Purification and properties of NADP‐isocitrate dehydrogenase from the unicellular cyanobacterium <i>Synechocystis</i> sp. PCC 6803

Muro‐Pastor, M. Isabel; Florencio, Francisco J.

doi:10.1111/j.1432-1033.1992.tb19833.x

Cited by 67 publications

(55 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For immunoblot experiments, protein samples were electrophoresed on non-denaturating or SDSPAGE and electrotransferred to nitrocellulose filters. The filters were blocked and washed as previously described [33] and incubated with anti-GSIII serum diluted 1 : 1000 in 50 mM Tris/HCl, pH 8.0, 0.15 M NaCl (TridNaCl). Filters were then washed four times with Tris/NaCl plus 0.1 % Tween 20 (Tris/NaCl/Tween 20) and incubated in the same buffer containing 1 : 1000-diluted horseradish-peroxidase-conjugated anti-(rabbit) serum.…”

Section: Methodsmentioning

confidence: 99%

Purification and Characterization of A New Type of Glutamine Synthetase from Cyanobacteria

García‐Domínguez

Reyes

Florencio

1997

European Journal of Biochemistry

View full text Add to dashboard Cite

The cyanobacterium Synechocystis sp. PCC 6803 contains two genes encoding two different types of glutamine synthetases (GS), glnA and glnN. The first codes for a typical prokaryotic GS type I and the second one codes for a GS type 111, different in amino acid sequence to the prokaryotic GSI and the eukaryotic GSI1. The glnN gene has been expressed in Escherichia coli and the corresponding protein purified almost to homogeneity (92%). The native enzyme (500 kDa) was composed of six identical subunits with an apparent molecular mass of 80 kDa. The protein was strongly stabilized in the presence of Mn2+ but not with other divalent cations. Biosynthetic activity of GSIII required the same substrates and cofactors as GSI and GSII enzymes. Apparent Kn, values for ATP, glutamate and ammonium were 0.43 mM, 0.9 mM and 0.19 mM, respectively. The enzyme was weakly inhibited by several amino acids and strongly inhibited by ADP. Synechocystis GSIIT was also inhibited by L-methionine sulfoximine and DL-phosphinotricin, two transition-state analogs of the GS reaction mechanism. GSIII has also been purified from nitrogen-starved Synechocystis 6803 glnA mutant cells, demonstrating that the GS activity, strongly induced under nitrogen starvation in these cells, corresponds to the glnN gene product. In addition, a Synechocystis 6803 glnN mutant lacks the corresponding 80-kDa protein (GSIII). Polyclonal antibodies specific for GSIII cross-react with GSIII from other cyanobacteria. In all the strains analysed, levels of GSIII protein increased under nitrogen deficiency. These data suggest that GSIII is specifically required under conditions of nitrogen starvation.

show abstract

Section: Methodsmentioning

confidence: 99%

Purification and Characterization of A New Type of Glutamine Synthetase from Cyanobacteria

García‐Domínguez

Reyes

Florencio

1997

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The lysate was centrifuged at 12,000 ϫ g for 15 min, and the resulting supernatant constituted the cell extract. NADP ϩ -specific IDH activity was measured as previously described (44). Units are expressed as micromoles of NADPH produced per minute.…”

Section: Methodsmentioning

confidence: 99%

“…strain PCC 7120 (45). In both cases, the enzymes show kinetic and physicochemical parameters similar to those of the NADP ϩ -IDH from Escherichia coli (44,45,48). The Anabaena icd gene has been cloned by complementation of an E. coli icd mutant (45).…”

mentioning

confidence: 99%

See 1 more Smart Citation

The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria

1996

View full text Add to dashboard Cite

Cyanobacteria are photosynthetic prokaryotes that have an incomplete tricarboxylic acid cycle, lacking ␣-ketoglutarate dehydrogenase and succinyl-coenzyme A synthetase activities (46, 54). The NADP ϩ -isocitrate dehydrogenase (IDH) (EC 1.1.1.42) reaction represents a terminal step in carbon flow; thus, the role of this enzyme is the provision of biosynthetic precursors rather than energy production. The ␣-ketoglutarate produced in the NADP ϩ -IDH reaction is required for ammonium assimilation through the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway (38) and is a key metabolite in the linking of nitrogen and carbon metabolism.IDHs have been purified and characterized from a variety of sources (8); most bacteria have only an NADP ϩ

show abstract

“…Western blot analysis of crude extract samples subjected to denaturby the glutamine synthetase-glutamate synthase (GS-GOGAT) ing or native electrophoresis were carried out as previously described pathway [4]. In the unicellular cyanobacterium Synechocystis [10]. Purified polyclonal monospecific antibodies obtained against sp.…”

mentioning

confidence: 99%

A novel mechanism of glutamine synthetase inactivation by ammonium in the cyanobacterium Synechocystis sp. PCC 6803. Involvement of an inactivating protein

