Amino Acid Metabolism of Lemna minor L.

Abstract

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by aminooxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of a-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.In a previous paper in this series (26), we have reported on the metabolic changes induced by the irreversible inhibitor of GS,2 MSO, in Lemna minor. It was proposed that glutamate, glutamine, asparagine, aspartate, alanine, and serine may be selectively catabolized to NH4+, either in the photorespiratory nitrogen cycle or by alternative routes (26). These alternative routes include dehydrogenase (e.g. GDH) and amidohydrolase (deamidase) (e.g. asparaginase) pathways (11,20,22,23,31).We have reasoned that if such alternative pathways of amino acid catabolism to NH4+ are in operation, then these should not be inhibited by the transaminase inhibitor, AOA (9,15,16,36 (20,23,31). In either case, asparagine catabolism should be inhibited by AOA since the initial step in asparagine catabolism entails transamination. Although inhibition of asparagine catabolism by AOA would rule out an asparaginase pathway, this result would not discriminate between asparagine amino-N transfer to glycine or other amino acids as major pathways of asparagine catabolism (12,16,(33)(34)(35). Similarly, inhibition of alanine catabolism by AOA would not discriminate between alanine amino-N transfer directly to glycine via alanine :glyoxylate aminotransferase or alanine amino-N transfer to glutamate by glutamate:pyruvate aminotransferase (2, 9, 24). Nevertheless, the responses of L. minor to AOA...

Amino Acid Metabolism of Lemna minor L.

“…In the comparative analyses of '4N-and '5N-grown fronds and the samples from experiments in which AOA was supplied, two internal standards were employed; L-a-amino-nbutyrate (for the neutral + basic amino acid fraction) and La-aminoadipic acid (for the acidic amino acid fraction), with both internal standards (250 nmol each) added to the samples after methanol:chloroform:H20 phase-separation of the methanol extracts. In the MSO experiments described, no internal standards were employed (11 ( 11) confirmed that greater than 95% of the asparagne was hydrolyzed by asparaginase in these treatments within 1 h. After 1 h, samples were applied to 2.5 x 1 cm columns of Dowex 50-H+, washed with 6 mL H20 and amino acids eluted with 6 mL 6 M NH40H, concentrated to dryness, derivatized to N-HFBI amino acid derivatives (2,13), and analyzed by GC (2, 13) to determine the relative proportions of N-HFBI-0-cyanoalanine and N-HFBI-aspartate.…”

“…Plant tissue (0.3-0.5 gfw) was harvested and extracted in methanol (10 mL) as described previously (2,11,13), and amino acids separated into neutral + basic and acidic amino acid fractions by Dowex 50-H+ and Dowex 1-acetate ion exchange chromatography as described previously (2,11,13). In the comparative analyses of '4N-and '5N-grown fronds and the samples from experiments in which AOA was supplied, two internal standards were employed; L-a-amino-nbutyrate (for the neutral + basic amino acid fraction) and La-aminoadipic acid (for the acidic amino acid fraction), with both internal standards (250 nmol each) added to the samples after methanol:chloroform:H20 phase-separation of the methanol extracts.…”

“…However, a serious limitation ofthis analytical methodology in '5N-tracer studies is that during derivatization, glutamine and asparagine undergo deamidation to glutamate and aspartate, respectively (12,14). Thus, independent quantification of glutamate, glutamine, asparagine, and aspartate requires prior separation of the neutral + basic and acidic amino acid fractions using ion exchange chromatography, and, more importantly, two key N groups (the amide moieties of glutamine and asparagine) are lost as NH4' during derivatization of the neutral + basic amino acid fraction (2,(11)(12)(13)(14). The latter problem has prompted us to seek methods for sensitively estimating the amide-['5N] abundance of glutamine and asparagine, using…”

“…This led us to consider an altemative procedure (G Fortier et a/. [1986] (N-HFBI3) derivatives of amino acids are convenient for routine quantification of amino acid pools in higher plant tissues by GC, and determination of the 'sN abundance of the majority of their aamino groups by GC-MS (2,(11)(12)(13)(14). However, a serious limitation ofthis analytical methodology in '5N-tracer studies is that during derivatization, glutamine and asparagine undergo deamidation to glutamate and aspartate, respectively (12,14).…”

Nucleoside intermediates in blasticidin S biosynthesis identified by the in vivo use of enzyme inhibitors

“…Attention was therefore focused on blocking three potential biosynthetic points. These points, and the specific inhibitors tested, were (a) blocking transamination with transaminase inhibitors (aminooxyacetic acid (AOAA), 11 (22)(23)(24), and 2-methylglutamate (MeGlu), 12 (25)) or amidotransferase inhibitors (azaserine, 13 (26), and 6-diazo-5-0x0-Lnorleucine (DON), 14 (27)); (b) blocking the availability of La-arginine with a L-a-arginine biosynthetic inhibitors (L-arginine hydroxamate (ArgH), 15 (28,29), and 12); and (c) blocking N-methylation with a methyltransferase inhibitor (L-ethionine (Eth), 16 (30, 3 1)).…”