Role of Inosine Monophosphate Oxidoreductase in the Formation of Ureides in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata L. Walp.)

Abstract

Cell-free extracts from nodules of cowpea (Vigna unguiculata L. (Walp.) cv Caloona:Rhizobium strain CB756) prepared in the presence of 15% (v/v) glycerol showed high rates (30 to 60 nanomoles NAD reduced per minute per gram fresh weight nodule) of inosine monophosphate oxidoreductase (EC 1.2.1.14) activity. The enzyme was labile (half-life of activity less than 3 hours) but could be stabilized for up to 18 hours by inclusion of the substrates NAD and inosine monophosphate in the breaking media. Activity showed a broad pH optimum between 8.5 and 9.5, had an apparent K_m (inosine monophosphate) of 4 and 12 micromolar at pH 7.5 and 9.0, respectively, and was largely (96%) associated with the plant cell cytosol fraction of the nodule.

Metabolism of [8-¹⁴C]inosine monophosphate and [1-¹⁴C]glycine by the cell-free system showed two pathways for purine base production from inosine monophosphate, one via xanthosine monophosphate, xanthosine, and xanthine, the other via inosine and hypoxanthine. The proportion of inosine monophosphate utilized by inosine monophosphate oxidoreductase and the xanthine-based pathway was increased from 30% at 0.5 millimolar to 80% at 0.01 millimolar inosine monophosphate. The data are interpreted to indicate that in vivo inosine monophosphate oxidation rather than dephosphorylation is the predominant metabolic route leading to ureide synthesis and that inosine monophosphate provides the link between de novo purine nucleotide synthesis in the plastid and ureide production in the plant cell cytosol.