Abstract
Iron regulatory protein (IRP) is a cytosolic bifunctional [Fe-S] protein which exhibits aconitase activity or binds iron responsive elements (IREs) in untranslated regions of specific mRNA. The modulators of these activities are the intracellular concentration of iron and, as recently described, NO synthase activity. In this study, we attempted to establish in in vitro experiments whether peroxynitrite (ONOO-, the product of the reaction between NO and O2-.), as well as oxygen-derived radicals (O2-. and H2O2) and various NO donors, allow IRP to bind IREs using cytosol extract of macrophagelike RAW 264.7 cells. Neither the addition of a bolus of ONOO- or H2O2 nor O2-. generation significantly affected IRE binding even though they inhibited its aconitase activity. Moreover, we show that 3-morpholinosydnonimine (SIN-1), a chemical which releases both NO and O2-. enhanced IRE binding activity of IRP only in the presence of superoxide dismutase (SOD). S-Nitrosothiols and the NONOate sper/NO plus gluthathione (GSH) activated IRE binding by IRP whereas oxyhemoglobin prevented enhancement of this binding by SIN-1/SOD and sper/NO plus GSH. cis-Aconitate, substrate, also abolished the effect of SIN-1/SOD on IRE binding by IRP. These results imply that neither O2-. nor ONOO- can convert [4Fe-4S] IRP into IRE-binding protein but rather suggest that an active redox form of NO converts IRP into its IRE binding form by targeting the [Fe-S] cluster.
Highlights
Iron regulatory protein (IRP) to bind iron-responsive elements (IREs) using cytosol extract of macrophagelike RAW 264.7 cells
IRE binding activity was analyzed by gel shift experiments, and, in some experiments, the aconitase activity of IRP was measured by spectrophotometry
We and others previously supplied evidence that endogenous and exogenous NO modulates the cellular iron metabolism by increasing IRP binding to specific sequence(s) in their mRNA, i.e. to IREs [7, 8, 10, 26, 43]
Summary
FURTHER INSIGHTS INTO THE ROLE OF NITROGEN- AND OXYGEN-DERIVED REACTIVE SPECIES*. (Received for publication, August 31, 1995, and in revised form, November 15, 1995). The functional behavior of IRP, either aconitase or IRE-binding protein, can be determined by the intracellular iron concentration [1, 2] and the NO flux into the cell [7,8,9,10] Another line of research showed previously that the activities of bacterial and mammalian aconitases are inhibited by O2. More recent findings support the view that peroxynitrite (ONOOϪ), a reactive molecule derived from NO, is responsible for inhibiting both cytosolic and mitochondrial aconitases, rather than NO itself [12, 17] All these results and considerations led us to investigate IRE binding by IRP to ascertain whether it is directly sensitive to O2., H2O2 (ROS), and ONOOϪ. We previously reported that induction of nitric-oxide synthase in activated murine macrophages results
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