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1.
Li X Wei Z Zhang M Peng X Yu G Teng M Gong W 《Biochemical and biophysical research communications》2007,354(1):21-26
CueO protein is a hypothetical bacterial laccase and a good laccase candidate for large scale industrial application. Four CueO crystal structures were determined at different copper concentrations. Low copper occupancy in apo-CueO and slow copper reconstitution process in CueO with exogenous copper were demonstrated. These observations well explain the copper dependence of CueO oxidase activity. Structural comparison between CueO and other three fungal laccase proteins indicates that Glu106 in CueO constitutes the primary counter-work for reconstitution of the trinuclear copper site. Mutation of Glu106 to a Phe enhanced CueO oxidation activity and supported this hypothesis. In addition, an extra alpha-helix from Leu351 to Gly378 covers substrate biding pocket of CueO and might compromises the electron transfer from substrate to type I copper. 相似文献
2.
Kunishige Kataoka Ryosuke Sugiyama Shun Hirota Megumi Inoue Kanae Urata Yoichi Minagawa Daisuke Seo Takeshi Sakurai 《The Journal of biological chemistry》2009,284(21):14405-14413
The mechanism of the four-electron reduction of dioxygen by a multicopper
oxidase, CueO, was studied based on reactions of single and double mutants
with Cys500, a type I copper ligand, and the noncoordinating
Asp112 and Glu506, which form hydrogen bonds with the
trinuclear copper center directly and indirectly via a water molecule. The
reaction of C500S containing a vacant type I copper center produced
intermediate I in an EPR-silent peroxide-bound form. The formation of
intermediate I from C500S/D112N was restricted due to a reduction in the
affinity of the trinuclear copper center for dioxygen. The state of
intermediate I was realized to be the resting form of C500S/E506Q and C500S of
the truncated mutant Δα5–7CueO, in which the 50 amino acids
covering the substrate-binding site were removed. Reactions of the recombinant
CueO and E506Q afforded intermediate II, a fully oxidized form different from
the resting one, with a very broad EPR signal, g < 2, detectable
only at cryogenic temperatures and unsaturated with high power microwaves. The
lifetime of intermediate II was prolonged by the mutation at Glu506
involved in the donation of protons. The structure of intermediates I and II
and the mechanism of the four-electron reduction of dioxygen driven by
Asp112 and Glu506 are discussed.CueO is a multicopper oxidase involved in a copper efflux system of
Escherichia coli
(1–3).
In contrast to other multicopper oxidases such as laccase and ascorbate
oxidase (4), CueO exhibits
strong activity toward cuprous ion but does not show activity toward most
organic substrates such as 2,6-dimethoxyphenol, catechol, and guaiacol, except
considerably low levels toward
2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS)2 and
p-phenylenediamine. This substrate specificity, unique to CueO,
originates in the methionine-rich helical region covering the
substrate-binding site
(5–7).
Nevertheless, CueO has the same catalytic copper centers as other multicopper
oxidases: a type I copper that mediates electron transfer and a trinuclear
copper center comprised of a type II copper and a pair of type III copper
atoms, where dioxygen is reduced to two water molecules
(5,
7). The type I copper is
responsible for the intense charge transfer band at 610 nm due to
Cys(S-)π → Cu2+ and the bands at 430,
∼500, and ∼750 nm due to the charge transfers His(N) →
Cu2+ and Cys(S-)σ →
Cu2+ and d-d transitions, respectively
(4). The type III copper atoms
bridged with a hydroxide ion afford an intense charge transfer band,
OH- → Cu2+ at ∼330 nm, whereas the type II
copper does not give a conspicuous band in the visible region. The type I and
II coppers give rise to EPR signals with the hyperfine splitting of small (6.7
milliteslas (mT)) and normal (18.5 mT) magnitudes, respectively, whereas the
type III copper atoms are EPR-silent because of the strong anti-ferromagnetic
interaction
(7–9).Special attention has been paid to the four-electron reduction of dioxygen
by multicopper and terminal oxidases because activated oxygen species such as
superoxide, peroxide, etc. are not formed or, if formed, are effectively
converted into water molecules without damage to protein molecules. Therefore,
this four-electron reduction of dioxygen by multicopper oxidases has been
expected to be applicable to biofuel cells
(10–12).
