Determination of pools of tricarboxylic acid cycle and related acids in bacteria

1984

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Glutamate synthase [L-glutamate:NADP+ oxidoreductase (transaminating); EC 1.4.1.13] (GItS) was purified to homogeneity from BaciUus licheniformis A5. The native enzyme had a molecular weight of approximately 220,000 and was composed of two nonidentical subunits (molecular weights,-158,000 and-54,000). The enzyme was found to contain 8.1 ± 1 iron atoms and 8.1 + 1 acid-labile sulfur atoms per 220,000-dalton dimer. Two flavin moieties were found per 220,000-dalton dimer, with a ratio of flavin adenine dinucleotide to flavin mononucleotide of 1.2. The UV-visible spectrum of the enzyme exhibited maxima at 263,380 and 450 nm. The GitS from B. Iicheniformis had a requirement for NADPH, oa-ketoglutarate, and glutamine. Classical hyperbolic kinetics were seen for NADPH affinity, which resulted in an apparent Km value of 13 ,uM. Nonhyperbolic kinetics were obtained for a-ketoglutarate and glutamine affinities, and the reciprocal plots obtained for these substrates were biphasic. The apparent Km values obtained for glutamine were 8 and 100 ,LM, and the apparent Km values obtained for a-ketoglutarate were 6 and 50 ,uM. GltS activity was found to be relatively insensitive to inhibition by amino acids, keto acids, or various nucleotides. L-Methionine-DLsulfoximine, L-methionine sulfone, and DL-methionine sulfoxide were found to be potent inhibitors of GltS activity, yielding 10.5 values of 150, 11, and 250 ,uM, respectively. GltSs were purified from cells grown in the presence of ammonia and nitrate as sole nitrogen sources and were compared. Both yielded identical final specific activities and identical physical (UV-visible spectra, flavin, and iron-sulfur composition) and kinetic characteristics.

Section: Methodsmentioning

confidence: 99%

Purification and properties of glutamate synthase from Bacillus licheniformis

Schreier¹,

1984

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“…Cell growth conditions. B. licheniformis A5 (52) was grown in the minimal salts A medium previously described (46) with the following modification. The A medium contained (per liter) 1 ml of an inorganic salts solution (A salts; 46) and 25 ml of a 2.6 M potassium phosphate (pH 7.2)-0.04 M MgSO4 solution (A buffer).…”

Section: Methodsmentioning

confidence: 99%

Properties of the Bacillus licheniformis A5 glutamine synthetase purified from cells grown in the presence of ammonia or nitrate

Donohue¹,

1981

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othermophilus (55) and a partially purified GS preparation from Bacillus licheniformis ATCC 9945A (22,23) indicate that the glutamine synthetases of Bacillus spp. are regulated differently from the GS of E. coli (49). Most significant are the findings that the GS enzymes of Bacillus spp. are not regulated by covalent modification and are more susceptible to feedback regulation than is the Escherichia coli GS. There are also reports of more than one form of GS in some systems (5,19,29,50), including the 589 on July 16, 2020 by guest http://jb.asm.org/ Downloaded from 590 DONOHUE AND BERNLOHR GS assays. Biosynthetic GS (L-glutamate-ammonia ligase, EC 6.3.1.2) activity was assayed by a modification of the radiochemical method of Prusiner and J. BACTERIOL.on July 16, 2020 by guest

“…The procedure used for sampling and preparation of perchloric acid extracts has been described previously (43). The procedure used for the preparation of trichloroacetic acid-EDTA extracts was based on the one recommended by Lundin and Thore (31).…”

Section: Methodsmentioning

confidence: 99%

“…Fluorimetric metabolite assays. The protocol used for the fluorimetric analyses of pyruvate, citrate, a-ketoglutarate, fumarate, and malate have been described previously (43).…”

Section: Methodsmentioning

confidence: 99%

Effect of cultural conditions on the concentrations of metabolic intermediates during growth and sporulation of Bacillus licheniformis

Donohue¹,

1978

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Intracellular concentrations of adenine nucleotides and intermediates of the Embden-Meyerhof pathway and the tricarboxylic acid cycle have been determined during growth and sporulation of Bacillus licheniformis in a variety of different media. The ATP pool was independent of growth rate and nitrogen source, but the use of glucose as a carbon source resulted in a twofold elevation in the ATP pool during exponential growth. The intracellular phosphoenolpyruvate pool was at least twofold higher during gluconeogenesis than during glycolysis. The finding that the use of glutamate as the sole nitrogen source resulted in at least a fivefold elevation of the alpha-ketoglutarate pool suggests a role for alpha-ketoglutarate in the repression of the enzymes of the tricarboxylic acid cycle responsible for alpha-ketoglutarate synthesis. Not one of the metabolites assayed appears to function as a signal of the nutrient deprivation which accompanies the initiation of sporulation.