Non-enzymatic Formation Research Articles

Isolation of Two Unusual Tetrahydroisoquinoline-Monoterpene Glucosides from Alangium lamarckii as Possible Intermediates in the Non-enzymatic Formation of Alangimarine from Alangiside

Isolation of Two Unusual Tetrahydroisoquinoline-Monoterpene Glucosides from Alangium lamarckii as Possible Intermediates in the Non-enzymatic Formation of Alangimarine from Alangiside

Tyrosinase-catecholic substrates in Vitro model: kinetic studies on the o-quinone/ o-semiquinone radical formation

The mechanism of o-semiquinone production was examined in the tyrosinase and peroxidase catalyzed oxidations of a series of catecholic compounds using the electron spin resonance (ESR) spin-stabilization approach and in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). In the tyrosinase process, the nonenzymatic o-semiquinone formation by inverse disproportion mechanism was clearly confirmed. Mechanistic and kinetic studies of o-semiquinone and o-quinone formation by mushroom tyrosinase were carried out by ESR spin stabilization and optical spectroscopy. Two different types of cyclizable catecholic substrates ( l-dopa and dopamine 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid) together with an uncyclizable substrate (3,4-dihydroxybenzoic acid) were examined. The reactive quinones were monitored by measuring the apparent initial rates of the o-quinone-MBTH adducts. The transient behaviour of the o-semiquinone was studied by determining the pseudo first-order formation constants (k values in the range 0.226–0.035 s −1), the relative second-order decay kinetic constants (k = 3.3·10 2 M −1 s −1 for dopamine o-semiquinone) and the maximum concentrations of the o-semiquinone complexes formed in situ with Mg 2+ ions. The o-semiquinone formation constants are not directly correlated with their maximum concentrations; in fact, the o-semiquinone maximum concentration of the uncyclizable substrate is comparable with that derived from l-dopa. Furthermore, the secondary semiquinone formation is slow and not competitive with the primary semiquinone generation. Then, in our model the limiting factor for the o-semiquinone formation, is not simply the substrate ability to cyclize, and, therefore, the potential toxicity of the secondary semiquinone is questionable.

Absolute Configuration and Conformation of 1,3-Dioxanes from Cider

In extracts obtained by liquid-liquid extraction from French cider (2S,4R)- and (2R,4R)-2-methyl-4-pentyl-1,3-dioxane, 1a and 1b as well as (2S,4R)- and (2R,4R)-2-methyl-4-(2'(Z)-pentenyl)-1,3-dioxane, 2a and 2b, were identified by capillary gas chromatography (HRGC) and capillary gas chromatography-mass spectrometry (HRGC-MS). Absolute configuration and conformation of the 1,3-dioxanes was determined by nuclear magnetic resonance (NMR) spectrometry techniques [ 13 C, 1 H, nuclear Overhauser enhancement (NOE), and H/H homonuclear decoupling], multidimensional gas chromatography (MDGC), and by comparison with synthesized reference compounds. A nonenzymatic formation of 1a and 1b and 2a and 2b during fermentation of apple juice was proposed leading to 22, 8, 2, and <1 mg/L of 1a, 2a, 1b, and 2b, respectively in cider.

Monofunctional NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae

This chapter describes purification of the native enzyme from yeast, as well as purification of recombinant yeast enzyme from an Escherichia coli expression system. The chapter presents a summary of kinetic, structural, metabolic properties of the enzyme, and its evolutionary relationship to other methylene-tetrahydrofolate (THF) dehydrogenases. NAD-dependent methylene-THF dehydrogenase is assayed by the production of 5,10-methenyl-THF. The assay is modified from an NADP-dependent methylene-THF dehydrogenase assay 4 by optimizing pH, buffer, and temperature conditions. Enzyme activity is determined by end-point assay of the production of 5,10-methenyl-THF by measuring absorbance at 350 nm after acidification of the reaction. Acidification reverses any non-enzymatic formation of 10-formyl-THF, which equilibrates with 5,10-methenyl-THF at neutral pH.

Non-enzymatic formation of formaldehyde in mouse oocyte freezing mixtures.