Two reaction intermediates have been detected during reactions of some
multicopper oxidases. One of them, intermediate I, could be trapped by the
following modified multicopper oxidases so as to interrupt the electron
transfer from the type I copper: a plant laccase whose type I copper was
substituted with mercury (13);
a mixed valent laccase in which the type I copper was oxidized, but the
trinuclear copper center was reduced
(14); and a Cys → Ser
mutant of bilirubin oxidase
(15) and Fet3p
(16) whose type I copper
center became vacant. Although the trinuclear copper center must be fully
reduced to produce intermediate I, it has been considered to be a two-electron
reduced form and, therefore, also called the peroxide intermediate
(13,
16). Another reaction
intermediate, II, also called the native intermediate, has been detected at
the final stage of a single turnover
(15,
17–19).
Four electrons have already been transferred to dioxygen in this intermediate,
and accordingly, intermediate II is in a fully oxidized form to give the
g < 2 EPR signal at cryogenic temperatures. Under catalytic
conditions, intermediate II is not detected because of its prompt conversion
to the fully reduced form for the next enzyme cycle without decaying to the
resting form. Both intermediates have a half-life in the order of seconds to
minutes, but information to directly show their structures has not been
obtained yet. They afford analogous absorption bands at ∼330–350,
450–470, and 680 nm, of which the former two bands have been assigned to
the charge transfer from a certain oxygen group to Cu2+ (σ
and π transitions) and the latter to the d-d transitions of the trinuclear
copper center in the cupric state. The d-d transitions of intermediate II are
masked by strong absorption due to the oxidized type I copper
(13–19).In the present study, we succeeded in trapping intermediates I and II from
reactions of a recombinant form of CueO (rCueO) and mutants altered at
Cys500, a ligand to the type I copper, and at Asp112 and
Glu506 located adjacent to the trinuclear copper center to modify
the dioxygen reduction process. The Asp residue is conserved in every
multicopper oxidase except for ceruloplasmin, which has Glu instead
(Fig. 1). According to the
x-ray crystal structures of rCueO
(5) and the truncated mutant,
Δα5–7CueO, missing the 50 amino acids covering the
substrate-binding site (Fig. 2)
(7,
20), Asp112 forms a
hydrogen bond with His448, a ligand to a type III copper, and
indirectly with the water molecule coordinating the type II copper through an
ordered water molecule. In a preliminary study on the Asp112
mutants (21), we showed that
this acidic amino acid functions in the binding of dioxygen at the trinuclear
copper center and may also be involved in the donation of protons to the
reaction intermediate(s). On the other hand, one to three acidic amino acids
are present in the spacers to connect the copper ligands of multicopper
oxidases, His-Cys-His-XXX-His-XXXX-Met-(Leu/Phe).
Fig. 2 shows that
Glu506 of CueO in this spacer is directly hydrogen-bonded with the
His143 ligand to one of the type III copper atoms and indirectly
with the hydroxide ion bridged between the type III copper atoms through an
ordered water molecule. Therefore, Glu506 is also speculated to
play a crucial role in the reduction of dioxygen. We singly and doubly mutated
Cys500, Asp112, and Glu506 of CueO to trap
intermediates I and II and to elucidate the mechanism behind the four-electron
reduction of dioxygen.Open in a separate windowFIGURE 1.Homology of amino acid sequence around the copper binding sites of
multicopper oxidase. The numbers 1, 2, and 3 represent
the type I, II, and III copper ligands, respectively. BO, Myrothecium
verrucaria bilirubin oxidase; RvLc, Rhus vernicifera laccase;
CpAO, Cucurbita pepo ascorbate oxidase; TvLc, Trametes
versicolor laccase; CcLc, Coprinus cinereus laccase;
Fet3p, multicopper oxidase from Saccharomyces cerevisiae;
CumA, multicopper oxidase from Pseudomonas putida;
CotA, multicopper oxidase from Bacillus subtilis;
SLAC, small laccase from Streptomyces coelicolor;
hCp, human ceruloplasmin. The single asterisk represents the
conserved acidic amino acid residue in all multicopper oxidases, and the
double asterisk represents Glu506 in CueO, which forms a
hydrogen bond with a His residue coordinating a type III copper and the
hydroxide ion bridged between type III coppers.Open in a separate windowFIGURE 2.Structure around the active site of the truncated mutant of CueO
(7). Type I,
II, and III coppers are represented as spheres. Small spheres, oxygen
atoms. The two networks of hydrogen bonds lead to the exterior of the protein
molecule, forming the pathway to let protons in and water molecules out.
