Participation of oxygen activated species in enzymatic lipid peroxidation in biomembranes]

The participation of oxygen activated species in the induction of lipid peroxidation (LPO) in the membrane systems containing cytochrome P-450 (liver microsomes) and in the membrane fragments devoid of this hemoprotein (brain and skeletal muscle microsomes) was studied. It was shown that the rate of NADH-dependent LPO does not depend on the presence of hemoproteins and the activity of NADH-specific flavoprotein in the membranes. On the other hand, the microsomal membranes of the liver with high specific contents of b5 and P-450 cytochromes and NADPH-specific flavoprotein, had the highest rates of NADPH-dependent LPO. It was found that the most effective inhibitors of free oxygen activated species in the case of NADPH- and NADH-dependent LPO in the microsomal fractions of liver, brain and skeletal muscles are the superoxide (O ./2) anion radical inhibitors. The singlet oxygen (1O2) quenchers inhibit only NADPH-dependent LPO in the liver, however, in a far lesser degree. The hydroxyl radical (OH) scavengers had no effect on enzymatic LPO in all systems studied.     

Kinetic mechanism and specificity of bovine milk sulphydryl oxidase

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.     

Effect of ionizing radiation on O2 generation by cytochrome P-450

A study was made of generation of superoxide anion-radical (O2-) by cytochrome P-450 in a microsomal membrane of rat liver. Using spectrophotometry (by oxidation of adrenaline to adrenochrome) and ESR (with a spin-trap, tiron) the authors showed the ability of O2- generation by P-450 through decomposition of organic peroxides. During the first 24 h following irradiation of rats with doses of 7 and 10 Gy, the generation of O2- by cytochrome P-450 of rat liver microsomes was increased. Mechanisms of the postirradiation modification of O2- generation rate are discussed.     

In vitro chemotropism of pearl millet pollen tubes to stigma tissue: a response to glucose produced in the medium by tissue-bound invertase

Summary Water-homogenized stigma pellets of pearl millet and precipitates resulting from dialysis of their salt extracts were observed to: (1) chemotropically attract pearl millet pollen tubes on a sucrose-containing pollen germination and growth medium, (2) have acid invertase activity as assayed by the arsenomolybdate method, (3) hydrolyze sucrose in the pollen germination and growth medium to glucose as assayed by coupled glucose oxidation with Nitro Blue Tetrazolium, and (4) lose chemotropic and invertase activities upon heat treatment. The results indicate that the in vitro chemotropic attraction of pearl millet pollen tubes to water-homogenized stigma pellets is a response to glucose produced by homogenate-pellet-bound invertase hydrolyzing the sucrose present in the pollen germination and growth medium. Yeast and tomato invertases used as controls verified this conclusion. Water extracts of whole stigmas contained water-soluble acid invertase. The results are discussed in relation to the identification of possible in vivo chemotropic factors of pearl millet and other plants by in vitro assays.Abbreviations dH₂O Deionized, house-distilled water - NBT Nitro Blue Tetrazolium, NBT-medium - PGG medium, pollen germination and growth medium (10% sucrose, 1 mM H₃BO₃, and 1% agarose); - WHS pellet, water-homogenized stigma pellet On Specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

PROTEOLYTIC MICRODISSECTION OF SMOOTH-SURFACED VESICLES OF LIVER MICROSOMES

Digestion of rabbit liver microsomal smooth vesicles with Bacillus subtilis protease released proteins and peptide fragments from the vesicles, without solubilizing phospholipids and cholesterol. The proteolysis was, however, limited when about 30% of the protein had been solubilized. The same limitation was observed when the vesicles were treated with trypsin, chymotrypsin, or their combinations with the bacterial protease. The limited proteolysis was accompanied by selective solubilization of cytochrome b₅ and microsomal NADPH-specific flavoprotein, leaving the CO-binding hemoprotein and some other enzymes still attached to the vesicular membranes. Sucrose density gradient centrifugation of protease-treated vesicles indicated that all the vesicles had been attacked by the protease to similar extents. The behavior of intact and digested vesicles in dextran density gradient centrifugation suggested that the vesicles, even after proteolytic digestion, existed in the form of closed sacs which were impermeable to macromolecules such as dextran and proteases. It was concluded that only the outside surface of the vesicles is susceptible to the proteolytic action and that cytochrome b₅ and the NADPH-specific flavoprotein are located in the susceptible area.     

