Article Figures & Data
Figures
- FIG. 1.
SDS-PAGE (10%) analysis of GOGAT purification fractions. Lane 1, CFE (27 μg); lane 2, butyl-Toyopearl fraction (23 μg); lane 3, DEAE-Toyopearl fraction (4 μg); lane 4, hydroxyapatite fraction (1.5 μg); lane 5, MonoQ fraction (1.5 μg); lane 6, molecular mass markers (1 μg of each).
- FIG. 2.
Glu production activities in reaction mixtures with all substrates and electron donors and in those lacking one of them. Error bars indicate standard errors for at least three independent experiments. OG, 2-oxoglutarate; MV, methylviologen; DN, dithionite; N.D., not detected.
- FIG. 3.
UV-visible spectra of Fd-GOGAT in the air-oxidized state (solid line) and in the dithionite-reduced state (dotted line).
- FIG. 4.
UV-visible spectra of recombinant Fd3 in the air-oxidized state (solid line) and in the dithionite-reduced state (dotted line).
- FIG. 5.
Low-temperature EPR spectrum of recombinant Fd3 in the dithionite-reduced state. Instrument settings for EPR spectroscopy were as follows: temperature, 10 K; microwave power, 0.05 mW; microwave frequency, 9.026 GHz; modulation frequency, 100 kHz; modulation amplitude, 0.2 mT. The g values are indicated.
- FIG. 6.
GOGAT activities with various electron carriers. GOGAT activity was calculated by subtracting the Glu production without electron carriers from that with an electron carrier. Error bars indicate standard errors for at least three independent experiments. MV, methylviologen; BV, benzylviologen; N.D., not detected.
- FIG. 7.
Relative activities for Glu synthesis under GOGAT assay conditions (A) and glutaminase assay conditions (B) when an organic acid was present in reaction mixtures. The activity of the mixture without any organic acids was designated 100% activity. The following organic acids were added to the mixtures: Suc, succinate; Oxa, oxaloacetate; Mal, malate; Cit, citrate; Ici, isocitrate; Fum, fumarate; and Pyr, pyruvate. Error bars indicate standard errors for at least three independent experiments.
- FIG. 8.
Relative GOGAT activities (circles) and glutaminase activities (squares) at various succinate concentrations. The activity of mixtures without succinate was designated 100% activity.
- FIG. 9.
Amino acid sequences in the vicinity of the Fd loop. Sequences from the following organisms were used: H. thermophilus, Synechococcus sp. strain WH 8102 (CAE08647), Synechocystis sp. strain 6803 (BAA11379), Arabidopsis thaliana (AAC78551), Spinacia oleracea (AAC26853), Azospirillum brasilense (AAA22179), and Escherichia coli (BAE77256). Asterisks indicate residues conserved in all seven sequences. The conserved region designated the Fd loop is highlighted in bold. Numbers indicate the positions of amino acid residues counted from the N-terminal Cys residue (position 1).
- FIG. 10.
Phylogenetic tree of Fd-GOGATs and the α subunits of NADPH-GOGAT homologues based on amino acid sequences. Sequences from the following organisms were used: Acinetobacter sp. strain ADP1 (CAG70018), Anabaena variabilis (ABA20918), Aquifex aeolicus (AAC07475), Arabidopsis thaliana, Azospirillum brasilense, Bacillus subtilis (CAB13728), Chlorobium tepidum (AAM71647), Clostridium acetobutylicum (AAK79639), Corynebacterium glutamicum (BAA75929), Escherichia coli, H. thermophilus, Gluconobacter oxydans (AAW61590), Paracoccus denitrificans (EAN66504), Prochlorococcus marinus (CAE21952), Pseudomonas aeruginosa (AAB39259), Rhodopseudomonas palustris (CAE26335), Rhodospirillum rubrum (ABC20824), Spinacia oleracea, Synechococcus sp. strain WH 8102, Synechocystis sp. strain 6803, Thermosynechococcus elongatus (BAC08920), Xanthomonas campestris (AAM39351), and Xylella fastidiosa (AAO29887).
Tables
- TABLE 1.
Purification of GOGAT from H. thermophilus TK-6
Fraction Total activity (U) Total amt of protein (mg) Sp act (U/mg) Purification factor (fold) Yield (%) CFE 19 2,443 0.0078 1 100 Butyl Toyopearl 9.0 197 0.046 6 47 DEAE Toyopearl 4.9 12 0.40 52 26 Hydroxyapatite 2.0 3.7 0.54 69 10 MonoQ 0.91 0.61 1.5 191 4.7