Mutated amino acid residues, Cys500, Glu506, and
Asp112, and the networks of hydrogen bonds are indicated. 相似文献
3.
ATP is synthesized by an enzyme that utilizes proton motive force and thus nature creates various proton pumps. The best understood proton pump is bacteriorhodopsin (BR), an outward-directed light-driven proton pump in Halobacterium salinarum. Many archaeal and eubacterial rhodopsins are now known to show similar proton transport activity. Proton pumps must have a specific mechanism to exclude transport in the reverse direction to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Although an inward proton pump has neither been created naturally nor artificially, we recently reported that an inward-directed proton transport can be engineered from a bacterial rhodopsin by a single amino acid replacement Anabaena sensory rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria, possessing little proton transport activity. When we replace Asp217 at the cytoplasmic domain (distance ~15 Å from the retinal chromophore) to Glu, ASR is converted into an inward proton transport, driven by absorption of a single photon. FTIR spectra clearly show an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to normal proton pumps. 相似文献
4.
The active site of mannitol 2-dehydrogenase from Pseudomonas fluorescens (PfM2DH) is connected with bulk solvent through a narrow protein channel that shows structural resemblance to proton channels utilized by redox-driven proton pumps. A key element of the PfM2DH channel is the "mobile" Glu(292), which was seen crystallographically to adopt distinct positions up and down the channel. It was suggested that the "down → up" conformational change of Glu(292) could play a proton relay function in enzymatic catalysis, through direct proton shuttling by the Glu or because the channel is opened for water molecules forming a chain along which the protons flow. We report evidence from site-directed mutagenesis (Glu(292) → Ala) substantiated by data from molecular dynamics simulations that support a role for Glu(292) as a gate in a water chain (von Grotthuss-type) mechanism of proton translocation. Occupancy of the up and down position of Glu(292) is influenced by the bonding and charge state of the catalytic acid base Lys(295), suggesting that channel opening/closing motions of the Glu are synchronized to the reaction progress. Removal of gatekeeper control in the E292A mutant resulted in a selective, up to 120-fold slowing down of microscopic steps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD(+)-mannitol complex is promoted by a corresponding position change of Glu(292), which at physiological pH is associated with obligatory deprotonation of Lys(295) to solvent. These results underscore the important role of conformational dynamics in the proton transfer steps of alcohol dehydrogenase catalysis. 相似文献
5.
Kataoka K Komori H Ueki Y Konno Y Kamitaka Y Kurose S Tsujimura S Higuchi Y Kano K Seo D Sakurai T 《Journal of molecular biology》2007,373(1):141-152
CueO is a multicopper oxidase (MCO) that is involved in the homeostasis of Cu in Escherichia coli and is the sole cuprous oxidase to have ever been found. Differing from other MCOs, the substrate-binding site of CueO is deeply buried under a methionine-rich helical region including alpha-helices 5, 6, and 7 that interfere with the access of organic substrates. We deleted the region Pro357-His406 and replaced it with a Gly-Gly linker. The crystal structures of a truncated mutant in the presence and in the absence of excess Cu(II) indicated that the scaffold of the CueO molecule and metal-binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and its spectroscopic and magnetic properties due to four Cu centers were also conserved after truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca 10% that of recombinant CueO owing to the decrease in the affinity of the labile Cu site for Cu(I) ions, although activities for laccase substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and 2,6-dimethoxyphenol increased due to changes in the access of these organic substrates to the type I Cu site. The present engineering of CueO indicates that the methionine-rich alpha-helices function as a barrier to the access of bulky organic substrates, which provides CueO with specificity as a cuprous oxidase. 相似文献
6.
