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PubMed1999

Antagonistic effect of nerve growth factor on neuronal injury induced by hypoxia in cultured cerebral cortical neurons of rats.

AIM: To observe the effects of nerve growth factor (NGF) on neuronal hypoxic injury induced by sodium dithionite in primary cultures from gestation of 17-d rat fetal cerebral cortex. METHODS: Neuronal death and lactate dehydrogenase (LDH) efflux in the bathing medium were measured. The homogenate of cortical cells was used to determine malonyldialdehyde (MDA) content and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. RESULTS: When cultures were incubated for 24 h with sodium dithionite, efflux of LDH and MDA content increased while the number of surviving neurons decreased. NGF 100 micrograms.L-1 increased the number of surviving neurons to 85% +/- 9% of normoxia level under hypoxia. NGF 1-100 micrograms.L-1 concentration-dependently attenuated hypoxia-induced increase of efflux of LDH and MDA content, with IC50 of 27 and 49 (95% confidence limits: 18-49 and 29-110) micrograms.L-1. NGF 30 micrograms.L-1 induced a 3-fold increase in SOD and GSH-Px activities. The levels of SOD and GSH-Px activities in hypoxia group were increased 2.7-fold by NGF 100 micrograms.L-1. CONCLUSION: NGF prevented hypoxic insults in cultured cerebral cortical neurons by suppressing the generation of lipid peroxides and increasing the activities of antioxidant enzymes.

Animals↗
PubMed1975

Cytochrome pigments in Mycobacterium leprae isolated from armadillos (Dasypus novemcinctus L.).

The bacilli were isolated from granulomata harvested from armadillos. Cytochrome systems in whole cell suspensions as well as in cell-free extracts were examined spectrophotometrically. The intact cells contained cytochromes of the a+b3, b and c type which were found to be present mainly in the reduced form. The cytochrome systems in cell-free extracts of M. leprae were in the oxidized form but contained the same type of cytochromes as the intact bacteria. The presence of cytochromes was easily detectable in the anaerobically-reduced (no substrate added) as well as in the dithionite- or succinate-reduced minus O2-oxidized difference spectra. The dithionite-reduced plus CO minus reduced difference spectra exhibited cytochromes a3 and o monoxide binding pigments.

Animals↗
PubMed1976

A new oxygenase, 2-nitropropane dioxygenase of Hansenula mrakii. Enzymologic and spectrophotometric properties.

2-Nitropropane dioxygenase, purified to homogeneity from Hansenula mrakii (IFO 0895), has a molecular weight of approximately 62,000 and consists of two subunits nonidentical in molecular weight (39,000 and 25,000). Stoichiometrical studies and the results obtained with 18O2 showed that 2 atoms of molecular oxygen are incorporated into 2 molecules of acetone formed from 2-nitropropane. In addition to 2-nitropropane, nitroethane, 3-nitro-2-pentanol, and 1-nitropropane are oxidatively dentrified. The enzyme, which exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm, contains 1 mol of FAD and 1 g atom of non-heme iron per mol of enzyme. The enzyme-bound FAD is reduced by 2-nitropropane under anaerogic conditions, but the enzyme-bound Fe3+ is not affected. The introduction of oxygen to the reduced form of enzyme causes reoxidation of the enzyme. The bound FAD and Fe3+ are reduced by the addition of nitromethane, which is not a substrate, under anaerobic conditions. The aerobic dialysis of the enzyme treated with nitromethane causes reoxidation of only the Fe2+. Sodium dithionite also reduces both the enzyme-bound FAD and Fe3+ under anaerobic conditions. When the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appeared with concomitant restoration of approximately 80% of the activity. The enzyme activity is significantly inhibited by pyrocatechol-3,5-disulfonate disodium salt, 8-hydroxyquinoline, reducing agents such as 2-mercaptoethanol, and HgCl2. The Michaelis constants are as follows: 2-nitropropane (2.13 X 10(-2) M), nitroethane (2.43 X 10(-2) M), 3-nitro-2-pentanol (6.8 X 10(-3) M), 1-nitropropane (2.56 X 10(-2) M), and oxygen (3.03 X 10(-4) M, with 2-nitropropane).

