A Metabolic Control Analysis of the Glutamine Synthetase/Glutamate Synthase Cycle in Isolated Barley (Hordeum vulgare L.) Chloroplasts

Abstract

Ammonia assimilation in chloroplasts occurs via the glutamine synthetase/glutamate synthase (CS/COCAT) cycle. To determine the extent to which these enzymes contribute to the control of ammonia assimilation, a metabolic control analysis was performed on isolated barley (Hordeum vurgare 1.) leaf chloroplasts. Pathway flux was measured polarographically as ammonium-plus-2-oxoglutarate-plus-glutamine-dependent O2 evolution in illuminated chloroplasts. Enzyme activity was modulated by titration with specific, irreversible inhibitors of CS (phosphinothricin) and COCAT (azaserine). Flux control coefficients (C:",) were determined (a) by differentiation of best-fit hyperbolic curves of the data sets (flux versus enzyme activity), and (b) from estimates of the deviation indices (/I&). Both analyses gave similar values for the coefficients. lhe control coefficient for CS was relatively high and the value did not change significantly with changes in 2-oxoglutarate concentration (Cc = 0.58 at 5 mM 2-oxoglutarate and 0.40 at 20 mM 2-oxoglutarate). lhe control coefficient for COCAT decreased with decreasing glutamine concentrations, from 0.76 at 20 mM glutamine to 0.19 at 10 mM glutamine. Thus, at high concentrations of glutamine, COCAT exerts a major control over flux with a significant contribution also from CS. At lower concentrations of glutamine, however, COCAT exerts far less control over pathway flux.Ammonia assimilation in higher plants proceeds via GS and GOGAT, together termed the GS/GOGAT cycle. In barley (Hordeum vulgare

Successive maturation and senescence of individual leaves during barley whole plant ontogeny reveals temporal and spatial regulation of photosynthetic function in conjunction with C and N metabolism

“…Underlying regulatory mechanisms were analyzed by determination of protein and transcript levels of two key enzymes of C and N anabolism. AGPase was selected because of its committing function in starch synthesis and chloroplastic GS was chosen as the regulatory end point of assimilation of reduced nitrogen (Kleczkowski et al, 1993;Baron et al, 1994). Levels of these proteins in the different leaves during growth of the barley plants were detected by immunoblot analyses (Figs.…”

Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine

“…AZA blocks transfer of nitrogen from glutamine to purine precursors (Aaronson 1959). It is also an effective inhibitor of glutamate synthase (GOGAT), a principal reaction along with glutamine synthetase in ammonia assimilation (Kusnan et al 1987;Baron et al 1994;Cogoni et al 1995). DON and AZA both react, forming covalent bonds, with glutamine-dependent enzymes involved in purine and pyrimidine biosynthesis and hence nucleotide synthesis.…”

Eucalyptus ESTs corresponding to the enzyme glutamine synthetase and the protein D1, sites of action of herbicides that cause oxidative stress

“…In addition, this compound is produced by fermentation and shows low toxicity and high safety to environment. The mechanism of action and the genes involved in resistance to this herbicide have been described by Kishi and Ishisuka (1992), Baron et al (1994), Baron et al (1994a), Downs et al (1994), Altenburger et al (1995), Bridges and Hess (2002) and Thill (2002). White et al (1990), Schwartz et al (1996) and Bridges and Hess (2002) presented two sequences that code for the production of the enzyme phosphinothricin-Nacetyltransferase, which has the ability to inactivate glufosinate: the PAT gene from Streptomyces hygroscopicus and the BAR gene from Streptomyces viridochromogenes.…”