Influence of coenzyme structure on the transient chemical intermediate formed during horse-liver alcohol-dehydrogenase-catalyzed reduction of aromatic aldehydes.

The influence of coenzyme structure on the transient chemical intermediate formed in the reaction between the horse-liver alcohol dehydrogenase-NADH complex and an aromatic aldehyde such as 4-trans-(N,N-dimethylamino)cinnamaldehyde or 4-(N,N-dimethylamino)benzaldehyde was investigated by substituting various adenylic dinucleotides for NADH. Two classes of dinucleotide were studied. (a) Dinucleotides which, in the presence of horse-liver alcohol dehydrogenase and either 4-(N,N-dimethylamino)benzaldehyde or 4-trans-(N,N-dimethylamino)cinnamaldehyde, lead to a chromophore structurally analogous to the transient chemical intermediate formed with NADH under the same experimental conditions. This includes dinucleotides with a neutral 1,4-dihydropyridine ring, analogues of NADH and adducts of NAD+ (or analogues) with enolizable carbonyl compounds. (b) Dinucleotides which, under the same experimental conditions, do not form any new chromophores when mixed with horse-liver alcohol dehydrogenase and either 4-trans-(N,N-dimethylamino)cinnamaldehyde or 4-trans-(N,N-dimethylamino)benzaldehyde. This includes oxidized coenzyme analogues, NADPH and NADP+ adducts. Our data suggest that a neutral 1,4-dihydropyridine ring is crucial for the formation of the transient chemical intermediate. When the NAD+-sulphite complex, which has a 1,4-dihydronicotinamide structure and a positive charge at position 4 neutralized by sulphite ions, was substituted for NADH, the transient chemical intermediate chromophore was observed. The implications of this phenomenon are examined by assuming the existence of intermediate-activated forms of substrates and coenzymes during the horse-liver alcohol dehydrogenase catalytic reduction of aldehydes.

Binding of adducts of NAD(P) and enolizable ketones to NAD(P)-dependent dehydrogenases.

Carbonyl compounds such as alpha-ketoglutarate, pyruvate, oxaloacetate, butyraldehyde, acetaldehyde or acetone react with NAD or NADP to give adducts. Binding studies of adducts to dehydrogenases are performed by means of ultraviolet differential spectroscopy, circular dichroism and spectrofluorimetry. The dehydrogenases show a high degree of binding specificity toward the adducts which contain their specific oxidized substrate and their specific coenzyme. The high selectivity of the dehydrogenases for adducts is evidenced by binding studies of NAD(P)-pyruvate and NAD(P)-alpha-ketoglutarate adducts on glutamate dehydrogenase at pH 7.6 and 8.9. Evidence is presented showing that adducts bind to the active site of the enzymes.

Nickel (II) as a temporary catalyst for hydroxyl radical generation.

Many in vivo studies show peroxidative damage during nickel toxicity, suggesting the generation of oxygen-activated species. Using the murexide (5,5'-nitrilodibarbituric acid ammonium salt) bleaching technique, we attempted to spectroscopically determine whether there are any histidylpeptides-Ni (II) complexes able to catalyze a nickel-dependent reduction of hydrogen peroxide leading to free oxygen radical production. We show that peptides containing the glycyl-glycyl-L-histidyl sequence trigger nickel-dependent production of oxygen radicals which can damage proteins, cause a rapid loss of tryptophan and a significant production of bityrosine and also induce peroxidation of polyunsaturated fatty acids. During the reaction, the histidine residue in the peptide is selectively damaged and breakdown of the peptide switches off hydroxyl-radical production.

Nitration of tyrosyl-residues from extra- and intracellular proteins in human whole blood.

