Modulation of nitrate reductase in vivo and in vitro: Effects of phosphoprotein phosphatase inhibitors, free Mg2+and 5′-AMP
✍ Scribed by Werner M. Kaiser; Steve Huber
- Publisher
- Springer-Verlag
- Year
- 1994
- Tongue
- English
- Weight
- 741 KB
- Volume
- 193
- Category
- Article
- ISSN
- 0032-0935
No coin nor oath required. For personal study only.
✦ Synopsis
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 reac-This paper is dedicated to Prof. O.H. Volk on the occasion of his 90th birthday