ADP, chloride ion, and metal ion binding to bovine brain glutamine synthetase

Maurizi, Michael R.; Pinkofsky, Harold B.; Ginsburg, Ann

doi:10.1021/bi00390a021

Cited by 30 publications

(29 citation statements)

References 28 publications

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“…In Vitro Transcription and Translation-The short and long transcripts of canine GS, covering the exon 1 to exon 7, were reverse transcribed with oligo(dT) 30 , amplified with primers cGSBamHI-f (5Ј-GGGGGATCCAGAGCCGAGAATGGGAGCGG-3Ј; AF544242, nt [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and cGSXhoI-r1 (5Ј-GGGCTCGAGTTAGTTTTTGTACTGGAAG-GG-3Ј; AF544242, nt 1369 -1349), and subcloned into the BamHI and XhoI sites of pcDNA3.1(ϩ) (Invitrogen), yielding pSGS and pLGS, respectively. The linearized templates of 5 g by digestion with XhoI restriction enzyme were used for in vitro transcription using the mMESSAGEmMACHINE TM kit (Ambion) to obtain capped transcripts.…”

Section: Methodsmentioning

confidence: 99%

“…Further addition of MnSO 4 and MgSO 4 to the chloridetreated SGS resulted in 85% quenching relative to the untreated. For LGS, quenching was 17% with MnSO 4 , whereas MgSO 4 increased fluorescence of about 5-6%.…”

Section: Figmentioning

confidence: 97%

“…When MSOX was titrated in the presence of 50 mM L-glutamate, the K i values were 0.067 and 0.124 mM for SGS and LGS, respectively ( Table I), indicating that the affinity for MSOX is lower in LGS than in SGS. The affinity changes for ATP and MSOX suggest that the binding sites for both (4) demonstrated that bindings of various effectors, such as chloride, manganese, and magnesium, to allosteric or activator site of the bovine brain GS cause a conformational change as monitored by fluorescence quenching, without affecting the oligomerization state. There are seven tryptophans and fifteen tyrosines in the short GS protein, whereas the long one contains an extra tryptophan in the N-terminal peptide (Fig.…”

Section: Figmentioning

confidence: 98%

“…2). To assess whether the extended peptide of LGS affects the conformation of GS, we monitored fluorescence quenching by the presence of KCl, MnSO 4 enzyme itself (Fig. 5, F o ).…”

Section: Figmentioning

confidence: 99%

“…The bindings of chloride and manganese/magnesium ions to allosteric and activator sites of GS, respectively, cause changes in GS conformation, without affecting its oligomerization (4). As an essential trace metal in vivo, the manganese ion is mostly (ϳ80%) bound to GS in astrocytes (5), although its concentration is variable in other mammalian tissues.…”

mentioning

confidence: 99%

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N-terminal Extension of Canine Glutamine Synthetase Created by Splicing Alters Its Enzymatic Property

Shin

Park

2004

Journal of Biological Chemistry

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It was found that an extra exon exists in the first intron of glutamine synthetase gene, generated by means of alternative splicing (Shin, D., Park, S., and Park, C. (2003) Biochem. J. 374, 175-184). Inclusion of this exon decreased the translation of glutamine synthetase (GS) in human, dog, and mouse. When translated in vitro with the canine GS transcript containing the exon, we obtained two different species of GS enzymes. Besides the known 45-kDa protein, the extended form of GS was identified with additional 40 amino acids on its N-terminal end. An upstream ATG in the extra exon served as a translation initiator for the long form of GS. When the long transcript was translated in vivo in animal cells, only the long GS was expressed. On the other hand, the long GS is less predominant relative to the short one in canine tissues including brain and liver. Subcellular fractionation of canine brain revealed that the long GS is present in all cellular compartments as is the short one, which is consistent with fluorescence microscopy data obtained with green fluorescent protein fused to GS. The short (SGS) and long (LGS) forms of canine GS were purified in Escherichia coli and shown to have similar K m values for L-glutamate and hydroxylamine. However, the K m values for ATP were slightly altered, 1.3 and 1.9 mM for the short and long GSs, respectively. The K i s for L-methionine-S-sulfoximine (MSOX), a highly potent ATP-dependent inactivator of GS, were considerably different such that the values are 0.067 and 0.124 mM for the short and long forms, respectively. When the intrinsic fluorescences of tryptophans were monitored upon bindings of chloride and metal ions without any effect on the oligomeric state, the pattern of quenching in LGS was significantly different from that of SGS. Taken together, the N-terminal extension in the long isoform of GS induces a conformational change of core enzyme, leading to a change in affinity to its substrates as well as in the effector-induced conformational alterations.Glutamine synthetase (GS 1 ; EC 6.3.1.2; L-glutamate ammonia ligase) is an enzyme that catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine, and thus plays a critical role in eliminating the excitotoxic glutamate in animal brains (1). The expression of eukaryotic GS is regulated at transcriptional and post-transcriptional levels and becomes unstable with oxidation mediated by metal or free radical peptides, generated from a fragmentation of ␤-amyloid, and by growth hormone or glutamine (2).The mammalian GS protein has been reported to form an eight-subunit oligomer (3) with unknown three-dimensional structure. The bindings of chloride and manganese/magnesium ions to allosteric and activator sites of GS, respectively, cause changes in GS conformation, without affecting its oligomerization (4). As an essential trace metal in vivo, the manganese ion is mostly (ϳ80%) bound to GS in astrocytes (5), although its concentration is variable in other mammalian tissues. Magnesium is also bound to m...

