Purification and properties of glutamate dehydrogenase from a thermophilic bacillus

Epstein, Israel; Grossowicz, N.

doi:10.1128/jb.122.3.1257-1264.1975

Cited by 24 publications

(10 citation statements)

References 19 publications

(17 reference statements)

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“…However, the molecular weight of mammalian brain glutamine synthetase (Denman & Wedler, 1984) depended upon Mn2+ and protein concentrations, and it could have a molecular weight as high as I X 106 and above. The molecular weight of C. pasteurianum glutamine synthetase is, however, comparable to that found for glutamate dehydrogenases from a thermophilic bacillus (Epstein & Grossowicz, 1975) and beef liver (Sund & Burchard, 1968), i.e., 2 X I06, and that of rabbit skeletal muscle phosphorylase b kinase, which is 1 X 106 (DcLangc et al, 1968).…”

Section: Resultsmentioning

confidence: 57%

Purification and characterization of glutamine synthetase from Clostridium pasteurianum

Krishnan¹,

Singhal²,

Dua³

1986

Biochemistry

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Glutamine synthetase from Clostridium pasteurianum grown on molasses as the sole carbon source and ammonium chloride as the nitrogen source has been purified to homogeneity (45-fold) with 32% recovery. The procedure involves ammonium sulfate precipitation and chromatography on a combined Sepharose 4B/DEAE-Sephadex A-50 column. The purified enzyme being very unstable was stabilized by the addition of 25% (v/v) glycerol. The enzyme has an unusually high molecular weight of 1 X 10(6) and 20 subunits of Mr 50 000 each, as determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively. It has an absorption maximum at 280 nm and a fluorescence emission maximum at 380 nm when excited at 280 nm. Its substrate binding pattern as studied by fluorescence quenching studies is different from that of the Escherichia coli enzyme. Both the gamma-glutamyltransferase and synthetase activities reside in the same protein as the ratio of the two activities at each step of purification remains constant and the enzyme exhibits optimal transferase and synthetase activities at the same pH (7.2) and temperature (50 degrees C). The thermal stabilities of both activities were also similar, and decay of both the activities at 50 degrees C ran parallel. The enzyme shows stabilization by substrates, as L-glutamate, Mg2+, and ATP + Mg2+ protected both the synthetase and gamma-glutamyltransferase activities against thermal inactivation. Storage in 25% (v/v) glycerol enhanced the thermal stability of glutamine synthetase. Metal ion requirement and substrate specificity of the enzyme have been examined. Maximum synthetase activity occurs when [Mg2+]: [ATP] = 2. The Km app values are as follows (in parentheses): ATP (0.34 mM), NH2OH (0.4 mM in the synthetase reaction and 4.1 mM in the transferase reaction), glutamine (14.7 mM), ADP (3.8 X 10(-4) mM), arsenate (2.5 mM), and L-glutamate (3.4 mM, 22.2 mM). The enzyme exhibits negative cooperativity in the binding of glutamate. Amino acids such as L-serine, glycine, L-alanine, and L-aspartic acid inhibit the enzyme.

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Section: Resultsmentioning

confidence: 57%

Purification and characterization of glutamine synthetase from Clostridium pasteurianum

Krishnan¹,

Singhal²,

Dua³

1986

Biochemistry

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“…The Km value of GDH obtained for ammonia (20.2 mM) is relatively high compared with that for 2-ketoglutarate. However, this is not unusual as GDHs from several other sources have similarly high Km values (11,13,14).…”

Section: Discussionmentioning

confidence: 95%

“…Attempts to reconstitute activity by recombining fractions from such columns were also unsuccessful. GDH enzymes from several other organisms have molecular weights in the region of 300,000 (10,13,14,23,24); but the NAD-dependent enzyme from P. aeruginosa has a molecular weight of 110,000 (15), and, on the other extreme, Epstein and Grossowicz reported that the enzyme from a thermophilic bacillus had a molecular weight of 2 x 106 (11). Denaturing SDS-electrophoresis revealed a minimum molecular weight of 50,000, indicating that the enzyme is composed of four subunits.…”

Section: Discussionmentioning

confidence: 99%

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NADP-dependent glutamate dehydrogenase from a facultative methylotroph, Pseudomonas sp. strain AM1

Bellion¹,

Tan²

1984

J Bacteriol

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The NADP-dependent glutamate dehydrogenase (EC 1.4.1.4.) elaborated by the methylotrophic bacterium Pseudomonas sp. strain AM1 when growing on succinate and ammonium chloride was studied. The enzyme, which has a pH optimum of 9.0, was purified 140-fold and shown to have Km values of 20.2 mM, 0.76 mM, 0.033 mM, and 31.6 mM for ammonia, a-ketoglutarate, NADPH, and glutamate, respectively. The native molecular weight was determined by polyacrylamide gel electrophoresis to be 190,000, and electrophoresis under denaturing conditions in the presence of sodium dodecyl sulfate revealed a minimum molecular weight of 50,000. The enzyme was highly specific; NADH was unable to replace NADPH in the reaction, various ot-keto acids could not replace a.-ketoglutarate, and neither methylamine nor hydroxylamine could substitute for ammonia. Glutamate dehydrogenase was synthesized by the bacteria only when ammonia was its nitrogen source and was repressed if methylamine or nitrate were provided as sources of nitrogen instead of ammonia.

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“…Intensive studies on nitrogen assimilation in this genus revealed the existence of a NAD-GIuDH in B. pasteurii [12], Bo subtilis strain 168 [13] and B. thuringiensis var. thuringiensis Berliner [14], while B. azotofvcans [15], B. macerans [16], B. megaterium [17], B. mycoides [11], B. polymyxa [18] or a thermophilic member of the genus [19] have been described to be devoid of NAD-GluDH activity. Traces of NAD-GluDH activity have been observed in B. fastidiosus under certain growth conditions [20] and were attributed to an unspecificity of NADP-GluDH for the co-enzyme.…”

Section: Introductionmentioning

confidence: 99%

Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species

Jahns

1992

FEMS Microbiology Letters

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A nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.3) inactivated by incubation at low temperatures was detected in several species of the genus Bacillus, including strains of B. cereus, B. laterosporus, B. lentus, B. panthotenicus, B. pasteurii, B. sphaericus, B. stearothermophilus, B. subtilis and B. thuringiensis. Incubation of cell-free extracts of these strains at 0 degrees C resulted in an 80-100% inactivation of NAD-GluDH activity within 120 min. The addition of 20% glycerol protected the enzyme from this inactivation in the cold. Strains of B. fastidiosus, B. licheniformis, B. macerans, B. megaterium and B. pumilus were found to lack NAD-GluDH activity.

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Purification and properties of glutamate dehydrogenase from a thermophilic bacillus

Cited by 24 publications

References 19 publications

Purification and characterization of glutamine synthetase from Clostridium pasteurianum

Purification and characterization of glutamine synthetase from Clostridium pasteurianum

NADP-dependent glutamate dehydrogenase from a facultative methylotroph, Pseudomonas sp. strain AM1

Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species

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