Regulation of glutamine synthetase.  XI.  The nature and implications of a lag phase in the Escherichia coli glutamine synthetase reaction

Kingdon, Henry S.; Hubbard, J. S.; Stadtman, Earl R.

doi:10.1021/bi00846a016

Cited by 135 publications

(94 citation statements)

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“…Transferase activity was measured by the y-glutamyl transferase reaction (Ferguson & Sims, 1971). One unit of enzyme activity is defined as the formation of 1 pmol yglutamylhydroxamate min-' at 37 "C. Biosynthetic activity was measured either according to Bender et al (1977), except that cetyltrimethylammonium bromide was omitted, or with the coupled assay system described by Kingdon et al (1968), or by the formation of radioactive glutamine, as described by Prusiner & Milner (1970). In the first assay, y-glutamylhydroxamate is measured using glutamate in the reaction mixture and the unit of activity is defined as in the case of the transferase reaction.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were removed at the times indicated and cell-free extracts were assayed for transferase (0) activity or for GSII immunoreactive material by Elisa (+). Values represent the means of duplicate or triplicate determinations The extracts were also assayed for biosynthetic activity, either measuring the formation of y-glutamylhydroxamate formation (Bender et al, 1977) or ADP formation (Kingdon et al, 1968). Data (not shown) superimpose the transferase and ELISA curves, respectively.…”

Section: Analysis Of Gsii In Cell-free Extractsmentioning

confidence: 99%

“…The half-lives of disappearance of transferase and biosynthetic activities measured according to Bender (1977) were 90 and 60 min, respectively. On the other hand, biosynthetic activity measured as ADP formation in the coupled assay of Kingdon et al (1968) decreased with a half-life of 4 h. GSII was partially purified from cells harvested at 0 and 3 h after NH4Cl treatment. The purification was as described in Methods except that the (NH4)*S04 precipitation and the final Mono Q Sepharose step were omitted.…”

Section: Analysis Of Gsii In Cell-free Extractsmentioning

confidence: 99%

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Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

Manco

Rossi²,

Defez³

et al. 1992

Journal of General Microbiology

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Glutamine synthetase I1 (GSII) was purified to homogeneity from Rhizobium leguminosarum biovar viceae and characterized.-The Sequence of 26 m i n o acid?esidues from the amino-terminal end bf the protein showed high similarity with the sequence of GSII from Bradyrhizobium japonicum or from Rhizobium meliloti. Non-denaturing PAGE showed that GSII, either in crude extracts or in the pure state, was a mixture of an octamer and a tetramer and that under specific conditions the octamerltetramer ratio could be modified in either direction. The pure enzyme was used to raise an antiserum which was highly specific. Addition of NH4CI to a bacterial culture derepressed for GSII caused a specific decrease in transferase activity, faster than the one observed when the amount of immunoreactive material was measured by different methods. On the other hand, biosynthetic activity, measured as the rate of ADP or glutamine formation, paralleled the rate of decrease in immunoreactive material. A partially purified enzyme preparation retained this dissociation of kinetic parameters, strongly suggesting a post-translational modification. These findings are discussed with respect to the possible role of GSII in the Rhizobium-legume symbiosis.

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Section: Methodsmentioning

confidence: 99%

Section: Analysis Of Gsii In Cell-free Extractsmentioning

confidence: 99%

Section: Analysis Of Gsii In Cell-free Extractsmentioning

confidence: 99%

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Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

Manco

Rossi²,

Defez³

et al. 1992

Journal of General Microbiology

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“…The uridylylated form of Pi, promotes the adenylyltransferase (ATase)-dependent deadenylylation of GS, while the unmodified form promotes the ATase-dependent adenylylation [5,6]. Under conditions of nitrogen limitation, covalent attachment of UMP to a tyrosyl residue at each of the four P,, subunits [6,7] is catalyzed by uridylyltransferase (UTase), while under conditions of nitrogen excess, hydrolysis of the bound UMP is catalyzed by the uridylyl-removing enzyme (UR) [6,8] Ahhrrwatzotzs. CETAB, cetyl trimethyl ammonium bromide; SVPD.…”

Section: Introductionmentioning

confidence: 99%

Uridylylation of the P_II protein in Rhizobium leguminosarum

et al. 1993

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Permeabilization with cetyl trimethyl ammonium bromide was used to study the post‐translational modification of the PII protein in Rhizobium leguminosarum. Upon incubation with radioactive UTP a single band was obtained after SDS‐PAGE and autoradiography. RNase resistance and snake venom phosphodiesterase sensivity showed that radioactivity was bound through a phosphodiester bond to a protein which was absorbed by an antiserum specific for the PII protein. Uridylylation of the PII protein was shown to be dependent on the modifications of the glutamine/α‐ketoglutarate ratio.

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“…Enzyme activities were determined by the standard yglutamyl transferase assay (Woolfolk et al, 1966) and by the biosynthetic assay as described by Kingdon et al (1968). Characterization of the mutant proteins is described elsewhere .…”

Section: Protein Purification and Characterizationmentioning

confidence: 99%

Time‐resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: Conformational analysis of intermediates and transition‐state complexes

Atkins

Villafranca

1992

Protein Science

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Single tryptophan-containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency-domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn-bound wild-type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp-57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp-158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry30,3406-3416). In addition, Trp-57 and Trp-158 both exhibit an ATP-induced increase in the relative fraction of the long lifetime component, whereas only Trp-57 is affected by this ligand at pH 7.5. The transition-state analogue L-methionine-(R,S)-sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp-158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the yglutamylphosphate-ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp-158, but has no effect on Trp-57. Similar results were obtained when ATP was added to the MSOX complex; Trp-57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp-158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX-ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp-57 loop and the central loop (Trp-158) during several intermediate stages of catalysis.Keywords: Escherichia coli; fluorescence studies; glutamine synthetase; transition-state complexes; tryptophan mutants Glutamine synthetase (GS) plays a central role in the nitrogen metabolism of both prokaryotes and eukaryotes, catalyzing the ATP-dependent condensation of glutamate with ammonia to form glutamine (Ginsburg, 1972;Stadtman & Ginsburg, 1974). The bacterial enzymes consist of a highly symmetrical dodecamer formed by two face-toReprint requests to: Joseph J. Villafranca, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.Abbreviations: W57L, the site-directed mutant that has Trp-57 replaced by leucine; W158S or WlSSF, the site-directed mutants that have Trp-158 replaced by serine or phenylalanine; glutamylphosphate-ADP-GS, the ternary complex formed after the addition of ATP and glutamate to glutamine synthetase.

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Regulation of glutamine synthetase. XI. The nature and implications of a lag phase in the Escherichia coli glutamine synthetase reaction

Cited by 135 publications

References 10 publications

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

Uridylylation of the P_II protein in Rhizobium leguminosarum

Time‐resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: Conformational analysis of intermediates and transition‐state complexes

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Regulation of glutamine synthetase. XI. The nature and implications of a lag phase in the Escherichia coli glutamine synthetase reaction

Cited by 135 publications

References 10 publications

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

Uridylylation of the PII protein in Rhizobium leguminosarum

Time‐resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: Conformational analysis of intermediates and transition‐state complexes

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Uridylylation of the P_II protein in Rhizobium leguminosarum