.Dr. M. Herrera-Marschitz M. Goiny Z. -B. You J. J. Meana E. Engidaworkl Y. Chen R. Rodriguez-Puertas C. Broberger K. Andersson L. Terenius T. Höpkfelt U. Ungerstedt 《Amino acids》1998,14(1-3):197-203
Summary There is immunohistochemical evidence suggesting that glutamate (Glu) is released from nerve terminals and acts, via several receptor subtypes, as a major excitatory neurotransmitter in the cortico-striatal pathway of the rat. Aspartate (Asp) is also present in cortico-striatal neurons, but its role as a neurotransmitter has been questioned, since, in contrast to Glu, it has not been demonstrated in presynaptic vesicles. Glu and Asp can be found at subM concentrations in the extracellular compartment of most areas of the basal ganglia. Their concentrations are largely regulated by transport mechanisms, but also by a synaptotagmin-dependent exocytotic release, and are sufficiently high to occupy junctional and extrajunctional receptors.We have investigated whether Glu and Asp release in the neostriatum can be selectively modulated by different neuronal systems. Dopamine (DA) and cholecystokinin (CCK) selectively stimulate Asp release, via D1 and CCKB receptor subtypes, respectively. Also opioid -agonists increase Asp release. We propose that the selective modulation of Asp release by D1–, CCKB- and agonists involves striatal neurons containing Asp, but not Glu. In contrast, local perfusion with the ,-opioid antagonist D-Phe-Cys-Tyr-D-Trp-Orn-ThrPen-Thr-NH2 (CTOP) increases both Glu and Asp release. This effect is probably exerted on cortico-striatal terminals, via presynaptic inhibitory -receptors. Thus, these results demonstrate that extracellular levels of Glu and Asp are modulated differentially by different neuronal systems, and suggest that in the neostriatum of the rat there are neuronal populations using Glu and/or Asp as messenger(s). 相似文献
7.
Melnik S. I. Lozovaya N. A. Tsintsadze T. Sh. Grebenuk S. E. Krishtal O. A. 《Neurophysiology》2002,34(2-3):102-105
Astroglia is capable of releasing glutamate (Glu) in concentrations sufficient to activate ionotropic Glu receptors. Provided the released Glu reaches the receptors in the postsynaptic density, it can desensitize them. We have tested this possibility in the hippocampal CA1 synapses of rats either by applying exogenous Glu to the CA1 neurons or activating Glu release by astroglia. We found that Glu does not reach the synapses due to the existence of a protective uptake cap, which is sensitive to dihydrokainate, an inhibitor of GLT-type Glu transporter(s). Our results suggest that extrasynaptic and postsynaptic densities of the membranes of CA1 neurons form separate compartments differing from each other in the mechanisms and efficiency of processing external Glu. This provides additional diversity to specialized regulation of synaptic transmission and electrical excitation of pyramidal neurons. 相似文献
8.
Nabedryk Eliane Breton Jacques Okamura Melvin Y. Paddock Mark L. 《Photosynthesis research》1998,55(2-3):293-299
In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon QB photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 Asn replacement on QB
–/QB FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 Asp, Arg M233 Cys, or Arg H177 His suppressor mutation. The proton uptake by Glu L212 upon QB
– formation, as reflected by the positive band at 1728 cm–1, is increased in the Asn M44 Asp and Arg H177 His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 Asn mutant RCs. Only the Arg M233 Cys suppressor mutation affected the 1728 cm–1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 Asn lesion. 相似文献
9.
The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a3/CuB centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different Em values of the electron acceptor in the binuclear site. 相似文献
10.