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.     

Random distribution of NADPH-specific flavoprotein and cytochrome P-450 in liver microsomes

The dilution of rabbit liver microsomes by soy-bean phospholipids was used as methodical approach to investigate the molecular organization of NADPH-dependent microsomal redox chain. The ultrastructural analysis of control and phospholipid diluted microsomes revealed that the incorporation of exogenous phospholipids into microsome membranes increased their surface area, as well as decreased the lateral density distribution and size of intramembrane particles. The dilution of microsome membranes by phospholipids slowed down the initial rate of cytochrome P-450 reduction by NADPH. The apparent second order rate constant of cytochrome P-450 reduction by NADPH: cytochrome P-450-reductase did not change in phospholipid-enriched microsomes. The results obtained provide strong evidence for the random distribution of NADPH-specific flavoprotein and cytochrome P-450 in liver microsome membranes.     

Reduction and catalytic properties of cytochrome P-450 LM2 in reconstituted system containing monomeric carriers

The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only. The addition of benzphetamine and/or flavoprotein to cytochrome P-450 monomers did not cause the spin equilibrium shift and the low-spin form content was higher than 85% in all cases. Investigation of the dependence of the initial rates of NADPH-dependent cytochrome P-450 reduction and benzphetamine oxidation on the stoichiometry of the flavoprotein and cytochrome P-450 at their constant total concentration showed that the molar ratio of 1:1 was required for maximal activity. Thus this system works in full accordance with the mass action law.     

The Copper Complex of Captopril is not a Superoxide Dismutase Mimic. Artefacts in DMPO Spin Trapping

The effect of captopril and of its copper complex on several superoxide-dependent reactions used to detect and assay superoxide dismutase activity was studied, including pyrogallol and hematoxylin autoxidation and Nitro Blue Tetrazolium reduction. ln none of these systems were superoxide dismutase-like properties of captopril/Cu apparent. Captopril/Cu decreased the yield of DMPO-OH adducts generated by KO₂ but this effect may be due to the acceleration of the decay of the adduct by captopril/Cu.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

A non-radioactive method for mapping restriction fragment length polymorphic genetic markers in Anopheles gambiae

A non-radioactive method for in situ hybridisation of Restriction Fragment Length Polymorphic (RFLP) markers to the polytene chromosome of Anopheles gambiae, the important malaria vector, which yielded good readable quality of chromosomal bands is reported. The methodology adopted was a Biotin-Streptavidin-Alkaline Phosphatase system which yielded fluorescent signals when stained with dyes such as Nitro Blue Tetrazolium and Bromo Chloro Indolyl Phosphate.     

Myeloperoxidase-oxidase oxidation of cysteamine.

Cysteamine oxidation was shown to be catalysed by nanomolar concentrations of myeloperoxidase in a peroxidase-oxidase reaction, i.e. an O2-consuming oxidation of a compound catalysed by peroxidase without H2O2 addition. When auto-oxidation of the thiol was prevented by the metal-ion chelator diethylenetriaminepenta-acetic acid, native, but not heat-inactivated, myeloperoxidase induced changes in the u.v.-light-absorption spectrum of cysteamine. These changes were consistent with disulphide (cystamine) formation. Concomitantly, O2 was consumed and superoxide radical anion formation could be detected by Nitro Blue Tetrazolium reduction. Both superoxide dismutase and catalase inhibited the reaction, whereas the hydroxyl-radical scavengers mannitol and ethanol did not. O2 consumption increased with increasing pH (between pH 6.0 and 8.0), and 50% inhibition was exhibited by about 3 mM-NaCl at pH 7.0 and by about 100 mM-NaCl at pH 8.0. Cysteamine was about 5 times as active (in terms of increased O2 consumption at pH 7.5) as the previously reported peroxidase-oxidase substrates NADPH, dihydroxyfumaric acid and indol-3-ylacetic acid. A possible reaction pathway for the myeloperoxidase-oxidase oxidation of cysteamine is discussed. These results indicate that cysteamine is a very useful substrate for studies on myeloperoxidase-oxidase activity.     

