Evidence for the involvement of superoxide anions in the oxygenase reaction of ribulose-1,5-diphosphate carboxylase.

The mechanism of oxygenase reaction catalysed by ribulose-1,5-diphosphate carboxylase was investigated using superoxide dismutase from bovine erythrocytes. Inclusion of superoxide dismutase in the assay mixture resulted in strong inhibition of oxygenase reaction. Ribulose-1,5-diphosphate was found to compete for superoxide anions with dismutase and nitroblue tetrazolium which also inhibited the oxygenase reaction. These observations indicate the possible involvement of superoxide anions in the oxygenase reaction.     

Microsomal hydroxylation of specifically deuterated monosubstituted benzenes. Evidence for direct aromatic hydroxylation

The aromatic hydroxylation of six pairs of selectively deuterated monosubstituted benzenes was investigated with rat liver microsomes of various induction states. The substrates studied included 3,5-D2C6H3X (1a-6a) and 2,4,6-D3C6H2X (1b-6b), where X = Br, CN, NO2, OCH3, CH3, or Ph, respectively. The deuterium content of the ortho, meta, and para hydroxylated metabolites, as well as side chain oxidation products from 4 and 5, was determined by capillary gas chromatography-mass spectroscopy. These data were analyzed according to a hypothetical model in which a molecule of substrate can undergo either direct aromatic hydroxylation (defined as obligatory and complete loss of deuterium from the site of hydroxylation) or indirect aromatic hydroxylation (defined as the obligatory and complete shift of deuterium to an adjacent position, followed by its partial loss as governed by a kinetic deuterium isotope effect). From this and other analyses of the data the following conclusions were reached. (1) The relative extent of meta hydroxylation increased and the total yield of metabolites decreased as the substituents X became more electron withdrawing. (2) The induction state of the microsomes altered the regioselectivity of hydroxylation (2, 3, 4, or side chain) noticeably and predictably but had little or no effect on the retention or loss of deuterium during each hydroxylation. (3) With each substrate and at each ring position hydroxylation was found to occur by a combination of direct and indirect mechanisms. (4) The relative importance of direct vs. indirect mechanisms did not vary in a simple manner with either the position of hydroxylation or the nature of the substituent X.(ABSTRACT TRUNCATED AT 250 WORDS)     

A para-site-specific hydroxylation of aromatic compounds by Mycobacterium sp. strain 12523: stabilization of the hydroxylation activity

Mycobacterium sp. strain 12523 has a para-site-specific hydroxylation activity, which produce para-substituted phenols from various aromatic compounds. However, the activity is unstable and the reactions are inactivated within 24 h. In order to extend the reaction period, the factors that affected reaction stability were examined. The hydroxylation activity of the cells incubated in buffer was significantly stabilized by the inclusion of an inducer such as methyl ethyl ketone. It is suggested that a regulatory mechanism is involved in controlling the activity. This study resulted in the development of a convenient method to stabilize the hydroxylation activity, involving the addition of an inducer, such as acetone, to the reaction system. This method permitted the hydroxylation reaction to continue for more than 67 h. Received: 27 January 1997 / Received revision: 18 March 1997 / Accepted: 13 April 1997     

The involvement of superoxide anions in the autoxidation of various cofactors of cysteamine-oxygenase

The reduction of tetranitroblue tetrazolium with cysteamine, mediated by a number of dyes, elemental sulphur, elemental selenium and selenide, under aerobic conditions, was inhibited to various extent upon addition of superoxide dismutase. A strict parallelism between the ability to produce O2- ions and the property of those compounds to act as cofactors for cysteamine-oxygenase, to yield hypotaurine, has been observed. Based on the fact that the autoxidation of cysteamine also gives rise to O2- formation, though to a minor extent, we propose a mechanism for cysteamine-oxygenase action. This mechanism was derived from the data obtained in the model system studied.     