We measured the amounts of tyrosine and 3-nitrotyrosine (NO2-tyrosine) in proteins of plasma and polymorphonuclear leukocytes (PMN) from human whole blood before and after activation with phorbol ester (PMA) or calcium ionophore (A 23187). In unstimulated blood, no significant nitration of tyrosine was detected into PMN proteins, but a NO2-tyrosine/tyrosine ratio of 0.7% was detected in plasma proteins. When blood was activated with PMA, the NO2-tyrosine/tyrosine ratio stayed at 0.7% in plasma proteins, but it increased to 1.4% in PMN proteins, indicating a peroxynitrite production within the cells. In blood activated with calcium ionophore, the NO2-tyrosine/tyrosine ratio was 1.2% in plasma proteins and 2.1% in PMN proteins. Incubation of blood with a NO-synthase inhibitor before stimulation inhibited such a protein tyrosine nitration. To ensure that NO2-tyrosine detected in intracellular proteins did not result from the enzymatic posttranslational tyrosylation of PMN proteins, the incorporation of 14C labeled tyrosine into PMN proteins after activation with PMA or A23187 was studied. The addition of a 10 fold excess of NO2-tyrosine did not modify the course of protein tyrosylation. Because tyrosine nitration is an irreversible reaction, NO2-tyrosine could be accumulated into proteins and could act as a cumulative index of peroxynitrite production.

[Affinity chromatography of NAD(P)+ dehydrogenases dependent on the concentration of NAD(P)+ bound to ketones or aldehydes]. / Utilisation pour la chromatographie d'affinité des déshydrogénases NAD(P)+ dépendantes de composes d'addition du NAD(P)+ immobilisé avec les cétones ou les aldéhydes.

The use of general ligands such as NAD(P)+ for affinity chromatography of dehydrogenase requires elution with pulses of oxidized or reduced cofactor at suitable concentrations. This method of elution can involve considerable effort before ideal eluent conditions are evolved. We report on a new method of immobilized NAD(P)+ modifications which increases the selectivity of the phase and may be of rather wide application. The immobilized NAD(P)+ is modified by addition of a ketone or an aldehyde substrate of the dehydrogenase which must be purified and becomes a very specific ligand of that dehydrogenase. The adduct of immobilized NAD+ and sodium pyruvate absorbs specifically the lactate dehydrogenase, whereas the adduct of immobilized NAD+ and cyclohexanone adsorbs specifically the horse liver alcohol dehydrogenase. Absorbed dehydrogenases are eluted with NaCl gradients.

Rapid separation of dehydrogenases by affinity chromatography with new induced specificity phases.

We describe a procedure using immobilized nicotinamide as an affinity chromatographic ligand for the binding of NAD(P)+-dependent dehydrogenases. The procedure involves preparation of nicotinamide N1-(N-(6-aminohexyl)-acetamide)-agarose and modification of the immobilized nicotinamide by the addition of a ketone or an aldehyde to form an adduct. The nicotinamide, which has no affinity for dehydrogenase, becomes a very specific ligand of dehydrogenase, which binds the ketone or the aldehyde as substrate or inhibitor. In tests, the adduct prepared with immobilized nicotinamide and sodium pyruvate bound specifically to lactate dehydrogenase (EC 1.1.1.27), whereas the adduct prepared with alpha-ketoglutarate bound to glutamate dehydrogenase (EC 1.4.1.3). This technique enables the rapid isolation of a given dehydrogenase.

[Simple compounds with high pharmacologic potential: beta-carbolines. Origins, syntheses, biological properties]. / Des structures simples aux potentialités pharmacologiques élevées: les beta-carbolines. Origines, synthèses, propriétés biologiques.

We reviewed the origins, the synthetic pathways and the biological properties of beta-carbolines, the condensation products of tryptophan and indole alkylamines with aldehydes. They were found in many plants, some of which have been used as hallucinogens. They also occur as minor constituents in tobacco smoke. In mammalian body, beta-carboline derivatives occur normally in plasma, platelets and urine, moreover it seems that some are formed in human body after alcohol intake. Due to interesting biological effects described in recent years (inhibition of monoamine oxidase, binding to benzodiazepine receptors, comutagenic and carcinogenic properties, 5-hydroxy tryptamine uptake inhibition), many attempts were made to prepare beta-carbolines starting from various indole derivatives. We reviewed the published methods up to 1975 and summarized the main patents related with pharmacological properties of synthetic beta-carbolines.

NAD(P) adducts as protective agents against glutamate dehydrogenase inactivation by pyridoxal 5'-phosphate: a tool for the study of oxidized coenzyme activated state in enzymatic evolutive and abortive complexes.