Ziemińska E Hilgier W Waagepetersen HS Hertz L Sonnewald U Schousboe A Albrecht J 《Neurochemical research》2004,29(11):2121-2123
Rat cerebral nonsynaptic mitochondria were incubated in medium containing 2 mM glutamine (Gln) or 2 mM glutamate (Glu), in the presence of a Gln uptake inhibitor histidine (His) as well as other basic amino acids, lysine and arginine (Lys, Arg) not inhibiting Gln uptake. Subsequently, the mitochondrial contents of Glu and Gln were determined by HPLC. Incubation in the presence of Glu alone increased the Glu content from 3.5 to 15 nmol/mg protein, without affecting the Gln content. On the other hand, incubation with Gln increased the content of Gln from 1.5 to 12 nmol/mg, and that of Glu to 10 nmol/mg. As expected, addition of His did not alter the Glu and Gln content resulting from incubation with Glu. However, His significantly decreased to almost the preincubation level the content of Glu in mitochondria incubated with Gln, without affecting the content of Gln. No other amino acid had any effect on these parameters. The results point to the existence of distinct Gln pools, one of which is accessible to external Gln via a His-sensitive transporter and is accessible for deamidation in the mitochondria.Special issue dedicated to Dr. Lawrence F. Eng. 相似文献
11.
S. Hanau L. Proietti d'EmpaireK. Montin C. CervellatiI. Capone F. Dallocchio 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(4):785-792
The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pKa of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pKa 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pKa, 7.17 and 9.64, in the enzyme–substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pKa from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pKa from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈ 6.1 kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240–325 cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pKa shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2–2.3 kcal/mol to set the enzyme into a catalytically competent form. 相似文献
12.
The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a3/CuB centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the "prepumped" proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different Em values of the electron acceptor in the binuclear site. 相似文献
13.
Summary The present study investigates the effects of phenylsuccinate (PS), an inhibitor of the mitochondrial ketodicarboxylate carrier (KCC), on release of-aminobutyric acid (GABA), glutamate (Glu), glutamine (Gln), and glycine (Gly), induced by potassium chloride (KCl) and by cardiac arrest caused by a halothane overdose. Microdialysates were collected from the hippocampus of anaesthetized rats, and analyzed by HPLC. Continuous perfusion of 50 mM PS through the dialysis probe, reduces release of GABA induced by KCl (50 mM for 10 min through the dialysis probe) by up to 72%. In addition, PS abolished KCl-induced release of Glu. Release of GABA during cardiac arrest was not reduced by PS, whereas PS reduced release of Glu in the early stage of cardiac arrest. PS furthermore increased the basal level of Gln, and reversed a decrease of Gln induced by cardiac arrest.It is proposed that the KCC is present in GABA'ergic neurons of the rat hippocampus, and that GABA, released by KCl, can be synthesized in a KCC dependent manner. It is also suggested that ischemia-induced release of GABA, to some extent, has a non-transmitter origin. The results furthermore indicate that uptake of Gln into GABA'ergic and Glu'ergic neurons is not regulated by simple demand mechanisms.Abbreviations PS
phenylsuccinate
- KCC
ketodicarboxylate carrier
- GABA
-aminobutyric acid
- Glu
glutamate
- Gln
glutamine
- Gly
glycine
-
-KG
-ketoglutarate
- Mal
malate
- KRB-buffer
Krebs-Ringer bicarbonate-buffer
- HPLC
high pressure liquid chromatography 相似文献
14.
Summary Nuclear Overhauser effects (NOE) were measured between water protons and protons of the glutamic acid side chain of the bicyclic decapeptide
in aqueous solution. Positive NOEs were observed between the CH2 group of Glu and the water resonance, with similar NOE intensities at pH 2.0 and pH 6.3 in both the laboratory frame and the rotating frame of reference. These results indicate that the residence times of the hydration water molecules near the side-chain methylene protons are shorter than 500 ps for both the charged form and the uncharged form of Glu, and hence comparable to the water residence times near uncharged amino acid side chains. Furthermore, this study shows that the acidic proton in protonated carboxylic acid groups is not likely to interfere with the observation of polypeptide-hydration water NOEs, which is in contrast to the hydroxyl protons of the side chains of serine, threonine and tyrosine.Abbreviations NOE
nuclear Overhauser effect
- NOESY
NOE spectroscopy in the laboratory frame
- ROESY
NOE spectroscopy in the rotating frame
- ID
one-dimensional
- 2D
two-dimensional
- HPLC
high-pressure liquid chromatography 相似文献
15.
