Phenazine Methosulphate Research Articles

Elimination of artifacts due to glutaraldehyde fixation in the histochemical detection of glucose oxidase with tetrazolium salts

High background staining due to glutaraldehyde fixation prevents phenazine methosulphate and a tetrazolium salt being used to visualize glucose oxidase activity in tissue slices prepared from mice injected with the enzyme. Experiments in solution showed that products formed during the reaction between amino groups and glutaraldehyde are, at least in part, responsible for the non-enzymatic reduction of tetrazolium salts. Experiments performed with artificial membranes chemically akin to glutaraldehyde-fixed sections and prepared by cross-linking albumin by glutaraldehyde, showed that double bonds in amino-glutaraldehyde products are mainly responsible for the background staining development, whereas thiol groups play only a minor role. A sequential treatment with sodium borohydride and N-ethylmaleimide greatly reduced the background staining, thus permitting the detection of glucose oxidase activity. Optimal conditions for glucose oxidase activity demonstration (maximum enzyme velocity for minimum 'nothing dehydrogenase' phenomenon) were studied: choice of the tetrazolium salt, nature, pH and molarity of the buffer used for the staining mixture. A procedure similar to that developed with artificial membranes was applied to tissue sections of mice in which glucose oxidase had been injected intravenously. It allowed detection of glucose oxidase activity without artifactual staining in control slices.

NAD(P)+-independent aldehyde dehydrogenase from Pseudomonas testosteroni. A novel type of molybdenum-containing hydroxylase.

Aldehyde dehydrogenase from Pseudomonas testosteroni was purified to homogeneity. The enzyme has a pH optimum of 8.2, uses a wide range of aldehydes as substrates and cationic dyes (Wurster's blue, phenazine methosulphate and thionine), but not anionic dyes (ferricyanide and 2.6-dichloroindophenol), NAD(P)+ or O2, as electron acceptors. Haem c and pyrroloquinoline quinone appeared to be absent but the common cofactors of molybdenum hydroxylases were present. Xanthine was not a substrate and allopurinol was not an inhibitor. Alcohols were inhibitors only when turnover of the enzyme occurred in aldehyde conversion. The enzyme has a relative molecular mass of 186,000, consists of two subunits of equal size (Mr 92,000), and 1 enzyme molecule contains 1 FAD, 1 molybdopterin cofactor, 4 Fe and 4 S. It is a novel type of NAD(P)+-independent aldehyde dehydrogenase since its catalytic and physicochemical properties are quite different from those reported for already known aldehyde-converting enzymes like haemoprotein aldehyde dehydrogenase (EC 1.2.99.3), quino-protein alcohol dehydrogenases (EC 1.1.99.8) and molybdenum hydroxylases.

The effect of water-miscible solvents on the ?1-dehydrogenase activity of free and PAAH-entrapped Arthrobacter simplex

The effect of water-miscible cosolvents on biotransformations of poorly water-soluble substrates by immobilized cells was investigated, using Δ1-dehydrogenation of hydrocortisone by Arthrobacter simplex as a model. Criteria for solvent selection on the basis of retention of enzymic activity were postulated and tested. Diols were considered to be the most suitable group of solvents. Substrate solubility increased tenfold in 30% (v/v) ethylene glycol, but reaction rates were significantly slower in such solutions. This was mainly caused by a decrease of oxygen solubility in the presence of the cosolvent and conformational changes imposed on the intracellular enzyme by cosolvent molecules penetrating the cell. The inhibition could be eliminated by the addition of an artificial electron acceptor, phenazine methosulphate (PMS). Reaction rates faster than those for substrate suspensions (no cosolvent added) could thus be achieved. Immobilization of Arthrobacter simplex in cross-linked polyacrylamide hydrazide gave high retentions of activity. PMS exhibited toxic effects on the entrapped cells, leading to reduced activity after extended use.

A fluorescent redox dye. Influence of several substrates and electron carriers on the tetrazolium salt-formazan reaction of Ehrlich ascites tumour cells.

This study was performed to elaborate the best conditions for measuring the redox activity of Ehrlich ascites tumour cells by using a new tetrazolium salt, cyantolyl tetrazolium chloride (CTC). This tetrazolium salt forms a fluorescent water-insoluble formazan on reduction on the surface of intact vital cells. The influences of fixation and of various substrates and electron carriers on the cellular reduction of CTC were investigated quantitatively using an elution technique. The amount of formazan obtained after incubating vital cells with Meldola Blue as electron carrier was greater than that obtained with Methylene Blue, menadione, 2,6-dichloroindophenol, 1-methoxyphenazine methosulphate or phenazine methosulphate. Using flow cytometry, the formazan production per cell and, after staining the nuclear DNA, the distribution of the redox activity in the cell population can be visualized with satisfactory resolution. We conclude from our findings that dehydrogenases are only partially involved in the reduction of tetrazolium salts by intact cells and that a redox activity, probably related to a cell membrane-bound NAD(P)H-oxidase system, is mainly measured.

