Synthesis of [¹⁵N]Glutamate from [¹⁵N]H₄⁺ and [¹⁵N]Glycine by Mitochondria Isolated from Pea and Corn Shoots

Abstract

ABSTRACT?Metabolically competent mitochondria were isolated from pea and corn shoots on Percoll discontinuous density gradients. Rates of synthesis of ['5Nlglutamate were measured by gas chromatography-mass spectrometry after the incubation of mitochondria with either 2 millimolar [I5N1 Glutamate dehydrogenase (EC 1.4.1.2.), which is localized in mitochondria, catalyzes both synthetic (amination of a-KG2) and catabolic (deamination of glutamate) reactions. With the identification of the GS/GOGAT cycle in green leaves, Miflin and Lea (5) postulated that GS (EC 6.3.1.2.) represented the sole port of entry for NH$, that glutamine served as the precursor of glutamate in the reaction mediated by GOGAT (EC 1.4.1.14), and that GDH served a catabolic function (5, 16). In green leaves, the major source of NH4 is the photorespiratory release of NH4 during the conversion of glycine to serine in mitochondria (9,17). According to the current model of the photorespir- 2Abbreviations: a-KG, ca-ketoglutarate; GDH, glutamate dehydrogenase; GS, glutamine synthetase; GOGAT, glutamate synthase; N-HFBI, N-heptafluorobutyryl isobutyl; AOA, aminooxyacetate; PALP, pyridoxal 5-phosphate; RCR, respiratory control ratio. atory nitrogen cycle, the NH' released in the mitochondria is totally reassimilated by the GS/GOGAT system (9, 17).The potential of GDH in leaves as another port of entry for NH' has however, not been ruled out. Our previous results show that mitochondria isolated from pea (20) and corn shoots (19) contain 3 to 5 mm of free NH4+ which is close to the Km of GDH. We also estimated the concentrations of other substrates and cofactors, which are required for both amination and deamination reactions of GDH, in mitochondria from corn shoots and with this information simulated its reaction in vitro. The results showed that GDH was potentially active in the direction of glutamate formation over a wide range of pH (6-9) and with NH4 concentrations ranging from 2 to 100 mm (19). We have also shown that a transaminase rather than GDH is probably active in the oxidation of glutamate since low concentrations of aminooxyacetate inhibit the oxidation of glutamate (8,20). Supporting this in vitro simulation, Neeman et al. (7) recently showed in their '5N-NMR study that nitrogen from ['5N]glycine is incorporated into both the amide group of glutamine and aamino group of glutamate. They incubated tobacco leaf protoplasts in the dark when the ferredoxin GOGAT of the chloroplasts should not be very active (5). Where GOGAT is inactive, a synthesis of ['5Njglutamate would not be expected unless another glutamate producing system were active. Therefore, both GS and GDH appear to be active in the reassimilation of NH4 in isolated protoplasts.In this paper, we describe evidence that mitochondria isolated from pea and corn shoots produce ['5N]

Estimation of Ammonium Ion Distribution between Cytoplasm and Vacuole Using Nuclear Magnetic Resonance Spectroscopy

“…Results of in vivo experiments have tended to support the view that GS is primarily responsible for NH4' assimilation (9,11,22), whereas in vitro mitochondrial GDH catalyzes NH4' assimilation in the presence of millimolar amounts of NH4' (31,32). The estimated cytoplasmic NH4' concentrations of approximately 10 uM in Table I contrast to the much lower concentrations estimated here.…”

Glutamine Synthetase-Glutamate Synthase Pathway and Glutamate Dehydrogenase Play Distinct Roles in the Sink-Source Nitrogen Cycle in Tobacco

“…Our in vivo 15 N-labeling data clearly contrast with the proposal that GDH and cytosolic GS1 play the major role for the synthesis and reallocation of amino acids in senescing leaves (for review, see Habash, 2002). Indeed, Glu synthesis from ammonium and 2-oxoglutarate in isolated plant mitochondria has been reported (Yamaya et al, 1986), but the rate of Glu formation was as low as 0.2% of photorespiratory [ 15 N]ammonium release (Yamaya et al, 1986) or 1.2% of the oxidative deamination rate of Glu (Aubert et al, 2001).…”

The Role of Glutamate Dehydrogenase in Plant Nitrogen Metabolism

“…complementary role to the GOGAT cycle (18,23,29). Alternatively, it could catalyze the oxidation of glutamate and furnish carbon to the TCA cycle (26).…”