Rat hepatic microsomal acetoacetyl-CoA reductase. A beta-ketoacyl-CoA reductase distinct from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system

The present study provides evidence for a new rat liver microsomal enzyme, a short chain beta-ketoacyl (acetoacetyl)-CoA reductase, which is separate from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system. This microsomal reductase converts acetoacetyl-CoA to beta-hydroxybutyryl-CoA at a rate of 70 nmol/min/mg of protein; the enzyme has a specific requirement for NADH and appears to obtain electrons directly from the reduced pyridine nucleotide without the intervention of cytochrome b5 and its flavoprotein reductase. The apparent Km of the enzyme of the acetoacetyl-CoA was 21 microM and for the cofactor, 18 microM. The pH optimum was broad, ranging from 6.5 to 8.0. The product formed is the D-isomer of beta-hydroxybutyryl-CoA. High carbohydrate fat-free diet resulted in a small but significant (35%) increase in microsomal acetoacetyl-CoA reductase activity. The cytosol also contains this enzyme activity, measuring approximately 57% of that found in the microsomes. The mitochondrial activity which is 20-25% higher than the microsomal activity appears to be due to L-beta-hydroxyacyl-CoA dehydrogenase which converts acetoacetyl-CoA to L-beta-hydroxybutyryl-CoA. The microsomal acetoacetyl-CoA reductase activity was extracted from the microsomal membrane by 0.4 M KCl, resulting in an 8- to 10-fold purification; in addition, the long chain fatty acid elongation system was unaffected by this extraction procedure. Employing beta- hydroxyhexanoyl -CoA as a substrate, evidence is also provided for a separate dehydratase which acts on short chain substrates. Lastly, the liver microsomes had no detectable acetoacetyl-CoA synthetase or acetyl-CoA acetyltransferase activities. Hence, the possible involvement of the rat hepatic microsomal short chain beta-ketoacyl-CoA reductase, short chain beta-hydroxyacyl-CoA dehydratase, and the previously reported short chain trans-2-enoyl-CoA reductase in the hepatic utilization of acetoacetyl-CoA and in the synthesis of butyryl-CoA for hepatic lipogenesis is discussed.     

THE CYTOCHEMICAL REDUCTION OF NITRO BLUE TETRAZOLIUM AS AN INDEX OF POLLEN VIABILITY

A new technique for estimating pollen viability utilizing Nitro Blue Tetrazolium (Nitro-BT) is given. Pollen of 16 taxa was studied and scored for stainability. A comparison was made between Nitro-BT-stained pollen samples and aniline-blue-lactophenol-stained samples, because the validity of the latter stain has been questioned in recent years. It is demonstrated that Nitro-BT can discriminate between pollens which have a capacity for oxidative metabolism, representing potential viability, and those which do not. Suggestions for pollen stainability studies are given.     

Histochemical studies of rat ovarian follicular cells in vitro

Summary Membrana granulosa cells were aspirated from large follicles of proestrous rat ovaries and were cultivated as monolayers. For histochemical identification of dehydrogenases, the monolayers were incubated in various steroid substrates, nicotinamide adenine dinucleotide, and Nitro Blue Tetrazolium. The presence of Δ^p5-3β-, 3α-, 17β- and 20α-hydroxysteroid dehydrogenases was demonstrated by the 4th day in vitro and was evident for as long as 20 days. Since none of these hydroxysteroid dehydrogenases is demonstrable in the membrana granulosa of intact follicles, it is concluded that the steroidogenic capacity of the cells, repressed in the preovulatory follicle in vivo, can be expressed upon mechanical removal from the follicle just as steroid synthesis occurs in these cells after normal ovulation. This research was supported in part by a Faculty Research Grant from the Horace H. Rackham School of Graduate Studies, and by United States Public Health Service Grants RR-05383-09 and AM-06918-06.     

Synergism between substrate and non-substrate thiols in peroxidase-oxidase reactions

Cysteamine and reduced glutathione were shown to act synergistically as peroxidase-oxidase substrates as measured by oxygen consumption and Nitro Blue Tetrazolium reduction. Cysteine methyl ester could be substituted for cysteamine and N-acetylcysteine and penicillamine could be substituted for glutathione. The involvement of reduced oxygen species and the effects of pH and chloride were studied. A possible mechanism of peroxidase-oxidase oxidation of cysteamine and glutathione is proposed. These studies show that peroxidase oxidase reactions can occur with close to physiological concentrations of peroxidase and thiols.     