Three cryoprotectant solvents, dimethylsulphoxide, 1,2-propanediol and glycerol, were investigated for a non-enzymatic reaction product, formaldehyde. All three cryoprotectants demonstrated a direct relationship between increasing solvent molarity and increasing formaldehyde concentration which was independent of temperature and protein (bovine serum albumin). Medium composition significantly influenced the formaldehyde concentration with HTF > T6 > M16 = M2. The formaldehyde could be effectively removed by reduced glutathione, cysteine and dithiothreitol with cysteine being the most effective scavenging agent. A reaction mechanism for this scavenging is proposed. The combination of cysteine and cryoprotectant reduced the zona pellucida 'hardening' effect in mouse oocytes.

Continuous measurement of gastric nitric oxide production.

With the use of a double-lumen catheter, nitric oxide (NO) was dialyzed across a Gore-Tex membrane into a gas phase and subsequently analyzed on-line by chemiluminescence. This new technique for the continuous measurement of NO was evaluated bench-side and applied in the human stomach in vivo to measure the nonenzymatic formation of NO generated from nitrite in an acidic milieu. A linear relation (r2 = 0.991, P < 0.0001) between concentrations of NO in aqueous solutions (2.5-52.5 mM) and NO in the corresponding gas phases obtained by the dialysis technique (50-1,000 parts per billion) validated the present method for quantitative analyses of NO. Interassay and intra-assay coefficients of variation at all concentrations of NO for six experiments were < 5%. High intragastric concentrations of NO (in the micromolar range) were found during basal conditions. The requirement of both nitrite from the saliva and an acidic environment for NO formation is indicated, since depletion of saliva as well as acid neutralization greatly reduced gastric NO concentrations. Furthermore, large amounts of gastric NO were formed after intake of sodium nitrate. With the use of this technique, NO can be continuously measured with accuracy experimentally and clinically in any organ accessible to intubation.

Detailed Mechanism of Phenol‐Inhibited Peroxidase‐Catalyzed Oxidation of Indole‐3‐Acetic Acid at Neutral pH*

ABSTRACTThe inhibition of horseradish peroxidase (HRP)‐cata‐lyzed oxidation of indole‐3‐acetic acid (IAA) by a phenol, caffeic acid (CA), was studied using both a kinetic approach and computer simulation. The presence of CA resulted in a lag period in IAA oxidation. The lag period increased slowly with increasing [CA] until a critical concentration, [CA]cr, was reached, then it increased much faster when [CA] was greater than [CA]cr. The [CA]cr was proportional to [IAA] and did not depend upon [HRP]. Caffeic acid was oxidized by compound I and compound II of HRP with bimolecular rate constants (6.8 ± 107 and 2.1 ± 107M‐1s−l), which were much higher than the corresponding rate constants for IAA oxidation (2.3 ± 103 and 2.0 ± 102M−1s−1). Our experimental data show that CA inhibits IAA oxidation because it is able to compete effectively as a peroxidase substrate. A model based on a detailed mechanism of IAA oxidation was investigated using computer simulation. A rate constant driving nonenzymatic hydroperoxide formation in IAA solution was determined, 3.0 × 10−‐7 s−1. The model quantitatively describes the experimental results of this work and also qualitatively explains data published earlier. The critical inhibitor concentration is approximately equal to twice the concentration of hydroperoxide in IAA solution at the time of inhibitor addition. Therefore hydroperoxide Concentration can be calculated from the determination of critical inhibitor concentration.

THE CELLULAR AND MOLECULAR MECHANISMS OF DIABETIC COMPLICATIONS

In this article, the cellular and molecular mechanisms of diabetic complications have been reviewed. Hyperglycemia-induced mechanisms that may induce vascular dysfunction in specific sites of diabetic microvascular damage include increased polyol pathway flux, altered cellular redox state, increased formation of diacylglycerol and the subsequent activation of specific PKC isoforms, and accelerated nonenzymatic formation of advanced glycation endproducts. Several of these mechanisms may be responsible for the potentially damaging overproduction of reactive oxygen species observed with hyperglycemia. Each of these mechanisms may contribute to the known pathophysiologic features of diabetic complications by a number of mechanisms, including the upregulation of cytokines and growth factors. Diabetic macrovascular disease may arise more from insulin resistance than from hyperglycemia, and the authors speculate that this may reflect a selective loss of insulin-dependent vascular homeostasis.