Cysteamine oxygenase: possible involvement of superoxide ion in the catalytic mechanism.

The reaction catalyzed by cysteamine oxygenase on cysteamine in the presence of phenazine methosulphate as cofactor like compound is inhibited by nitroblue tetrazolium, a scavenger of superoxide ions. The reaction is not inhibited by superoxide dismutase and allyl alcohol and it is not activated by superoxide ions produced in solution. Nitroblue tetrazolium is reduced by cysteamine or mercaptoethanol and phenazine methosulphate. This reaction is completely inhibited by superoxide dismutase. In the presence of cysteamine oxygenase the reduction with mercaptoethanol is greatly enhanced and it is only partially inhibited by superoxide dismutase. According to these data a reaction mechanism is proposed in which superoxide ions and thiyl radicals are produced at the active site during catalysis.

Glucose flux through the hexose monophosphate shunt and NADP(H) levels during in vitro ageing of human skin fibroblasts.

In cultured human skin fibroblasts the glucose flux through the hexose monophosphate shunt (HMS) amounts to 4% of the glucose flux through the glycolytic pathway. Upon in vitro ageing the rate of glucose utilization through the HMS is decreased more than 50%. This decrease in HMS was not caused by a limiting enzymatic capacity since glucose utilization through the HMS could be raised at least 30-fold in both 'young' and 'aged' fibroblasts upon stimulation with phenazine methosulphate. This effect of in vitro ageing upon glucose metabolism was also not due to differences in proliferation rate between 'young' and 'aged' human fibroblasts, since there was no difference in glucose utilization between proliferating and growth-inhibited (confluently cultured) fibroblasts. The NADPH/NADP ratio was found to be decreased by 12% in 'aged' cells.

Effect of oxygen on the tetrazolium reaction for glucose 6-phosphate dehydrogenase in cryosections of human breast carcinoma, fibrocystic disease and normal breast tissue.

We here report a method for the characterization of carcinoma cells in cryosections of human breast tissue. Cryosections from 37 biopsies including carcinomas, fibrocystic disease specimens and reduction mammoplasties were incubated in an atmosphere of either pure nitrogen or 99.5% oxygen. The incubation medium contained neotetrazolium, phenazine methosulphate and the colloid stabilizer Polypep 5115. A marked overlap in formation of reaction product was found between carcinomas, benign tissue and normal breast tissue when sections were incubated in pure nitrogen. However, following 5 min of oxygen incubation no reaction was seen in normal breast tissue, whereas carcinoma cells consistently showed reaction. Moreover, a statistically significant difference was seen between the level of reaction in carcinoma cells and that of ductal epithelial cells in fibrocystic disease not containing apocrine metaplasia. Apocrine metaplasia, on the other hand, showed the highest activity recorded at all.

Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.

Oxidation of nicotinamide coenzyme dimers by one-electron-accepting proteins.

The nicotinamide nucleotide dimers (NAD)2 and (NADP)2, obtained by electrochemical reduction of NAD+ and NADP+, are able to reduce such single-electron acceptors as the proteins cytochrome c, azurin and methaemoglobin, though at different rates. Under the same conditions the reduced nicotinamide coenzymes NADH and NADPH are not able to reduce these proteins at measurable rates unless a catalyst (phenazine methosulphate or NADH-cytochrome c reductase in the case of cytochrome) is present. The redox mechanism seems to involve the formation of an NAD(P). radical that in the presence of O2 gives rise to superoxide (O2.-), since superoxide dismutase inhibited these reactions.

Survey of p‐cresol assimilating bacteria: Characterization and identification of a p‐cresol utilizing bacterium

A survey of 110 bacterial strains drawn from various ecosystems yielded 10 capable of growth on p‐cresol. Of these, eight were Gram‐negative and two Gram‐positive. One Gram‐negative and both Gram‐positive strains metabolized p‐cresol via 4‐methylcatechol, the others initiated attack by hydroxylation of the methyl group and used the protocatechuate pathway. These included Pseudomonas alcaligenes, Ps. testosteroni, four other Pseudomonas species and an unidentified organism, the crude extract of which oxidized p‐cresol without requirement for either electron donor or acceptor, although inclusion of phenazine methosulphate stimulated activity. The conversion of p‐cresol to p‐hydroxybenzylalcohol catalysed by the PMS‐dependent p‐cresol methylhydroxylase was linked to the electron transport chain with molecular oxygen acting as the ultimate electron acceptor. Successive subculturing in the absence of the aromatic substrate led to the eventual loss of the organism's ability to metabolize p‐cresol. The organism was assigned to the genus of Alcaligenes.

