Isolation and characterisation of glutamate dehydrogenase from Mycobacterium smegmatis CDC 46

Sarada, K; Rao, N. Appaji; Venkitasubramanian, T. A.

doi:10.1016/0005-2744(80)90498-2

Cited by 18 publications

(11 citation statements)

References 11 publications

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“…strain PCC 6803 enzyme (208 kDa) (10) but is of the same order of magnitude as that of other GDHs characterized from different sources (1,20,34 In contrast with the NADPH-GDH activity characterized from other sources (21,38), the P. laminosum enzyme was affected by neither thiol reagents nor thiol compounds. As with the enzyme from bacterial (28,45) or higher plant (39) sources, only high concentrations of glutaric acid or L-Glu inhibited the enzyme to some extent.…”

Section: Discussionmentioning

confidence: 82%

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

et al. 1988

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The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-flxing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30°C it showed a specific activity of 32.9 gimol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (K,m = 1.25 mM) and NADPH (Km = 64 ,uM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [SJ0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60°C (Ea = 5.1 kcal/ mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.

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Section: Discussionmentioning

confidence: 82%

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

et al. 1988

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“…Glutamate dehydrogenase (EC 1.4.1.4) activity was measured in the forward (reductive amination activity) direction as described elsewhere (72). The reaction followed the oxidation of NADPH 2 spectrophotometrically at 340 nm and was monitored for 3 min at room temperature in a Genesys 10-S spectrophotometer (Thermo Scientific).…”

Section: Methodsmentioning

confidence: 99%

Transcriptome of Proteus mirabilis in the Murine Urinary Tract: Virulence and Nitrogen Assimilation Gene Expression

et al. 2011

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The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In this study, microarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7 days postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across nine microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded mannose-resistant Proteus-like (MR/P) fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism enzymes, and a portion of the tricarboxylic acid (TCA) cycle enzymes. Flagella were downregulated. Ammonia assimilation gene glnA (glutamine synthetase) was repressed in vivo, while gdhA (glutamate dehydrogenase) was upregulated in vivo. Contrary to our expectations, ammonia availability due to urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant was growth deficient in minimal medium with citrate as the sole carbon source, and loss of gdhA resulted in a significant fitness defect in the mouse model of urinary tract infection. Unlike Escherichia coli, which represses gdhA and upregulates glnA in vivo and cannot utilize citrate, the data suggest that P. mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential of P. mirabilis in the urinary tract.

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“…The mammalian enzymes seem to exhibit a dual specificity: thus at pH 8.5, glutamate is oxidized optimally, whilst there is a second pH optimum (around 10) for the oxidation of a variety of monocarboxylic amino acids, including alanine and norvaline (Struck & Sizer, 1960;Markau & Steinhubel, 1972). The prokaryotic GDHs, although generally poorly characterized, seem not to exhibit this 'alanine' activity: a possible exception is the NADP-linked GDH from Mycobacteriurn srnegrnatis (Savada et al, 1980).…”

Section: Asaccharolyticus Gdh On Dye-linked Columnsmentioning

confidence: 99%

Characterization of Peptostreptococcus asaccharolyticus Glutamate Dehydrogenase Purified by Dye-ligand Chromatography

Hornby

Engel

1984

Microbiology

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Glutamate dehydrogenase (L-glutamate : NAD + oxidoreductase (deaminating); EC 1.4.1.2) has been purified from Peptostreptococcus asaccharolyticus in a single step using dye-ligand chromatography. The enzyme (GDH) was present in high yields and was stabilized in crude extracts. A subunit molecular weight of 49000 & 500 was determined by SDS polyacrylamide gel electrophoresis and six bands were obtained after cross-linking the subunits with dimethyf suberimidate. This bacterial GDH was predominantly NAD +-linked, but was able to utilize both NADP+ and NADPH at 4% of the rates with NAD+ and NADH, respectively. An investigation of the amino acid specificity revealed some similarities with GDH from mammalian sources and some clear differences. The values of apparent K , for the substrates ammonia, 2-oxoglutarate, NADH, NAD + and glutamate were 18.4, 0.82, 0.066, 0.03 1 and 6 mM, respectively. The P. asaccharolyticus GDH was not regulated by purine nucleotides, but was subject to strong inhibition with increasing ionic strength.

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Isolation and characterisation of glutamate dehydrogenase from Mycobacterium smegmatis CDC 46

Cited by 18 publications

References 11 publications

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

Transcriptome of Proteus mirabilis in the Murine Urinary Tract: Virulence and Nitrogen Assimilation Gene Expression

Characterization of Peptostreptococcus asaccharolyticus Glutamate Dehydrogenase Purified by Dye-ligand Chromatography

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