Bicarbonate requirement for the water-oxidizing complex of photosystem II.
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It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII. (+info)
Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease.
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The accumulation of amyloid beta protein (Abeta) in the Tg2576 mouse model of Alzheimer's disease (AD) was evaluated by ELISA, immunoblotting, and immunocytochemistry. Changes in Abeta begin at 6-7 months as SDS-insoluble forms of Abeta42 and Abeta40 that require formic acid for solubilization appear. From 6 to 10 months, these insoluble forms increase exponentially. As insoluble Abeta appears, SDS-soluble Abeta decreases slightly, suggesting that it may be converting to an insoluble form. Our data indicate that it is full-length unmodified Abeta that accumulates initially in Tg2576 brain. SDS-resistant Abeta oligomers and most Abeta species that are N-terminally truncated or modified develop only in older Tg2576 mice, in which they are present at levels far lower than in human AD brain. Between 6 and 10 months, when SDS-insoluble Abeta42 and Abeta40 are easily detected in every animal, histopathology is minimal because only isolated Abeta cores can be identified. By 12 months, diffuse plaques are evident. From 12 to 23 months, diffuse plaques, neuritic plaques with amyloid cores, and biochemically extracted Abeta42 and Abeta40 increase to levels like those observed in AD brains. Coincident with the marked deposition of Abeta in brain, there is a decrease in CSF Abeta and a substantial, highly significant decrease in plasma Abeta. If a similar decline occurs in human plasma, it is possible that measurement of plasma Abeta may be useful as a premorbid biomarker for AD. (+info)
Active-site characterization of Candida boidinii formate dehydrogenase.
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NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii was cloned and expressed to a high level in Escherichia coli (20% of soluble E. coli protein). Molecular modelling studies were used to create a three-dimensional model of C. boidinii FDH, based on a known structure of the Pseudomonas sp. 101 enzyme. This model was used for investigating the catalytic mechanism by site-directed mutagenesis. Eleven forms of C. boidinii FDH were characterized by steady-state kinetic analysis: the wild type as well as 10 mutants involving single (Phe-69-Ala, Asn-119-His, Ile-175-Ala, Gln-197-Leu, Arg-258-Ala, Gln-287-Glu and His-311-Gln) and double amino acid substitutions (Asn-119-His/His-311-Gln, Gln-287-Glu/His-311-Gln and Gln-287-Glu/Pro-288-Thr). The kinetic results of the mutant enzymes provide the first experimental support that hydrophobic patches, formed by Phe-69 and Ile-175, destabilize substrates and stabilize products. Also, the key role of Arg-258 in stabilization of the negative charge on the migrating hydride was established. Asn-119, besides being an anchor group for formate, also may comprise one of the hinge regions around which the two domains shift on binding of NAD+. The more unexpected results, obtained for the His-311-Gln and Gln-287-Glu/His-311-Gln mutants, combined with molecular modelling, suggest that steric as well as electrostatic properties of His-311 are important for enzyme function. An important structural role has also been attributed to cis-Pro-288. This residue may provide the key residues Gln-287 and His-311 with the proper orientation for productive binding of formate. (+info)
Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail.
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Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme.substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs. (+info)
Fomepizole for the treatment of methanol poisoning.
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BACKGROUND: Methanol poisoning may result in metabolic acidosis, blindness, and death. The inhibition of alcohol dehydrogenase is fundamental to the treatment of methanol poisoning. We performed a multicenter study to evaluate fomepizole, an inhibitor of alcohol dehydrogenase, in the treatment of patients with methanol poisoning. METHODS: We administered intravenous fomepizole to 11 consecutive patients who presented with methanol poisoning at a participating center. Serial clinical and laboratory studies, including measurements of plasma formic acid and fomepizole, were performed. The outcomes measured were the preservation of visual acuity, the resolution of metabolic acidosis, the inhibition of formic acid production, the achievment of therapeutic plasma concentrations of fomepizole with the dosing regimen, residual illness or disability, and death. RESULTS: Plasma formic acid concentrations were detectable in eight patients, and these concentrations were closely correlated with the initial arterial pH values (r=0.92, P<0.001). In response to fomepizole, plasma formic acid concentrations fell and metabolic abnormalities resolved in all patients. Nine patients survived. Seven patients initially had visual abnormalities, but at the end of the trial no surviving patient had any detectable visual deficits related to methanol poisoning. Fomepizole had few adverse effects. The two patients who died had anoxic brain injury that was present at the time of enrollment. During treatment, methanol had an elimination half-life of 54 hours. CONCLUSIONS: Fomepizole appears to be safe and effective in the treatment of methanol poisoning. (+info)
Differential recovery of retinal function after mitochondrial inhibition by methanol intoxication.
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PURPOSE: The authors' laboratory has previously documented formate-induced retinal toxicity in a rodent model of methanol intoxication. These studies determined functional, bioenergetic, and structural recovery of the retina after methanol intoxication. METHODS: Rats were intoxicated with methanol, and retinal function was assessed by electroretinography 72 hours after the initial dose of methanol and after a 72-hour recovery period. Retinal energy metabolites, glutathione (GSH) concentrations, and histology were determined at the same time points. RESULTS: Both rod-dominated and UV-cone-mediated electroretinogram responses were profoundly attenuated in methanol-intoxicated rats. In rats allowed to recover from methanol intoxication, there was significant, although incomplete, recovery of rod-dominated retinal function. However, there was no demonstrable improvement in UV-cone-mediated responses. Retinal adenosine triphosphate (ATP), adenosine diphosphate (ADP), and GSH concentrations were significantly reduced after intoxication. Although retinal energy metabolites returned to control values after the recovery period, retinal GSH remained significantly depleted. Histopathologic changes were apparent in the photoreceptors after methanol intoxication, with evidence of inner segment swelling and mitochondrial disruption. In animals allowed to recover from methanol intoxication, there was no evidence of histopathology at the light microscopic level; however, ultrastructural studies revealed subtle photoreceptor mitochondrial alterations. CONCLUSIONS: These findings support the hypothesis that formate inhibits retinal mitochondrial function and increases oxidative stress. They also provide evidence for a differential sensitivity of photoreceptors to the cytotoxic actions of formic acid, with a partial recovery of rod-dominated responses and no recovery of UV-cone-mediated responses. (+info)
Presence of cytochrome and menaquinone in Clostridium formicoaceticum and Clostridium thermoaceticum.
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Cytochrome b and menaquinone have been demonstrated in the homoacetate-fermenting Clostridium formicoaceticum and Clostridium thermoaceticum. (+info)
Long-lived glycosyl-enzyme intermediate mimic produced by formate re-activation of a mutant endoglucanase lacking its catalytic nucleophile.
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The mutant E134A 1,3-1,4-beta-glucanase from Bacillus licheniformis, in which the catalytic nucleophilic residue has been removed by mutation to alanine, has its hydrolytic activity rescued by exogenous formate in a concentration-dependent manner. A long-lived alpha-glycosyl formate is detected and identified by (1)H-NMR and matrix-assisted laser desorption ionization-time-of-flight-MS. The intermediate is kinetically competent, since it is, at least partially, enzymically hydrolysed, and able to act as a glycosyl donor in transglycosylation reactions. This transient compound represents a true covalent glycosyl-enzyme intermediate mimic of the proposed covalent intermediate in the reaction mechanism of retaining glycosidases. (+info)