Amino Acids↗
PubMed2002

Purification and some properties of a blue copper protein from Methylobacillus sp. strain SK1 DSM 8269.

A blue protein was purified from the Methylobacillus sp. strain SK1 that is grown on methanol in the presence of copper ion. This protein was found to be a monomer with a molecular weight of 13,500. The Isoelectric point of the protein was estimated to be 8.8. The spectrum of the protein that was treated with ferricyanide showed a broad peak around 620 nm, but that of the dithionite-treated protein revealed no peaks. It contained 0.83 mol of EDTA-stable copper per mol protein. Under air, the protein accelerated the inactivation of methanol dehydrogenase (MDH). The protein was reducible by phenazine methosulfate or by active MDH that was prepared from cells that were grown in the absence of added copper, but not by methanol, dichlorophenol indophenol, or inactive MDH that was prepared from cells that were grown in the presence of added copper. It was also reducible by active MDH in the presence of methanol. The absorption peak at 340 nm of the active MDH disappeared after the enzyme was treated with ferricyanide, hydrogen peroxide, or the purified blue protein. The inactive MDH also showed no peak at 340 nm. The 340-nm peak was not recovered after incubation of the inactive MDH and blue protein-treated active MDH with dithionite or methanol. The inactive MDH and blue protein-treated active MDH co-migrated with the active MDH preparation on nondenaturing polyacrylamide gel, and contained two non-identical subunits with molecular weights that were identical to those of the active MDH. The N-terminal amino acid sequence of the protein was Ala-Gly-Cys-Ser-Val-Asp-Val-Glu-Ala-Asn-Asp-Ala-Met-Gln-Phe. An analysis of the amino acid composition revealed that the protein contained no tryptophan. It contained three cysteines per mol protein. The blue protein in Methylobacillus sp. strain SK1 was produced only in the cells that were grown in the copper-supplemented medium.

Alcohol Oxidoreductases↗
PubMed1992

Direct spectroscopic evidence for the presence of a 6Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans (ATCC 27774)

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. Mössbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (delta EQ = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic Mössbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.

Amino Acid Sequence↗
PubMed1992

The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.

The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.

Alcaligenes↗
PubMed1991

Formate dehydrogenase from Methylosinus trichosporium OB3b. Purification and spectroscopic characterization of the cofactors.

NAD(+)-coupled formate dehydrogenase has been purified to near-homogeneity from the obligate methanotroph Methylosinus trichosporium OB3b. The inclusion of stabilizing reagents in the purification buffers has resulted in a 3-fold increase in specific activity (98 microM/min/mg; turnover number 600 s-1) and as much as a 25-fold increase in yield over previously reported purification protocols. The enzyme, (molecular weight 400,000 +/- 20,000) is composed of four subunit types (alpha, 98,000; beta, 56,000; gamma, 20,000; delta, 11,500) apparently associated as 2 alpha beta gamma delta protomers. The holoenzyme contains flavin (1.8 +/- 0.2), iron (46 +/- 6), inorganic sulfide (38 +/- 4), and molybdenum (1.5 +/- 0.1). The flavin is optically similar to the common flavin cofactors, but it is chromatographically distinct. Anaerobic incubation of the enzyme with formate, NADH, or sodium dithionite, resulted in approximately 50% reduction of the iron and elicited an electron paramagnetic resonance (EPR) spectrum (approximately 2.5 spins/protomer) from which the spectra of five distinct EPR-active centers could be resolved in the g = 1.94 region. Four of these spectra were characteristic of [Fe-S]x clusters. The fifth (gave = 1.99; approximately 0.1 spins/protomer) was similar to that observed for the molybdenum cofactor of xanthine oxidase, and it exhibited the expected hyperfine splitting when the enzyme was enriched with 95Mo (I = 5/2). Mössbauer spectroscopy showed that all of the iron in the enzyme became reduced upon the addition of a redox mediator, proflavin, to the dithionite reduced enzyme at pH 8.0. Nevertheless, a decrease in the EPR-active spin concentration in the g = 1.94 region of the spectrum occurred and was attributed to the reduction of the molybdenum center to the EPR-silent Mo(IV) state (S = 1). The fully reduced enzyme also exhibited a new species with an S = 3/2 ground state (1-2 spins/protomer). Addition of 50% ethylene glycol to the fully reduced enzyme revealed no new species, but caused an increase in the EPR-detectable spin quantitation to 5-6 spins/protomer. This suggests that cluster spin-spin interactions may occur in both the partially and fully reduced native enzyme.