Glutamate dehydrogenase is reversibly inhibited by the reaction of 1 mole of pyridoxal 5'-phosphate per mole of subunit of the enzyme polypeptide chain. We have shown that NAD(P) adducts as well as NMNH protect the glutamate dehydrogense against this reversible inactivation in the same way as reduced coenzymes. These data lead to the conclusion that it is the 1,4-dihydronicotinamide structure that is responsible for protecting the enzyme. NAD+ and NMN+ do not protect the enzyme, but in the presence of the oxidized substrate NAD+ became a good protecting agent whereas NMN+ remained ineffective. To explain the protection exerted by NAD+ in the presence of oxidized substrate, a transient activated form of the oxidized coenzyme with a 1,4-dihydronicotinamide structure and a positive charge on the C-4 atom is postulated.

Murexide bleaching: a new direct assay method for characterizing reactive oxygen species.

The murexide (5,5'-nitrilodibarbituric acid, monoammonium salt) is an efficient scavenger for superoxide and hydroxyl radicals. When exposed to oxygen radicals, murexide is converted to a colorless alloxan derivative and its absorbance at 520 nm decreases in proportion to the radicals produced. It is used to detect these reactive oxygen species in biochemical systems such as acetaldehyde oxidation by xanthine oxidase and the respiratory burst of polymorphonuclear leukocytes induced by phorbol 12-myristate, 13-acetate. The method was sensitive enough to allow direct monitoring of the production of superoxides from 10(6) phorbol 12-myristate, 13-acetate polymorphonuclear leukocyte-stimulated cells. Moreover, murexide bleaching is inhibited in the presence of radical scavengers, allowing a comparison of their scavenging activities.

The structure of the abortive nicotinamide adenine dinucleotide-pyruvate-lactate dehydrogenase complex as determined by circular dichroism.

Both ternary complexes consisting of lactate dehydrogenase-NAD+ and pyruvate or alpha ketobutyrate were compared, by means of circular dichroism spectra, with the corresponding binary complexes formed between lactate dehydrogenase and both NAD-pyruvate and NAD-alpha ketobutyrate adducts. Strong differences were observed. They disappeared when the ternary complexes were dissociated by addition of urea. This behaviour is explained by assuming the existence, in the abortive ternary complexes of a NAD+ activated transient form having a partial carbonium ion at position 4. Such a structure may account for the abortive and for the evolutive ternary complexes of the dehydrogenases as well as for the chemical synthesis mechanism of adducts.

[Structure of adducts prepared from nicotinamide adenine dinucleotide and their oximes]. / Structure des composés d'addition formés par le nicotinamide adénine dinucléotide et les oximes.

Preparation of adducts from nicotinamide adenine dinucleotide and a number of oximes is described; these include acetoxime, pyruvatoxime, cyclohexanoxime, cyclopentanoxime. These adducts are closely related to the corresponding NAD-ketone adducts in their spectra properties, but they are stable in acid solutions (pH 5).

Studies of covalent adducts of NAD(P) and enolizable ketones as specific glutamate dehydrogenase inhibitors.

Structural analogues of the reduced coenzymes, NADH or NADPH, of dehydrogenases are prepared by addition of carbonyl compounds including: pyruvate, alpha ketoglutarate, oxaloacetate, butyraldehyde, acetaldehyde and acetone, to the oxidized coenzymes NAD(P). Some of the adducts obtained are specific inhibitors of the glutamate dehydrogenase. The specificity is related to the carbonyl compound used. The high selectivity of the dehydrogenases for adducts is evidenced by inhibition studies of NAD(P)-pyruvate and NAD(P)-alpha ketoglutarate adducts on both activities of glutamate dehydrogenase. The inhibitions are competitive with the reduced coenzymes and the oxidized substrates: adducts could be considered as structures closely related to the ternary complexes of the dehydrogenase.

Peroxidase-release associated with phagocytosis in Mytilus galloprovincialis haemocytes.

Fluorescence spectra of the supernatant of adherent haemocyte monolayers from Mytilus galloprovincialis, supplemented with homovanillic acid or with a tyrosyl peptide glycylglycyltyrosine (GGY), were recorded before and after stimulation by zymosan. The formation of fluorescent derivatives was observed to have spectral characteristics similar to those of fluorescent compounds generated by the exposure of homovanillic acid or GGY to a horseradish peroxidase/hydrogen peroxide system in vitro. Lucigenine-enhanced chemiluminescence (CLluc) of M. galloprovincialis haemocytes stimulated by zymosan or by phorbol ester (PMA) was measured in the presence and absence of sodium azide, a peroxidase inhibitor. Sodium azide inhibited the CLluc of haemocytes stimulated by zymosan, an effective stimulus for myeloperoxidase secretion in human polymorphonuclear leukocytes, but not the CLluc of haemocytes stimulated by PMA, indicating the presence of peroxidases with some properties of myeloperoxidase, in adherent haemocytes from M. galloprovincialis.