Role of uncoupled and non-coupled oxidations in maintenance of safely low levels of oxygen and its one-electron reductants

AbstractTo proceed at a high rate, phosphorylating respiration requires ADP to be available. In the resting state, when the energy consumption is low, the ADP concentration decreases so that phosphorylating respiration ceases. This may result in an increase in the intracellular concentrations of O2as well as of one-electron O2reductants such asThese two events should dramatically enhance non-enzymatic formation of reactive oxygen species, i.e. of, and OHׁ, and, hence, the probability of oxidative damage to cellular components. In this paper, a concept is put forward proposing that non-phosphorylating (uncoupled or non-coupled) respiration takes part in maintenance of low levels of both O2and the O2reductants when phosphorylating respiration fails to do this job due to lack of ADP.In particular, it is proposed that some increase in the H+leak of mitochondrial membrane in State 4 lowers, stimulates O2consumption and decreases the level ofwhich otherwise accumulates and serves as one-electron O2reductant. In this connection, the role of natural uncouplers (thyroid hormones), recouplers (male sex hormones and progesterone), non-specific pore in the inner mitochondrial membrane, and apoptosis, as well as of non-coupled electron transfer chains in plants and bacteria will be considered.

Effect of Strychnos nux vomica alcohol extract on lipid peroxidation in rat liver

This report investigates the antioxidant properties of the purified Strychnos nux vomica L. seed extract for protection against non-enzymatic formation of lipid peroxides and on reduced glutathione (GSH) levels in rat liver homogenate. Results were compared with the natural antioxidant vitamin E. For the first time it could be reported that this drug protects against cumene hydroperoxide (CHP) induced lipid peroxidation in a dose dependent manner. It also inhibits the process of the lipid peroxidation, once induced. The drug significantly maintains the hepatic content of glutathione in a dose and time dependent manner, even in the presence of the above toxin (CHP). Thus it appears that S. nux vomica is a potent antioxidant and the mechanism of action could be through the scavenging of free radicals.

Nonenzymatic acetolactate oxidation to diacetyl by flavin, nicotinamide and quinone coenzymes

Acetolactate nonenzymatically reduced flavins, quinones and nicotinamide coenzymes in a time-dependent manner at physiological pH and moderate temperature. In the presence of excess acetolactate, the reduction of FAD and NAD + followed pseudo-first-order kinetics. The rate of reduction was proportional to the concentration of acetolactate, and the rate constants at 37°C and pH 7.5 were 4.8 · 10 −2 M −1 s −1 and 7.4 · 10 −3 M −1 s −1 for FAD and NAD +, respectively. In contrast, ubiquinone reduction followed pseudo-zero-order kinetics in the presence of excess acetolactate. At 37°C and pH 7.5, the rate of reduction was proportional to the acetolactate concentration, and the apparent rate constant was 8.3 · 10 −6 s −1. In contrast to FAD, the rate of reduction of ubiquinone was higher at low pH. The kinetics of ubiquinone reduction suggested that the rate-limiting step was acetolactate decarboxylation and formation of the enolate anion, whereas the rate of FAD reduction was governed by the second-order reaction of the enolate anion. Following the oxidation, acetolactate was converted to diacetyl. Reduced FAD formed by the reaction with acetolactate generated a low rate of O 2 consumption during assays of the oxygenase activity of acetohydroxy acid synthase. The reaction of acetolactate with quinones may provide a mechanism for the nonenzymatic formation diacetyl in whole milk.

Release of volatile halogenated organic compounds by unialgal cultures of polar macroalgae

The release of volatile halogenated organic compounds (VHOC) by different cultivated polar brown, red and green macroalgae was monitored in laboratory experiments. Isolation and identification of VHOC was performed by a purge and trap technique and capillary gas chromatography with electron capture detection. By using a column with high stationary phase thickness, 11 compounds from bromomethane to diiodomethane were separated and their release rates determined. Of all compounds investigated, bromoform is released in highest quantities from all species studied. Significant linear correlations of bromoform with the halogenated methanes and ethanes investigated, suggest an enzymic formation for each single compound. No significant linear correlation is found for bromomethane with the other VHOC investigated. Therefore, a non-enzymic formation is assumed. The results are discussed with relation to a possible influence of VHOC on atmospheric ozone depletion.