O'Shea RD Fodera MV Apricó K Dehnes Y Danbolt NC Crawford D Beart PM 《Neurochemical research》2002,27(1-2):5-13
Removal of L-glutamate (Glu) from the synapse is critical to maintain normal transmission and to prevent excitotoxicity, and is performed exclusively by excitatory amino acid transporters (EAATs). We investigated the effects of substrates and blockers of EAATs on extracellular Glu and cellular viability in organotypic cultures of rat hippocampus. Seven-day treatment with a range of drugs (L-trans-pyrrolidine-2,4-dicarboxylate, (2S,4R)-4-methyl-glutamate, (±)-threo-3-methylglutamate and DL-threo--benzyloxyaspartate), in the presence of 300 M added Glu, resulted in increased extracellular Glu and a significant correlation between Glu concentration and cellular injury (as indicated by lactate dehydrogenase release). In contrast, (2S,3S,4R)-2-(carboxycyclopropyl)glycine (L-CCG-III) exerted a novel neuroprotection against this toxicity, and elevations in extracellular Glu were not toxic in the presence of this compound. Similar results were obtained following two-week treatment of cultures without added Glu. Whilst blockade of GLT-1 alone was relatively ineffective in producing excitotoxic injury, heteroexchange of Glu by EAAT substrates may exacerbate excitotoxicity. 相似文献
16.
Phenytoin was observed to inhibit competitively the sodium dependent high affinity synaptosomal transport of both glutamate (Glu) and -aminobutyric acid (GABA) withK
i values of 66±10 and 185±65 M, respectively. This constrasted with a previous report that the uptakes of Glu and GABA were enhanced by phenytoin. The degree of inhibition is dependent on the concentrations of the competing drug and substrate present. Taking the therapeutic levels of phenytoin and the overall brain Glu and GABA contents, the degrees of inhibition obtainble appear to be negligible. However, as most of the high levels of Glu and GABA in the brain are intracellular, Glu, and GABA concentrations in the microenvironment of the uptake sites may be sufficiently small so that the ability of phenytoin to inhibit Glu and GABA transport may contribute significantly to the anticonvulsant property of this drug. 相似文献
17.
Zielińska M Hilgier W Borkowska HD Oja SS Saransaari P Goryński P Albrecht J 《Neurochemical research》2002,27(1-2):37-42
Accumulation of taurine (Tau), glutamate (Glu) and glutamine (Gln) was measured in vivo in microdialysates of the rat striatum following a direct application to the microdialysis tube of 60 mM ammonium chloride which renders the final ammonia concentration in the extracellular space to 5 mM. The following compounds were coadministered with ammonia to distinguish between the different mechanisms that may underlie the accumulation of amino acids: ion transport inhibitors, diisothiocyanostilbene-2,28-disulfonate (DIDS) and furosemide, a Glu transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an NMDA receptor antagonist dizocilpine (MK-801) and an 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). Ammonia stimulated Tau accumulation in the microdialysates to 250% of the basal value. Furosemide did not significantly affect the stimulation by ammonia and DIDS only moderately depressed the effect. The ammonia-dependent Tau accumulation was increased by 50% in the presence of PDC and reduced by 35% in the presence dizocilpine and DNQX. In the microdialysates ammonia stimulated Glu and Gln accumulation somewhat less than Tau accumulation. Except for stimulation of Gln accumulation by DNQX, the effects were not modified by any of the cotreatments. The results are consistent with the assumption that ammonia stimulates Tau efflux mainly via activation of ionotropic Glu receptors. 相似文献
18.