Rubidium uptake by mouse pancreatic islets exposed to 6-hydroxydopamine, ninhydrin, or other generators of hydroxyl radicals.

The purpose was to study the toxicity of drugs known to generate free radicals on isolated pancreatic islets. The accumulation of 86Rb+ by mouse pancreatic islets was measured in vitro. Exposing the islets to 6-hydroxydopamine, ninhydrin, or phenazine methosulphate+NADH inhibited the Rb+ uptake, whereas paraquat or acetylphenylhydrazine had no effect. This effect of 6-hydroxydopamine was prevented by either of the hydroxyl radical scavengers, sodium benzoate and mannitol, but not by the non-scavenger, urea; ninhydrin was partially protected against by mannitol but not by benzoate. Protection against 6-hydroxydopamine was also afforded by D-glucose but not by L-glucose or 3-O-methyl-D-glucose; none of the sugars protected against ninhydrin. In damaging islet beta-cells and in being protected against by D-glucose, 6-hydroxydopamine closely resembles the diabetogenic drug, alloxan. It is suggested that protection against alloxan may involve both glucose metabolism and the interaction of glucose with its membrane-located carrier, while protection against 6-hydroxydopamine appears to be unrelated to the hexose carrier mechanism.

Changes in decoated spores of Clostridium perfringens caused by treatment with some enzymatic and non-enzymatic systems

Decoated and Cu2+-binding spores of Clostridium perfringens underwent germination-like changes followed by lysis when incubated with the following non-enzymatic and enzymatic systems: ascorbic acid under aerobic conditions, a mixture of NADH and phenazine methosulphate, glucose with glucose oxidase, and NADH with NADH oxidase. Hydrogen peroxide formed from these systems in common is responsible for lysis of treated spores, because catalase strongly inhibited lysis. Superoxide radical is not directly involved in the reaction, because superoxide dismutase did not inhibit lysis.

Purification and properties of an amine dehydrogenase from Pseudomonas K95 grown on 1,12-diaminododecane (DAD).

The cell-free extract of Pseudomonas K95 gwown on 1,12-diaminododecane (DAD) could oxidize all tested alkyl diamines, from C4 to C12. The cell-free extract contained an amine dehydrogenase. The amine dehydrogenase was purified 1700-fold to homogeneity from Pseudomonas K95 grown on DAD as the sole carbon source. The molecular weight of the enzyme was found to be 56,000, on gel filtration, and 47,000, on sodium dodecyl sulfate gel electrophoresis; no evidence was obtained for subunits. The enzyme is able to utilize only phenazine methosulphate as an electron acceptor. The enzyme is markedly nonspecific, readily oxidizing both short and long chain primary monoamines and diamines, polyamines, l-noradrenaline, histamine, benzylamine and di-n-hexylamine. The enzyme was inhibited by the carbonyl reagents, semicarbazide and isoniazid. The optimum pH for the oxidation of DAD was 7.0 and that for 12-aminododecanoic acid (ADA) was 8.0. The Km value for DAD at pH 7.0 was 3 μ and that for ADA was 33μm. The difference ...

Metabolism of 3-Butyn-1-ol by Pseudomonas BB1

Summary: A degradation pathway for the acetylenic compound 3-butyn-1-ol by a bacterium showing strong resemblance to the physiologically well characterized Pseudomonas AM1 is presented. Enzyme studies revealed that the pathway of 3-butyn-1-ol metabolism involves an initial oxidation of the alcohol group by a phenazine methosulphate dependent alcohol dehydrogenase. Subsequently, the aldehyde is probably oxidized to yield 3-butynoic acid. The latter product is hydrated in the absence of cofactors to give acetoacetate which, in turn, is further degraded via acetoacetyl-CoA to acetyl-CoA. The inducible hydration reaction was catalysed by a nonspecific enzyme; hydration of propynoic acid resulted in the formation of malonic semialdehyde.

Single-turnover flash-induced ATP synthesis in photosystem I-enriched subchloroplast vesicles

Flash-induced ATP synthesis coupled to cyclic electron flow in photosystem I-enriched subchloroplast vesicles was studied by continuous registration of luciferin-luciferase luminescence. Cyclic electron flow was elicited in the presence of the artificial mediator phenazine methosulphate or of native ferredoxin, the latter redox-poised by NADPH and oxygen. ATP hydrolysis is observed in dark-adapted vesicles in the presence of ferredoxin without requirement for previous activation of the ATPases. Ferredoxin-catalyzed ATP synthesis proceeds at a constant (maximal) rate between NADPH/O 2; ratios of 1.5 and 11. It was sensitive to FCCP, DCCD, DBMIB, DNP-INT and HQNO, but not to antimycin A.