Light-dependence of paraquat-initiated membrane deterioration in bean plants. Evidence for the involvement of superoxide

Treatment of primary bean leaves with 10 mg/I paraquat induces the formation of gel phase lipid in microsomal and chloroplast membranes and enhances the activity of superoxide dismutase, but only if the leaves are exposed to light. These light-dependent changes in membrane lipid phase properties show a close temporal correlation with enhanced O₂⁷ production by illuminated chloroplasts and the onset of lipid peroxidation. Malondialdehyde and ethane, which are both formed during lipid peroxidation, are produced in large amounts by paraquat-treated leaves exposed to light, but not by those maintained in the dark. Electron spin resonance measurements indicate that production of O₂⁷ by illuminated chloroplasts is more than 2-fold greater in the presence of paraquat than in its absence. The identity of the radical formed by illuminated chloroplasts in the presence of paraquat was confirmed by using the diagnostic spin trap 5,5'-dimethyl-l-pyrroline-l-oxide and by establishing that its formation is sensitive to superoxide dismutase. The observations collectively indicate that paraquat-mediated membrane deterioration is light-dependent and attributable to enhanced O₂⁷-production.     

Mechanism of aromatic hydroxylation. Properties of a model for pyridine nucleotide-dependent flavoprotein hydroxylases

A model (NADH-phenazine methosulfate-O₂) formally similar to pyridine nucleotide-dependent flavoprotein hydroxylases catalyzed the hydroxylation of several aromatic compounds. The hydroxylation was maximal at acid pH and was inhibited by ovine Superoxide dismutase, suggesting that perhydroxyl radicals might be intermediates in this process. The stoichiometry of the reaction indicated that a univalent reduction of oxygen was occurring. The correlation between the concentration of semiquinone and hydroxylation, and the inhibition of hydroxylation by ethanol which inhibited semiquinone oxidation, suggested the involvement of phenazine methosulfate-semiquinone. Activation of hydroxylation by Fe³⁺ and Cu²⁺ supported the contention that univalently reduced species of oxygen was involved in hydroxylation. Catalase was without effect on the hydroxylation by the model, ruling out H₂O₂ as an intermediate. A reaction sequence, involving a two-electron reduction of phenazine methosulfate to reduced phenazine methosulfate followed by disproportionation with phenazine methosulfate to generate the semiquinone, was proposed. The semiquinone could donate an electron to O₂ to generate O₂ which could be subsequently protonated to form the perhydroxyl radical.     

The mechanisms of hemoglobin autoxidation. Evidence for proton-assisted nucleophilic displacement of superoxide by anions

Human oxyhemoglobin (HbO₂) in the presence of excess nucleophile (e.g., N₃^?, SCN^?, F^?, Cl^?) is shown by visible and Soret spectra to form cleanly the oxidized metHb with the nucleophile as ligand. The rates, sensitive to pH and to both the concentration and the nucleophilicity of anionic nucleophile (N^?), follow the rate law: rate = k[HbO₂][N^?][H⁺]. This autoxidation process thus appears to involve the nucleophilic displacement of superoxide from a protonated intermediate and can reasonably account for normal metHb formation in the erythrocyte where chloride can serve as the nucleophile. MetHb formation due to electron transfer agents (e.g. nitrite) which are normally not present can follow a different course such as direct electron transfer to bound dioxygen to form iron (III) peroxide. Abnormal amino acids or denaturation can provide increased access of nucleophile or electron transfer reactant and thus promote autoxidation.     