Amino Acids↗
PubMed1991

Spectroscopic studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans.

Absorption and EPR spectroscopic properties of purified dimethyl sulfoxide (Me2SO) reductase from Rhodobacter sphaeroides f. sp. denitrificans have been examined. The absence of prosthetic groups other than the molybdenum center in the enzyme has made it possible to study its absorption properties. The enzyme displays multiple absorbance peaks in both the oxidized and the dithionite-reduced forms. The oxidized enzyme has absorbance peaks at 280, 350, 470, 550, and 720 nm while the dithionite-reduced enzyme has peaks at 280, 374, and 645 nm with a shoulder at 430 nm. A comparison of the absorbance spectrum of oxidized Me2SO reductase with that of the molybdenum fragment of rat liver sulfite oxidase shows that the 350 and 470 peaks are common to both proteins. EPR studies of the Mo(V) form of Me2SO reductase show a rhombic signal with g1 = 1.988, g2 = 1.977, g3 = 1.961, and g(ave) = 1.975. The signal shows evidence of coupling to an exchangeable proton with A1 = 1.05, A2 = 1.13, A3 = 0.98, and Aave = 1.05 millitesla. These parameters are similar to those of other Mo enzymes, however, the epr signal of this enzyme differs from those of other Mo hydroxylases in showing only a slight sensitivity to pH and no detectable anion effect. EPR potentiometric titrations of Me2SO reductase gave midpoint potentials of +144 mV for the Mo(VI)/Mo(V) couple and +160 mV for the Mo(V)/Mo(IV) couple at room temperature and +141 mV for the Mo(VI)/Mo(V) couple and +200 mV for the Mo(V)/Mo(IV) couple at 173 K.

Chromatography, Ion Exchange↗
PubMed1979

Cytochrome c peroxidase. Interconversion of chemically and enzymatically reactive and unreactive forms of the ferric protein.

Ferric yeast cytochrome c peroxidase in the presence of different anions may assume a number of forms which differ in optical spectra and chemical properties. In solutions whose only anion is acetate, two spectral forms are present together in an equilibrium. Each of these spectral species is believed to bear bound acetate anion. A form characterized by an intense absorption maximum at 620 nm is unreactive enzymatically and does not react with hydrogen peroxide or with dithionite. A form characterized by a less intense absorption near 645 nm is enzymatically and chemically reactive. Increasing temperature and increasing pH displace the equilibrium toward the 645 nm form. Increasing cytochrome c peroxidase concentration favors the 620 nm form. In kinetic experiments in which the 645 nm form is removed by rapid reaction with H2O2 or dithionite, the 620 nm form is converted in a first order reaction (k = 0.36 s-1, 15 degrees C) to the 645 nm form. In solutions whose sole anion is phosphate a 645 nm form is the only demonstrable spectral species. The enzymatic activity and rates of chemical reaction of 645 nm spectral forms occurring in acetate and in phosphate buffers are the same.

Cytochrome-c Peroxidase↗
PubMed1989

Purification and some characteristics of a cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes.