The role of auxiliary oxidants in the maintenance of a balanced redox poise for photosynthesis in bacteria

Carotenoid absorbance changes were used to monitor the development of membrane potential in intact cell suspensions of Rhodopseudomonas capsulata strain N22. Low concentrations of phenazine methosulphate almost completely inhibited the generation of membrane potential in the light by anaerobic cells. The light-dependent reactions were restored by addition of either trimethylamine N-oxide, dimethylsulphoxide, nitrous oxide, or oxygen. In Rhodopseudomonas capsulata strain N22 DNAR + addition of nitrate was also effective. The inhibition by phenazine methosulphate and restoration by auxiliary oxidant were observed in the presence of sufficient rotenone to block NADH dehydrogenase or with low concentrations of uncoupling agent to dissipate the membrane potential under dark, anaerobic conditions. It is suggested that in intact cells of these organisms there are mechanisms which operate to maintain the electron-transport chain at an optimal redox poise for efficient photosynthesis. Phenazine methosulphate perturbs the optimal redox poise by hastening equilibrium of the photosynthetic electron-transport chain with low-potential couples in the cell. The addition of auxiliary oxidants restores the optimal redox poise. This suggests a role in photosynthesis for the pathways of respiratory electron flow to nitrate, nitrous oxide, trimethylamine N-oxide/dimethylsulphoxide and oxygen.

The histochemical demonstration of fructose diphosphate aldolase activity using a semipermeable membrane technique

In this communication an enzyme histochemical multistep technique for the demonstration of class 1 fructose-1,6-diphosphate aldolase in heart and skeletal muscle sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the enzyme into the medium during incubation. In the histochemical system the enzyme cleaves the substrate D-fructose-1,6-diphosphate to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triose phosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase and the electrons are transported concomitantly via NAD+, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.

Destruction and reconstitution of the activity of hydrogenase from Chromatium vinosum

The specific activity and the EPR spectra of purified hydrogenase from Chromatium vinosum vary from preparation to preparation. It has been found that both properties are dependent on the redox state of specific redox groups, probably thiols, in the enzyme. In defect enzyme molecules the proposed thiol groups are oxidized to an -S-S- bridge. As a result, the activity of the enzyme in the assay with viologens is minimal and nickel and the iron-sulphur cluster in the oxidized enzyme are present as two non-interacting S = 1 2 systems, Ni(III) and a [3Fe-4S] 1+ cluster. In intact enzyme the -S-S- bridge does not exist. Such enzyme molecules are 15- (H 2-production reaction) or 65- (H 2-uptake reaction) times as active as defect molecules. Intact oxidized enzyme can accomodate a spin-coupled Ni(III)/[4Fe-4S] 3+ pair. It can be converted to defect molecules by partial reduction with phenazine methosulphate plus ascorbate in air at 40–50°C. The reverse transition is induced by anaerobic incubation with a disulphide-reducing agent at 50°C.

Amino acid transport by prosthecae of Asticcacaulis biprosthecum: evidence for a broad-range transport system.

Prosthecae purified from cells of Asticcaulis biprosthecum possess active transport systems that transport all 20 amino acids tested. Using ascorbate-reduced phenazine methosulphate in the presence of oxygen, all 20 amino acids are accumulated against a concentration gradient by isolated prosthecae. Results of experiments testing the inhibition of transport of one amino acid by another, and of experiments testing the exchange of exogenous amino acids with those preloaded in prosthecae, along with characteristics of mutants defective in amino acid transport, suggest the presence in prosthecae of three amino acid transport systems. One, the general or G system, transports at least 18 of the 20 amino acids tested. Another system, referred to as the proline or P system, transports seven amino acids (including proline) that are also transported by the G system. The third system transports only glutamate and aspartate, and is referred to as the acidic amino acid transport system or A system.

Electrogenic reduction of Rhodospirillum rubrum reaction centre bacteriochlorophyll P870 + by redox dyes : Indication of intraprotein electron transfer

Reduction of laser flash-oxidized bacteriochlorophyll in Rhodospirillum rubrum reaction centre proteoliposomes or in cytochrome c 2-deficient R. rubrum chromatophores by artificial redox dyes like N,N,N',N'-tetramethyl- p-phenylenediamine or phenazine methosulphate gives rise to an electrogenic phase in the millisecond range. This phase contributes as much as 20% to the total photoelectric response and is ascribed to vectorial electron transfer from the reaction centre protein surface to the protein-embedded Mg-porphyrin rings of P870 +. Rhodospirillum rubrum Membrane potential Proteoliposome Reaction center bacteriochlorophyll Intramolecular electron transfer Rapid kinetics