Adsorption of 5'-AMP and catalytic synthesis of 5'-ADP onto phosphate surfaces: correlation to solid matrix structures.

A non-enzymatic formation of 5'-ADP starting from phosphorylation of 5'-AMP in the presence of either calcium phosphate or calcium pyrophosphate precipitates is reported. This reaction is taken as a model for the study of heterogeneous catalysis of transphosphorylation in prebiotic conditions. Experiments were performed in completely aqueous media and in media containing dimethyl sulfoxide (Me2SO), to simulate periods of dehydration in primitive aquatic environments. It has been observed that the nucleotide is adsorbed onto both calcium phosphate and calcium pyrophosphate in accordance with Langmuir isotherms. Adsorptive capacity and affinity of the precipitates for nucleotide are changed by the presence of Me2SO, suggesting that the interaction between biomonomers and surfaces can be modulated by the degree of hydration of the anionic components of these compounds. In completely aqueous environments, formation of 5'-ADP from 5'-AMP adsorbed on precipitates of calcium phosphate and calcium pyrophosphate is very small. However, in the presence of 60% Me2SO this synthesis increases by factors of 3 and 6 for surfaces of calcium phosphate and calcium pyrophosphate, respectively, and follows first-order kinetics. Determinations of free energy changes show that phosphorylation of 5'-AMP adsorbed to these precipitates is thermodynamically favorable. Depending on the precipitation time of the samples and the composition of the medium, structural analysis of these precipitates by electron and X-ray diffraction shows changes in their cristallinity grade. It is proposed that these changes are responsible for the modulation of the quantity of adsorbed nucleotides to the surface of solid matrices as well as the catalytic activity of the precipitates.

Hepoxilins: A review on their enzymatic formation, metabolism and chemical synthesis

This article reviews published evidence describing the enzymatic and nonenzymatic formation and the routes of metabolism of the hepoxilins. Also treated are the major approaches used for the chemical synthesis of these compounds and for some of their analogs.

Hydrogen Peroxide Formation during Catechin Oxidation Is Inhibited by Superoxide Dismutase.

We established a simple method to quantify hydrogen peroxide (H2O2) generated during the aerobic heating process of (+)-catechin solution. Using this method, we found that non-enzymatic H2O2 formation from catechin depended on pH, temperature, incubation time, and the presence of O2 in the solution. The formation of oxidized products of (+)-catechin, which was estimated by measuring the absorbance of the solution at 430 nm, also depended on these factors. The H2O2 formation was inhibited by various kinds of superoxide dismutase (SOD) with almost the same dose dependency. Although the oxidation of (+)-catechin was enhanced by superoxide (O2−), neither catalase nor H2O2 had any effects on the oxidation. These results suggest that O2−, rather than H2O2, participated in the autoxidation of (+)-catechin, which was coupled with the H2O2 formation.

In vitro Reaction of L-Tryptophan and Vitamin B6. Synthesis of the Corresponding .beta.-Tetrahydrocarbolines

The L-tryptophan load test is used to study vitamin B6 deficiency in animals. This test might suffer from an inherent defect due to the possible nonenzymatic formation of cyclic compounds between L-tryptophan and pyridoxal or pyridoxal 5'-phosphate. In this paper the synthesis, under physiological conditions (pH 7.0 and 37 o C), of the cyclic compounds (β-tetrahydrocarbolines) formed from L-tryptophan and pyridoxal or pyridoxal 5'-phosphate is reported. The in vitro formation of these compounds has been implied for many years through evidence from biological experiments and ultraviolet absorption spectra

Lanthanide ions for the first non-enzymatic formation of adenosine 3',5'-cyclic monophosphate from adenosine triphosphate under physiological conditions.