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Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 97%

Section: Figmentioning

confidence: 98%

“…2). To assess whether the extended peptide of LGS affects the conformation of GS, we monitored fluorescence quenching by the presence of KCl, MnSO 4 enzyme itself (Fig. 5, F o ).…”

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

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N-terminal Extension of Canine Glutamine Synthetase Created by Splicing Alters Its Enzymatic Property

Shin

Park

2004

Journal of Biological Chemistry

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Deciphering interactions of the aminoglycoside phosphotransferase(3′)‐IIIa with its ligands

Wu¹,

Serpersu²

2009

Biopolymers

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Aminoglycoside phosphotransferase(3')-IIIa (APH) is the enzyme with broadest substrate range among the phosphotransferases that cause resistance to aminoglycoside antibiotics. In this study, the thermodynamic characterization of interactions of APH with its ligands are done by determining dissociation constants of enzyme-substrate complexes using electron paramagnetic resonance and fluorescence spectroscopy. Metal binding studies showed that three divalent cations bind to the apo-enzyme with low affinity. In the presence of AMPPCP, binding of the divalent cations occurs with 7-to-37-fold higher affinity to three additional sites dependent on the presence and absence of different aminoglycosides. Surprisingly, when both ligands, AMPPCP and aminoglycoside, are present, the number of high affinity metal binding sites is reduced to two with a 2-fold increase in binding affinity. The presence of divalent cations, with or without aminoglycoside present, shows only a small effect (<3-fold) on binding affinity of the nucleotide to the enzyme. The presence of metal-nucleotide, but not nucleotide alone, increases the binding affinity of aminoglycosides to APH. Replacement of magnesium (II) with manganese (II) lowered the catalytic rates significantly while affecting the substrate selectivity of the enzyme such that the aminoglycosides with 2'-NH(2) become better substrates (higher V(max)) than those with 2'-OH.

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Manganese potentiates nuclear factor‐κB‐dependent expression of nitric oxide synthase 2 in astrocytes by activating soluble guanylate cyclase and extracellular responsive kinase signaling pathways

Moreno

Sullivan

Carbone

et al. 2008

J of Neuroscience Research

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Inflammatory activation of glial cells is associated with neuronal injury in several degenerative movement disorders of the basal ganglia, including manganese neurotoxicity. Manganese (Mn) potentiates the effects of inflammatory cytokines on nuclear factor-κB (NF-κB)-dependent expression of nitric oxide synthase 2 (NOS2) in astrocytes, but the signaling mechanisms underlying this effect have remained elusive. It was postulated in the present studies that direct stimulation of cGMP synthesis and activation of mitogen-activated protein (MAP) kinase signaling pathways underlies the capacity of Mn to augment NF-κB-dependent gene expression in astrocytes. Exposure of primary cortical astrocytes to a low concentration of Mn (10 µM) potentiated expression of NOS2 mRNA and protein along with production of NO in response to interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα), which was prevented by overexpression of dominant negative IκBα. Mn also potentiated IFNγ- and TNFα-induced phosphorylation of extracellular response kinase (ERK), p38, and JNK, as well as cytokine-induced activation of a fluorescent NF-κB reporter construct in transgenic astrocytes. Activation of ERK preceded that of NF-κB and was required for maximal activation of NO synthesis. Independently of IFNγ/TNFα, Mn-stimulated synthesis of cGMP in astrocytes and inhibition of soluble guanylate cyclase (sGC) abolished the potentiating effect of Mn on MAP kinase phosphorylation, NF-κB activation, and production of NO. These data indicate that near-physiological concentrations of Mn potentiate cytokine-induced expression of NOS2 and production of NO in astrocytes via activation of sGC, which promotes ERK-dependent enhancement of NF-κB signaling.

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ADP, chloride ion, and metal ion binding to bovine brain glutamine synthetase

Cited by 30 publications

References 28 publications

N-terminal Extension of Canine Glutamine Synthetase Created by Splicing Alters Its Enzymatic Property

N-terminal Extension of Canine Glutamine Synthetase Created by Splicing Alters Its Enzymatic Property

Deciphering interactions of the aminoglycoside phosphotransferase(3′)‐IIIa with its ligands

Manganese potentiates nuclear factor‐κB‐dependent expression of nitric oxide synthase 2 in astrocytes by activating soluble guanylate cyclase and extracellular responsive kinase signaling pathways

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