Reyes

Florencio

1995

FEBS Letters

View full text Add to dashboard Cite

s (in 15 s periods). The homogenated was centrifuged at 40,000 × g for Cyanobacteria; Ammonium assimilation; Enzyme inactivation 15 min and the resulting supernatant constituted the cell-free extract. Polyacrylamide gel electrophoresis and Western blot analysisSamples of crude extract were run in polyacrylamide SDS gel, using 6% (w/v) to 10% acrylamide gradient gel, according to Laemmli [8]. IntroductionMarker proteins were Prestained SDS-PAGE Standards (low and high range) from Bio-Rad Laboratories. Non-denaturing gel electrophoresis was carried out in 6.25% (w/v) acrylamide gels and run at 4°C. IdentiGlutamine synthetase (GS) (EC 6.3.1.2) is a key enzyme in fication of the glutamine synthetase activity in non-denaturing gel elec-2+ the nitrogen assimilation process in most microorganisms. This trophoresis was carried out by the Mn -dependent y-glutamyl-transcentral role of GS requires the synthesis and the activity of this ferase assay [9], modified as follows: after non-denaturing PAGE, the gel was incubated at 30°C in a reaction mixture containing, in a final enzyme to be finely regulated in response to the available nitrovolume of 10 ml, 600/zmol of HEPES-NaOH buffer (pH 7.0), 400/zmol gen source [1,2]. GS from Escherichia coli and other GramofL-glutamine,40/lmolofMnCl:, 600/zmolofhydroxylamine, 10/lmol negative bacteria is modulated at the activity level by a mechaof ADP and 200/~mol of sodium arsenate. After 20 rain the reaction nism of adenylylation/deadenylylation of the enzyme, being was stopped by transferring the gel to FeCI 3 in acid solution that stains active the deadenylylated form [3]. the bands where y-glutamylhydroxamate was formed by the GS activityIn cyanobacteria, ammonium assimilation takes place mainly [9]. Western blot analysis of crude extract samples subjected to denaturby the glutamine synthetase-glutamate synthase (GS-GOGAT) ing or native electrophoresis were carried out as previously described pathway [4]. In the unicellular cyanobacterium Synechocystis [10]. Purified polyclonal monospecific antibodies obtained against sp. PCC 6803 we have previously reported a short-term ammoSynechococcus sp. PCC 6301 glutamine synthetase were used [11].nium-promoted GS inactivation, in a process that comprises 2.4. Cross-linking treatment the non-covalent binding of a phosphorylated factor, whose Cross-linking reactions were performed at 25°C in 50 mM HEPESnature was then unknown [5,6]. The inactive GS could be reacNaOH buffer, pH 7.0, by addition of l-ethyl-3-(3-dimethylaminoprotivated in vitro by different treatments that include alkaline pyl)carbodiimide (EDC) to a final concentration of 4 mM. Final volphosphatase, increasing pH or high ionic strength [6]. In this.ume of cross-linking reaction was 200 ~tl with about 2 mg of total protein. Aliquots of 20/zl were taken at various intervals and mixed study we show for the first time, by using Western blot analysis with an equal volume of dissociation buffer (1% SDS, 1% 2-mercapand cross-linking techniques, that the ammonium-promoted toethanol, 30/...

show abstract

Purification and properties of NADP‐isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803

Cited by 67 publications

References 34 publications

Purification and Characterization of A New Type of Glutamine Synthetase from Cyanobacteria

Purification and Characterization of A New Type of Glutamine Synthetase from Cyanobacteria

The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria

A novel mechanism of glutamine synthetase inactivation by ammonium in the cyanobacterium Synechocystis sp. PCC 6803. Involvement of an inactivating protein

Contact Info

Product

Resources

About