Reconstitution of liver monooxygenase system in solution from cytochrome P-450 and NADPH-specific flavoprotein monomers

Microsomal monooxygenase system was reconstituted in the presence of non-ionic detergent Emulgen 913 from cytochrome P-450 and NADPH-specific flavoprotein isolated from phenobarbital-induced rabbit liver microsomes. At Emulgen 913 concentration of 0.05 g/l mixed complex between flavoprotein and cytochrome was formed with 5: 5 protein molar ratio and molecular weight of 700 kD. The 2-hour incubation of the enzymes with 0.25 g/l Emulgen 913 at 4 degrees C was accompanied by dissociation of protein oligomers to monomers. The reconstituted systems containing flavoprotein and cytochrome as mixed complexes or monomers were able to catalyze NADPH-dependent cytochrome P-450 reduction and benzphetamine N-demethylation. Taking into consideration the effective concentrations of the enzymes the apparent second order rate constants of these reactions with monomers were 100 times those with complexes.     

Microsomal hemoprotein reduction by superoxide radical formed on NADPH-specific flavoprotein]

The superoxide radicals formed on NADPH-specific flavoprotein of liver microsomes can reduce cytochromes c, b5, and P-450. This reaction is inhibited under aerobic conditions by a low molecular weight analog of superoxide dismutase, e.g. the copper-tyrosine complex. The inhibitory effect of the complex is not observed under anaerobic conditions. Based on the results obtained a scheme of the electron transfer between the flavoprotein and haemoproteins involving superoxide radicals is proposed.     

Interrelationship between the generation of oxygen anion-radicals and the reduction of artificial acceptors and cytochrome P-450 by NADPH-cytochrome c reductase]

The interaction of NADPH-cytochrome c reductase with oxygen, artificial acceptors and cytochrome P-450 is investigated. It is found that generation of oxygen anion-radicals (O2-), determined from the reaction of adrenaline oxidation into adrenochrome, proceeds independently on the reactions of interaction with artificial "anaerobic" acceptors-cytochrome c, dichlorophenolindophenol. Propylgallate competitively inhibits the reaction of adrenaline oxidation by isolated DADPH-cytochrome c reductase and non-competitively suppress the reaction of cytochrome c reduction. In contrast to the process of electron transfer on cytochrome c, there is a direct correlation between the rate of cytochrome P-450 reduction and the rate of adrenaline oxidation in liver microsomes. Hexobarbital increases V of the adrenaline oxidation reaction and does not affect the Km value, while metirapon, a metabolic inhibitor, decreases the Vmax and does not change Km. On the basis of the data obtained it is suggested that the reactions of NADPH-cytochrome c reductase interaction with oxygen and artificial "anaerobic" acceptors are connected with different redox-states of flavoprotein or with different flavine coenzymes, and that the electron transport on cytochrome P-450 and directly on oxygen takes place in interrelated redox-states of flavoprotein.     

Purified rat lens aldose reductase. Polyol production in vitro and its inhibition by aldose reductase inhibitors.

The production of polyols in vitro by highly purified aldose reductase (EC 1.1.1.21) was monitored by g.l.c. In the presence of NADPH aldose reductase reduced glucose, galactose and xylose to the respective polyols sorbitol, galactitol and xylitol. The rates of formation of these polyols closely mirrored the Km values for the substrates obtained from kinetic measurements that monitored the rate of disappearance of NADPH. No polyol production occurred in the absence of purified aldose of purified aldose reductase, and analysis by g.l.c. revealed only the presence of unchanged monosaccharides. Addition of the aldose reductase inhibitor sorbinil to purified rat lens aldose reductase incubated with xylose in the presence of NADPH resulted in decreased xylitol production. However, aldose reductase inhibitors produced no effect in altering the rate of Nitro Blue Tetrazolium formation from either glucose or xylose, indicating that the observed inhibition in vitro does not result from a free-radical-scavenger effect.