Reciprocal effects between opioid peptides and human polymorphonuclear leukocytes--I. Chemical modifications of Leu-enkephalin by phorbol myristate acetate-stimulated polymorphonuclear leukocytes.

When L-tyrosyl-glycyl-L-phenylalanyl-L-leucine (Leu-enkephalin) is exposed to the activated oxygen species produced by phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMNs), hydroxylation of the phenylalanyl residue in position 4 of the peptide occurs, producing hydroxy-phenylalanyl derivatives which are identified by HPLC analysis and mass spectrometry. Attack of hydroxyl radicals generated by the Cu (II)/ascorbate system upon Leu-enkephalin also produces isomeric o-, m- and p-hydroxy-phenylalanyl derivatives. When PMNs are incubated with a synthetic peptide, L-tyrosyl-glycyl-glycyl-L-tyrosyl-L-leucine used as a model of hydroxylated Leu-enkephalin, their chemiluminescence response to PMA activation is higher than that of PMNs incubated with Leu-enkephalin.

Is malonaldehyde a valuable indicator of lipid peroxidation?

Malonaldehyde (MDA), a decomposition product of lipid hydroperoxides which is used as an indicator of oxidative damage to cells and tissues, reacts, in vitro, with hydrogen peroxide to form undetermined degradation products. Since human polymorphonuclear leukocytes (PMNs) release reactive oxygen species including hydrogen peroxide when stimulated with phorbol myristate acetate (PMA), we incubated specific amounts of MDA with resting PMNs and PMA-stimulated PMNs. The amount of MDA recovered after 30 min incubation with stimulated cells, as determined by MDA-thiobarbituric acid assay, was 25% lower than that recovered with resting cells. In the presence of catalase 18% of MDA disappeared and in the presence of superoxide dismutase 15% disappeared. This indicates that measurements of MDA production in living systems, in the presence of reactive oxygen species, could be underestimated.

Reciprocal effects between opioid peptides and human polymorphonuclear leukocytes--II. Enhancement of phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocyte by opioid peptides previously exposed to activated oxygen species.

Activated oxygen species (AOS) have often been shown to promote strong modifications in peptide structures and thus in their biological functions. In the present study, the immunomodulatory effects of Leu-enkephalin, beta-endorphin, dynorphin and some fragments are evaluated, before and after exposure of peptides to AOS, by studying their influence on human polymorphonuclear leukocyte (PMN) respiratory burst. None of the tested opioid peptides (modified or not) were shown to affect resting oxidative metabolism in the PMNs. The effects of peptides on phorbol myristate acetate (PMA)-stimulated production of AOS were measured in a lucigenin-enhanced chemiluminescence assay. Before AOS exposure, the opioid peptides suppressed the PMA-stimulated respiratory burst in human PMNs and a U-shaped dose-response relationship was observed. Conversely, after AOS exposure the opioid peptides enhanced the PMA-stimulated respiratory burst in human PMNs and an inverted U-shaped dose-response relationship was observed. In both cases, the maximal effect was reached at peptide concentrations of 10(-10)M-10(-12) M.

Potential role of the peroxidase-dependent metabolism of serotonin in lowering the polymorphonuclear leukocyte bactericidal function.

Serotonin (5-hydroxytryptamine, 5-HT) significantly and dose-dependently suppressed the luminol-enhanced chemiluminescence (CL) signal generated by polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate (PMA), but did not modify either lucigenin-enhanced CL or the reduction of superoxide dismutase-inhibitable cytochrome c. Moreover, stimulation of PMNs previously incubated with 5-HT resulted in a threefold increase in 5-HT equivalents bound to the proteins of PMN. The addition of catalase or sodium azide substantially reduced this binding. The present results suggest that 5-HT metabolism is mediated by H2O2 and myeloperoxidase (MPO) released by activated PMNs. Hence 5-HT could lower the bactericidal function of these cells by competition with hypochlorite formation from halides and MPO/H2O2.

Lipid peroxidation by peroxidase-catalyzed bioactivation of tyrosine.