Nitrous oxide reductase from Wolinella succinogenes was purified very nearly to homogeneity. The enzyme was found to be dimeric, with Mr = 162,000 and subunit Mr = 88,000, and to contain three copper atoms and one iron atom (as cytochrome c) per subunit. The oxidized enzyme exhibited absorption bands at 410 and 528 nm, and the dithionite-reduced enzyme, at 416, 520, and 550 nm. The isoelectric point was 8.6; specific activity was at 25 degrees C and pH 7.1, 160 mumol x min-1 x mg-1; and Km was 7.5 microM N2O under the same conditions. alpha-Chymotrypsin cleaved the enzyme into cytochrome c-depleted dimers with an average Mr = 134,000 and cytochrome c-enriched fragments with an average Mr = 13,000. The enzyme was stable at 4 degrees C for at least 100 h under air and 3 h in the presence of 5 mM EDTA. It exhibited a dithionite-N2O oxidoreductase as well as a BV+-N2O oxidoreductase activity. During turnover with BV+ at 25 mM N2O, the enzyme was observed to undergo an initial activation and a subsequent inactivation. The kinetics of inactivation were approximately first-order in remaining activity, and the first-order rate constant was essentially independent of the initial enzyme concentration. These characteristics are consistent with the occurrence of turnover-dependent inactivation. Acetylene was a relatively weak inhibitor, but cyanide and azide were rather strong inhibitors. The nitrous oxide reductase of W. succinogenes is quite different from that of denitrifying bacteria. The amount of activity in cell extracts and the absence of O2-labile nitrous oxide reductase suggested that the cytochrome c containing enzyme may be the only one produced by W. succinogenes.

Bacteroidaceae↗
PubMed1989

Studies of crystalline trimethylamine dehydrogenase in three oxidation states and in the presence of substrate and inhibitor.

Crystals of trimethylamine dehydrogenase have been examined by difference Fourier methods at 6.0-A resolution after partial reduction by substrate and by dithionite in the presence of inhibitor. Similar studies of the inhibited oxidized enzyme and of the enzyme reduced fully by dithionite alone were also carried out. In all cases ligand binding at the active site occurred. In addition, there were small structural changes, possibly side chain movements, in the inhibited oxidized enzyme and somewhat larger changes in the partially reduced crystals. The largest changes occurred with the fully reduced enzyme. However, in no cases were subunit or domain movements observed nor were changes observed in the position of the FMN or [4Fe-4S] cofactors. Parallel studies of crystalline trimethylamine dehydrogenase were carried out by EPR spectroscopy. The results show that the electronic states of the crystalline enzyme under the conditions of the difference Fourier studies are comparable to those which occur in solution under similar conditions.

Crystallization↗
PubMed1987

Porphobilinogen oxygenase. Purification and evidence of its hemoprotein structure.

Porphobilinogen oxygenase oxidizes porphobilinogen to 2-hydroxy-5-oxo-porphobilinogen. This enzyme isolated from wheat germ has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis under both nondenaturing and denaturing conditions. The molecular weight of the enzyme formed from two identical (or very similar) polypeptide chains is 70,000. It has a pI of 9.0 indicating its cationic nature. The pure enzyme contains 1 mol of high-spin heme and 2 mol of non-heme iron. It requires both of these as well as molecular O2 and a reducing agent for catalytic activity. Although the enzyme has many characteristics of a peroxidase, hydrogen peroxide cannot substitute for oxygen and dithionite for catalysis. The catalytic reaction is not affected by catalase, superoxide dismutase, or by hydroxyl radical scavengers. A comparison between porphobilinogen oxygenase and a commercial preparation of horseradish peroxidase was made. The latter also catalyzes aerobic porphobilinogen oxidation, with dithionite as electron donor. Here the oxidation of porphobilinogen is inhibited by superoxide dismutase and was not affected by catalase.

Amino Acids↗
PubMed1988

The ratio of two isozyme groups in microsomal cytochrome P-450 under exogenous influence of carbon tetrachloride and cyclophosphamide.