Mikko Nikinmaa Frank B. Jensen 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1992,162(5):424-429
Summary The effects of nitrite-induced methaemoglobinaemia on adrenergic proton extrusion from rainbow trout red blood cells were studied using the pH-stat method. In control conditions adrenergic proton extrusion was completely inhibited by amiloride and was greater in deoxygenated than in oxygenated erythrocytes. Nitrite-induced methaemoglobinaemia was associated with a pronounced reduction in the catecholamine-stimulated proton efflux from both deoxygenated and oxygenated erythrocytes. In deoxygenated erythrocytes the initial proton efflux upon catecholamine stimulation decreased by 60–70%, while the percentage of methaemoglobin in the red cells increased from the control level of 1–3% to 20%. In oxygenated erythrocytes the decrease was 30% at the same methaemoglobin percentage range. It is suggested that the pronounced influence of nitriteinduced methaemoglobinaemia on adrenergic proton efflux results from an inhibition of the red cell sodium/proton exchanger by the R-like haemoglobin conformations.Abbreviations DIDS
4,4-diisothiocyanostilbene-2,2-disulfonic acid
- DMO
5,5-dimethyloxazolidine-2,4-dione
- RBC
red blood cell 相似文献
19.
N. S. Novichkova A. K. Romanova A. R. Ignat’ev V. A. Mudrik S. E. Permyakov B. N. Ivanov 《Russian Journal of Plant Physiology》2008,55(2):201-210
Effect of surplus glucose on physiological and biochemical parameters of leaves of different age was investigated in sugar beet (Beta vulgaris L., subsp. saccharifera) plants in the stages of vegetative growth (SVG). Early and late SVG were differentiated by the ratio between the weights of roots and aboveground organs (0.10 and 0.35, respectively). The excess of Glu was produced by incubation of the disks excised from detached leaves in water or 0.1 M Glu at radiant flux density of 250 μmol/(m2 s) with the light regime pattern described as night/day/night/light (8/16/8/3 h). In all the leaf disks incubated in water and glucose solution, the content of Glu and other soluble carbohydrates considerably increased as compared with their content in the leaves they were taken from. After disk incubation in water and glucose solution, the content of chlorophyll (a + b) rose as compared with its level in respective leaves in early SVG; in late SVG, it declined. In early SVG, the rate of the O2 photosynthetic evolution (Ph) in the ageing leaves under saturating concentration of NaHCO3 after incubation in water and Glu solution declined more considerably than in young leaves. In late SVG, incubation of leaf disks in water and Glu solution weakly affected P n. The rate of O2 dark consumption in the leaf disks of all the types of treatment increased after incubation in water and especially in Glu solution. Activity of soluble carbonic anhydrase (sCA) in the extracts from young leaves in early SVG after their incubation in water and Glu solution was essentially the same, but after the incubation of ageing leaves in Glu solution, it reliably decreased. In late SVG, sCA activity sharply decreased after incubation in water and Glu solution irrespective of the leaf age. In late SVG, activity of Rubisco in the young leaves did not change after their incubation in water but decreased after incubation of the leaves of the three ages in Glu solution. In early SVG, nonphotochemical fluorescence quenching (NPQ) in the young intact leaf was lower than in the ageing leaf, and after leaf incubation in water and Glu solution, it rose. In late SVG, the value of NPQ was greater than in early SVG and, in contrast to the leaves of early SVG, it declined after leaf incubation; in water, this decline was more pronounced than in the Glu solution. In early SVG, efficient quantum yield of photosystem II (PSII) was much greater than in late SVG and it declined in the leaves incubated with Glu. It was concluded that surplus Glu can maintain biosynthetic processes in the young leaves of young sugar beet plants (trophic function). A decline in the level of chlorophyll and the activities of sCA and Rubisco in the course of leaf development and senescence is considered as a symptom of the suppression of biosynthesis of proteins of chlorophyll-protein complexes and the enzymes (Rubisco and sCA). 相似文献
20.
Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga 总被引:1,自引:0,他引:1
Wada T Shimono K Kikukawa T Hato M Shinya N Kim SY Kimura-Someya T Shirouzu M Tamogami J Miyauchi S Jung KH Kamo N Yokoyama S 《Journal of molecular biology》2011,411(5):986-8898
Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light-dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 Å resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the “late proton release”. 相似文献