Possible involvement of an enzymatic system for superoxide generation in lepidopteran larval haemolymph

The superoxide generative system in Pseudaletia separata larval haemolymph plasma was shown to consist of at least two components, low (LMF) and high (HMF) molecular weight factors using ultrafiltration, dialysis, and gel filtration. The LMF was concluded to be a molecule(s) smaller than 5 kDa while the HMF to be a protein(s) larger than 100 kDa. The total amount of superoxide produced depended on the amount of LMF and was independent of that of HMF added to the reaction mixture. Both the LMF and HMF were required for O₂^? generation. From results in the present article, it was hypothesized that the LMF was a substrate(s) discharging electrons and HMF was an enzyme(s) to mediate the electron transfer to O₂ forming O₂^?. Superoxide production was detected in the haemolymph plasma of 5 lepidopteran species in addition to P. separata. It was concluded that superoxide production is a common phenomenon at least in these lepidopterans. © 1995 Wiley-Liss, Inc.     

A para-site-specific hydroxylation of various aromatic compounds by Mycobacterium sp. strain 12523

A screening of microorganisms with para-site-specific hydroxylation activity of aromatic compounds has been carried out by a three-step screening procedure involving a newly established micro-plate assay method. About 1300 strains isolated from about 5000 soils showed the activity. The hydroxylation of aromatic compounds by one of the isolates, designated Mycobacterium sp. strain 12523, which had the highest activity, was examined in detail. It produced p-phenols with high selectivity without o-or m-site hydroxylation. The yield of hydroquinone from phenol by strain 12523 was 97 mol%.     

Evidence for the possible involvement of the superoxide radicals in the photodegradation of bilirubin

The photodecomposition of bilirubin follows first order kinetics with ak _B value of 12.5 × 10^-3 min^-1. In the presence of a model system generating superoxide anions, such as xanthine-xanthine oxidase, thek _B value was 103 × 10^-3 min^-1 This ten-fold enhancement ofk _B value by xanthine-xanthine oxidase was abolished when the reaction mixture was supplemented with a superoxide ion scavenger— superoxide dismustase. Further, known singlet oxygen quenchers like Β-carotene and bistidine did not prevent the enhancement of bilirubin oxidation by xanthine-xanthine oxidase, thereby ruling out the obligatory conversion of Superoxide anion to singlet oxygen. It is concluded that radical oxygen mediated bilirubin degradation might be a natural catabolic route for the bile pigment degradation during oxygen stress. deceased May 1977.     

Evidence for the involvement of superoxide in vitamin K-dependent carboxylation of glutamic acid residues of prothrombin.

The formation of vitamin K epoxide and the vitamin K-dependent carboxylation of glutamic acid residues present in synthetic substrates and decarboxyprothrombin are both inhibited by superoxide dismutase. Catalase only inhibits the generation of vitamin K epoxide, suggesting that the carboxylation and epoxidation reactions are not inter-dependent.     

Superoxide-dependent formation of hydroxyl radicals: Detection of hydroxyl radicals by the hydroxylation of aromatic compounds

A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O₂^$\text{?}$, and H₂O₂. In the presence of iron salts, O₂^$\text{?}$ and H₂O₂ can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described.     

Synaptic repression at crayfish neuromuscular junctions. II. Evidence for calcium involvement

The inability of synaptic junctions to generate normalsized postsynaptic potentials under normal physiological conditions was studied at crayfish neuromuscular synapses. Synaptic repression in the superficial flexor muscle system of the crayfish was induced by surgery: the nerve was cut in the middle of the target field, and the lateral muscle fibers were removed. After this surgery, the remaining medial synapses were unable to generate normal-sized junction potentials (jp) over the medial muscle population. In an attempt to study the mechanism underlying this response, we varied the extracellular calcium concentration of the Ringers solution bathing the preparation, in both repressed and control animals, while monitoring the size of the same junction potential. The junction potential generated by the spontaneous activity of the nerve increased in size with increasing calcium concentrations in control animals, but failed to do so in repressed animals, that is, changes in external calcium concentrations did not affect repressed synapses. However, in the presence of the calcium ionophore A23187, control and repressed synapses both show an increase in the junction potential sizes they generate. Our data suggest that calcium is involved in the mechanisms that underlie synaptic repression in this crustacean neuromuscular system. © 1993 John Wiley & Sons, Inc.