A method for measuring the content of two groups of microsomal cytochrome P-450 isozymes--cytochromes P-450W and P-450L--with the active sites directed into the water phase and membrane lipids, respectively, has been developed. The method is based on the ability of the xanthine oxidase-menadione complex to reduce microsomal cytochromes b5 and P-450 under anaerobic conditions by transferring electrons to hemoproteins with the active sites directed into the water phase. Cytochrome b5 is completely reduced (to the dithionite level) and cytochrome P-450 is reduced partially (only a group of cytochromes P-450W). The amount of cytochromes P-450L is estimated using the difference between the total content of cytochrome P-450 reduced by sodium dithionite and the content of cytochromes P-450W. The possibility of controlling the ratio of these two isozyme groups in cytochrome P-450 in vivo in membranes of the endoplasmic reticulum by pretreatment of animals with a variety of chemicals has been demonstrated. The ratio of cytochromes P-450W and P-450L has been shown to decrease two-fold 18 days after three injections of phenobarbital into mice. Carbon tetrachloride and cyclophosphamide also decrease this ratio in vivo.

Animals↗
PubMed1988

Structure and function of vanadium-containing bromoperoxidases.

The properties of the vanadium-containing bromoperoxidases from the seaweeds Ascophyllum nodosum, Laminaria saccharina and the lichen Xanthoria parietina were studied. Upon reduction with sodium dithionite, these bromoperoxidases show EPR spectra which are typical of a vanadyl cation (VO2+). From the spectral parameters and a comparison with inorganic vanadyl complexes, we conclude that the ligand environment largely consists of oxygen donors. The data also show that the structure of the active sites in these enzymes is very similar. Since EPR spectra of vanadium(IV) bromoperoxidase are only obtained after reduction, the metal ion is present in the native enzymes in the 5+ oxidation state. All these enzymes loose their enzymic activity upon dialysis against citrate-phosphate (PO4(3-)) buffer at pH 3.8, containing EDTA. The brominating activity could be reconstituted by the addition of vanadate (VO4(3-)). The experiments suggest that vanadate is incorporated into these enzymes. In line with the EPR data, we propose a structure of the active site in which at least 4 oxygen atoms are present as donors for the central vanadium(V) ion. Since several inorganic peroxovanadium(V) complexes have been described, we suggest that the vanadium ion in bromoperoxidases serves as a binding site for H2O2. Upon subsequent binding of bromide this ion is oxidized by the peroxo-intermediate to form hypobromite. This model does not require valence state changes of the metal ion itself and indeed no changes in the EPR spectrum of reduced bromoperoxidase are observed upon addition of H2O2 or Br-. Further, bromoperoxidase reduced with a small excess of sodium dithionite is not active in the bromination reaction. The bromoperoxidases from the various sources show similarity in the amino-acid composition with a predominance of acidic amino acids. Distinct pH optima are observed in the bromination reaction catalysed by the bromoperoxidases. Despite the presence of the same prosthetic group in these enzymes with comparable vanadium ligand-field environment, the enzymic properties are very different. The specific activity as well as the Km for bromide differ greatly. Unlike the enzymes from the seaweeds A. nodosum and L. saccharina the bromoperoxidase from the lichen X. parietina is inhibited by low concentrations (1-5 mM) of nitrate. These bromoperoxidases have a remarkable resistance towards organic solvents such as methanol, ethanol and propanol.

Amino Acids↗
PubMed1978

Biosynthesis of a prostaglandin by a plant enzyme.

Lipoxygenase-2 from soybeans catalyzes the oxygenation of arachidonic acid to form significant amounts of a prostaglandin product. Results obtained with the dithionite-reduced derivatized product upon combined gas chromatography-mass spectroscopy are consistent with structures of the stereoisomers of 9,11,15-trihydroxyprosta-5,13-dienoic acid. A portion of the material formed by reduction of the enzyme product with dithionite reacts positively in a radioimmunoassay against rabbit anti-prostaglandin F2alpha antibody. This appears to be the first report of the synthesis of a prostaglandin by a non-animal enzyme.

Arachidonic Acids↗
PubMed1977

Hypotonic exchange-loading of erythrocytes. II. introduction of hemoglobins S and C into normal red cells.