Tyrosyl free radicals generated by the peroxidase-catalyzed oxidation of peptide tyrosyl residues are known to yield the stable cross-linked product dityrosine. In the present report, horseradish peroxidase is used as a model of peroxidase to study oxidative modifications of non-protein cellular components. Tyrosyl free radicals promote, as many free radicals, the decay of ß-phycoerythrin fluorescence emission, they oxidize NADH and ascorbic acid and initiate arachidonic acid peroxidation with formation of hydroperoxides and dienes. These results suggest that tyrosyl free radicals generated when tyrosine residues in protein and peptides are activated in vivo by peroxidase-H2O2 might undergo the peroxidation of membrane lipids.

[Does nitric oxide stress exist?]. / Peut-on parler d'un stress NO?

Ten years ago, the term "oxidative stress" (sigma -O2) was created to define oxidative damage inflicted to the organism. This definition brings together processes involving reactive oxygen species production and action such as free radical production during univalent reduction of oxygen within mitochondria, activation of NADPH-dependent oxidase system on the membrane surface of neutrophils, flavoprotein-catalyzed redox cycling of xenobiotics and exposure to chemical and physical agents in the environment. Since the discovery of the nitric oxide biosynthetic pathway, the deleterious effects of uncontrolled nitric oxide generation are generally classified as oxidative stress. Indeed, products of the reaction of NO and superoxide lead to oxidants such as peroxinitrite, nitrogen dioxide and hydroxyl radical, which are involved in mechanisms of cell-mediated immune reactions and defence of the intracellular environment against microbiol invasion. However NO can also regulate many biological reactions and signal transduction pathways that lead to a variety of physiological responses such as blood pressure, neurotransmission, platelet aggregation, endothelin generation or smooth muscle cell proliferation. Then the uncontrolled NO production can lead to a variety of physiological and pathophysiological responses similar to a Nitric Oxide Stress: activation of guanylate cyclase and production of cGMP: overstimulation of the inducible L-arginine to L-citrulline and NO pathway by bactericidal endotoxins and cytokines has been shown to promote undesired increases in vasodilatation, which may account for hypotension in septic shock and cytokine therapy. stimulation of auto-ADP-ribosylation and modification of SH-groups of glyceraldehyde-3-phosphate dehydrogenase in a cGMP-independent mechanism: by this way, NO in excess can strongly inhibits this important glycolytic enzyme and reduce the cellular energy production. inhibition of ribonucleotide reductase: extensive inhibition of this key enzyme in DNA synthesis in the presence of large amounts of NO could lead to important antiproliferative effects; inhibition of cytochrome P450-dependent metabolism: in Kupffer cells and hepatocytes, LPS-induced overproduction of NO has been shown to inhibit cytochrome P450-dependent metabolism and to mediate the suppression of hepatic metabolism. Moreover, NO synthetized in the peripheral nervous system is known to mediate nonadrenergic noncholinergic (NANC) neurotransmission. Overstimulation of NO synthases might therefore contribute to pathophysiological states such as: gastrointestinal motility, reflux oesophagitis, asthma, adult respiratory distress syndrome (ARDS) and chronic pulmonary artery hypertension. To these NO-mediated biological functions, one could add the biological effects of NO-derivatives such as N-nitrosocompounds, which act as carcinogenic agents, or C-nitrosocompound which were recently used as "zinc-ejecting" agents to inhibit HIV-1 infectivity of human T-lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

[Casein-derived peptides can promote human LDL oxidation by a peroxidase-dependent and metal-independent process]. / Des peptides issus de la caséine peuvent provoquer l'oxydation des LDL humaines par un processus dépendant des peroxydases et indépendant des métaux.

The results of the present study revealed that peptides derived from bovine casein hydrolysates can promote peroxidase-dependent oxidation of human low-density lipoproteins (LDL). The reaction was independent of the free metal ions but required casein-derived peptides with tryosyl-residues, implying that the tyrosyl radical is a diffusible catalyst that conveys oxidizing potential from the active site of the heme enzyme to LDL lipids. This mechanism is independent of the peroxidase used to oxidize tyrosyl residues since myeloperoxidase and horseradish peroxidase mediate a similar LDL peroxidating process. Vitamin E, ascorbic acid, butylated hydroxytoluene and reduced glutathione delayed LDL oxidation and were consumed during the reaction, they transfered hydrogen to repair tyrosine.