Adenosine 3',5'-cyclic monophosphate (cAMP) is formed from adenosine triphosphate at pH 8 and 50 degrees C by use of lanthanide ions. Pr3+ and La3+ are the most active. The cAMP formation is more efficient at higher pH, where the mixture is made homogeneous by the addition of beta-cyclodextrin. The potential functioning of lanthanide ions as the catalytic center of an artificial adenylate cyclase is indicated.

Oxidation Products Arising from the Action of Monoamine Oxidase B on 1‐Methyl‐4‐Benzyl‐1,2,3,6‐Tetrahydropyridine, a Nonneurotoxic Analogue of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine

1-Methyl-4-benzyl-1,2,3,6-tetrahydropyridine (MBzTP), an analogue of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, despite its rapid oxidation by monoamine oxidase B (MAO B), is not neurotoxic. The pyridinium expected to arise from the four-electron oxidation of MBzTP inhibits mitochondrial respiration and the oxidation of NADH in inner membranes and is only moderately less inhibitory than 1-methyl-4-phenylpyridinium. It is also a competitive inhibitor of dopamine uptake by the dopamine transporter and hence likely to be taken up into neurons, despite its relatively high Ki value (Ki = 21 microM). Incubation of MBzTP with purified MAO B yields first the dihydropyridinium form, then a mixture of the pyridinium form and another unidentified product, in proportions that depend on the concentrations of MAO B and oxygen. At low MAO B concentration and moderate oxygen concentration, nonenzymatic formation of the unidentified product predominates. The lack of neurotoxicity of MBzTP appears to be due to the oxidative destruction of the dihydropyridine and consequent failure of accumulation of 1-methyl-4-benzylpyridinium.

Aspirin induces non-enzymatic formation of platelet-activating factor from lyso platelet-activating factor

Substantial amounts of platelet-activating factor (PAF) were formed when lysoPAF was mixed with aspirin (e.g. 0.04% of added lysoPAF (200 nmol) was converted to PAF when mixed with aspirin (2 μmol) for 1 h). Non-enzymatic formation of PAF from aspirin and lysoPAF also occurs in the aqueous solution or in organic solvents in time-dependent and dose-dependent manners. Possible meanings of the non-enzymatic formation of PAF are discussed.

Interactions of glutathione S-transferase- π with ethacrynic acid and its glutathione conjugate

Ethacrynic acid, a diuretic drug known to be an inhibitor of glutathione S-transferases (GSTs), has been shown to enhance the cytotoxicity of the alkylating agent class of chemotherapeutic drugs in cultured cancer cells resistant to alkylating agents. This action of ethacrynic acid is presumably mediated by inhibition of GSTs which are implicated in detoxification of alkylating agents. In addition to being an inhibitor of GSTs, ethacrynic acid also interacts with GSTs as a substrate for conjugation with GSH to yield an ethacrynic acid-GSH conjugate. This conjugate is formed both enzymatically and non-enzymatically and itself is a GST inhibitor. Since ethacrynic acid-GSH conjugate is itself likely to be able to mediate reversal of alkylating agents through GST inhibition, we have synthesized and purified the ethacrynic acid-GSH conjugate, studied the kinetics of inhibition of human lung π-class GST by ethacrynic acid and the conjugate, and compared the kinetics of the enzymatic and non-enzymatic formation of the conjugate using an HPLC method. Results of our studies showed that the ethacrynic acid-GSH conjugate was a more potent inhibitor of human lung GST-π than ethacrynic acid ( K i = 1.5 vs. 11.5 μM, respectively) and that their mechanisms for GST inhibition were distinct (competitive and non-competitive, respectively). Comparison of enzymatic and non-enzymatic rates of conjugate formation in vitro indicated that GST-γ catalyzed a rapid conjugation of ethacrynic acid with GSH at a concentration of ethacrynic acid an order of magnitude above that required to nearly completely inhibit GST catalyzed conjugation of 1-chloro-2,4-dinitrobenzene. However, because of the rapid non-enzymatic reaction, and the inhibition of GST-π with the accumulation of the conjugate in the reaction mixture, the overall quantity of the conjugate formed after 150 min was nearly identical in the presence or absence of GST-π. Results of these studies suggest that inhibition of GSTs by ethacrynic acid-GSH conjugate may be the main mechanism through which ethacrynic acid reverses alkylating agent resistance.