A rapid, simple technique whereby erythrocytes transiently exposed to hypotonic hemoglobin preparations undergo exchange of endogenous for exogenous hemoglobin has now been applied to normal, hemoglobin A-containing erythrocytes into which hemoglobin S or hemoglobin C have been introduced. Erythrocytes containing an average of 12 to 61 percent of hemoglobin S undergo sickling when incubated in isotonic dithionite solution and are morphologically indistinguishable from natural sickle cells. Ultrastructural studies of thin sections of these artificial sickle cells show microfialments and fascicles which appear typical of microtubular tactoids of hemoglobin S seen in natural sickle cells. Erthrocytes containing 43 percent introduced hemoglobin C developed all of the morphologic alterations associated with intra-erythrocytic hemoglobin C, including hemoglobin aggregation, targetting, and intracellular crystallization, after inclbation for 24 hours in isotonic saline: dithionite. The results support the concept that the sickling process, and morphologic erythrocyte alterations occurring in hemoglobin C disorders are primary to the respective hemoglobins rather than the membrane. They also suggest that any role played by the inner surface of the erythrocyte membrane in the development of these alterations is probably secondary and not specific to the inner membrane surface of the natural sickle cell or hemoglobin C-containing cell.

Cell Membrane↗
PubMedDate not supplied

The release of inorganic fluoride from halothane and halothane metabolites by cytochrome P-450, hemin, and hemoglobin.

Halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) releases inorganic fluoride when incubated anaerobically with rat hepatic microsomes reduced with either NADPH or sodium dithionite. Boiled microsomes (cytochrome P-420), hemoglobin, and hemin, each reduced with sodium dithionite, also catalyze the release of inorganic fluoride from halothane, suggesting that the protein portion of cytochrome P-450 is not required for reductive halothane defluorination. 2-Chloro-1,1-difluoroethylene and its bromo analog 2-bromo-1,1-difluoroethylene undergo metabolism in NADPH-reduced microsomes with optimal release of fluoride occurring under air compared to an atmosphere of nitrogen. Neither 2-chloro-1,1,1-trifluoroethane nor 2-bromo-1,1,1-trifluoroethane liberate fluoride in microsomes under air or nitrogen. 2-Chloro-1,1-difluoroethylene and 2-bromo-1,1-difluoroethylene are metabolized predominantly by oxidative cytochrome P-450 metabolism, while reductive pathways utilizing reduced cytochrome P-450, hemoglobin, or hemin liberate fluoride from halothane.

Animals↗
PubMed1982

Evidence for the inorganic nature of the cyanolyzable sulfur of molybdenum hydroxylases.

Activation of the desulfo forms of milk xanthine oxidase, chicken liver xanthine dehydrogenase, and aldehyde oxidase with S2- is greatly facilitated in the presence of reducing agents. Upon anaerobic incubation with 1 mM S2- and 1 mM dithionite, desulfo xanthine oxidase and chicken liver xanthine dehydrogenase prepared by cyanide treatment of active enzymes, are activated to the specific activity predicted by their molybdenum content. Routine preparations containing desulfo molecules are also similarly activated to the extent predicted. Cyanide-inactivated chicken liver xanthine dehydrogenase was reconstituted with 35S2- in the presence of dithionite. 85% of enzyme-bound radioactivity was shown to be in the form of cyanolyzable sulfur, by comparison of enzyme activity, bound radioactivity, and 35SCN- yields from exposure of labeled enzyme to cyanide. This radiolabeled enzyme allowed the determination of the following. 1) The cyanolyzable sulfur is largely removed from the polypeptide by incubation at 37 degrees C for one hour in 1% sodium dodecyl sulfate, pH 7, or for 15 min in 6 M guanidinium chloride, pH 6.2. 2) The cyanolyzable sulfur is "acid labile." [35S]Methylene blue is formed in the theoretical quantity from oxidized or substrate-reduced enzyme under the standard conditions for labile sulfur analysis by the methylene blue method. These data strongly support the conclusion that the cyanolyzable sulfur is a terminal sulfur ligand of the Mo atom, and is not part of an organic moiety.

Aerobiosis↗