion of his 90th birthday Abbreviations: DTT = dithiothreitol; Mops = 3-(N-morpholino)-propanesulfonic acid; NR = NADH-nitrate reductase; NRA = nitrate-reductase activity; PP = protein phosphatase Correspondence to: W.M. Kaiser; FAX: 49 (931)71446 tions. Evidence was also obtained that divalent cations may regulate NR-protein phosphatase activity in vivo. When spinach leaf slices were incubated in MgZ+-and Ca2+-free buffer solutions in the dark, extracted NR was inactive. After addition of the CaZ+/MgZ+-ionophore A 23187 plus EDTA to the leaf slices, NR was activated in the dark. It was again inactivated upon addition of divalent cations (Mg 2+ or Ca2+). It is tentatively suggested that Mg 2+ fulfills several roles in the regulatory system of NR: it is required for active NR-protein kinase, 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, suggesting that two different protein phosphatases may be involved. At pH 6.5, the activation of NR was relatively slow and the addition or removal of Mg 2+ had no effe...

No abstract

Abstract:A Met residue is located adjacent to phosphorylation site 1 in the sequences of mitochondrial pyruvate dehydrogenase E1α subunits. When synthetic peptides including site 1 were treated with H 2 O 2 , the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no longer phosphorylated by E1α-kinase. Isolated mitochondria were incubated under state III or IV conditions, lysed, the pyruvate dehydrogenase complex (PDC) immunoprecipitated, and tryptic peptides analyzed by MALDI-TOF mass spectrometry. In all instances both Met and MetSO site 1 tryptic-peptides were detected. Similar results were obtained when suspension-cultured cells were incubated with chemical agents known to stimulate production of reactive oxygen species within the mitochondria. Treatment with these agents had no effect upon the amount of total PDC, but decreased the proportion of P-PDC. We propose that the redox-state of the Met residue adjacent to phosphorylation site 1 of pyruvate dehydrogenase contributes to overall regulation of PDC activity in vivo.