Use of tetrazolium salts for electron transport studies in meningopneumonitis. II. Reduced nicotinamide adenine dinucleotide phosphate system

Allen, Emma G. (Downstate Medical School, State University of New York, Brooklyn). Use of tetrazolium salts for electron transport studies in meningopneumonitis. II. Reduced nicotinamide adenine dinucleotide phosphate system. J. Bacteriol. 92:1041-1046. 1966.-The conditions of electron transfer from nicotinamide adenine dinucleotide phosphate in meningopneumonitis (MP) are described and compared with electron transfer from nicotinamide adenine dinucleotide by this organism. The observations suggest that, in either system, there may be more than one pathway of electron flow, and that these pathways differ from those in normal membrane particulates. It was also found that after trypsin treatment, particulates from pools of normal allantoic fluids and membranes retain the normal characteristics, whereas those from pools of MP-infected fluids and membranes assume the characteristics of MP particles from fluids.     

Nicotinamide adenine dinucleotide -- independent formate dehydrogenase in Mycobacterium phlei.

Formate dehydrogenase activity (EC 1.2.1.2) has been demonstrated in cell-free preparations of Mycobacterium phlei by following the reduction of 2,6 dichlorophenolindophenol. thiazolyl blue tetrazolium, or equine cytochrome c. The reduction of equine cytochrome c was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. Neither nicotinamide adenine dinucleotide nor nicotinamide adenine dinucleotide phosphate were reduced by this formate dehydrogenase. The enzyme was constitutive and associated with the particular fraction. The greatest level of activity was observed at pH 9.0, with 8 mM formate, and with extracts of cells taken from the log phase of growth. Formaldehyde, hypophosphite, nitrate, and bicarbonate all inhibited the oxidation of formate.     

Effects of selected inhibitors on electron transport in Neisseria gonorrhoeae.

The electron transport system of Neisseria gonorrhoeae was partially characterized by using spectrophotometric, spectroscopic, and oxygen consumption measurements. The effects of selected electron transport inhibitors (amytal, rotenone, 2-heptyl-4-hydroxyquinoline, antimycin A1, and potassium cyanide [KCN]) on electron transfer in whole-cell and sonically treated whole-cell preparations of N. gonorrhoeae were examined. The oxidation of reduced nicotinamide adenine dinucleotide, measured as a decrease in absorbance at 340 nm, was inhibited by each of the compounds tested. Oxygen consumption stimulated by reduced nicotinamide adenine dinucleotide was also inhibited, whereas oxygen uptake stimulated by succinate and malate was inhibited by KCN alone, suggesting the presence of a KCN-sensitive terminal oxidase. Room temperature optical difference spectra indicate an operational electron bypass around the amytal-rotenone-binding site. Difference spectra in the presence of 2-heptyl-4-hydroxyquinoline suggest a possible site of interaction of this compound at the substrate side of cytochrome b. Reduced-minus-oxidized spectra of ascorbate-tetramethyl-p-phenylenediamine suggest the participation of b-, a-, and d-type cytochromes in terminal oxidase activity. Hence, N. gonorrhoeae appears to have an electron transport chain containing cytochrome c, two b-type cytochromes (one of which has an oxidase function), and possibly a- and d-type cytochromes. An abbreviated chain exists through which succinate and malate can be oxidized directly by a KCN-sensitive component.     

Preparation and some properties of submitochondrial particles from tightly coupled mung bean mitochondria

Osmotic shock was found to be better than freezing and thawing, a French press, or sonic oscillation for the preparation of submitochondrial particles from mung bean (Phaseolus aureus) hypocotyl mitochondria. Particles prepared by osmotic shock rapidly oxidize reduced nicotinamide adenine dinucleotide and succinate, but they oxidize malate slowly. NADH oxidation was slightly stimulated by cytochrome c, ATP, and ADP; succinate oxidation was markedly increased by ATP, slightly by ADP and cytochrome c; and malate oxidation required the addition of NAD⁺ NADH oxidation is inhibited weakly by amytal, completely by antimycin A and KCN, but not by rotenone. Chlorsuccinate, malonate, antimycin A, and KCN inhibit succinate oxidation. The action of antimycin A and KCN is incomplete, while chlorsuccinate and malonate were competitive inhibitors. Antimycin A combined stoichiometrically with particle protein in the ratio of 0.23 millimicromole per milligram of protein.     

α-α′-Dipyridyl or Ortho-Phenanthroline Stimulation of the Soluble Reduced Nicotinamide Adenine Dinucleotide Oxidase from Bacillus subtilis Spores and Dipicolinic Acid Inhibition of the Stimulated Enzyme

Soluble reduced nicotinamide adenine dinucleotide oxidase activity in extracts of Bacillus subtilis spores was stimulated by the addition of not only flavine mononucleotide (FMN) or flavine adenine dinucleotide (FAD) but also alpha-alpha'-dipyridyl or o-phenanthroline. These chelating agents showed stronger effect on the enzyme from spores than on that from vegetative cells. Activity stimulated by alpha-alpha'-dipyridyl or o-phenanthroline was inhibited by atabrine or dipicolinic acid, whereas FMN or FAD stimulation was inhibited only by atabrine.     

Adenosine 5''-monophosphate-stimulated cyanide-insensitive respiration in mitochondria of Moniliella tomentosa.

Mitochondria of the yeastlike fungus Moniliella tomentosa oxidize reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate, succinate, isocitrate, and lactate. These oxidations are completely inhibited by cyanide or antimycin A in mitochondria isolated from cells grown in the standard medium. On the other hand, the oxidation of all substrates, except lactate, is almost completely insensitive to cyanide or antimycin A in mitochondria from cells grown in the presence of ethidium bromide. In this instance, the oxidation is mainly mediated by an alternate oxidase which can be blocked by salicyl hydroxamic acid. The alternate oxidase can be specifically stimulated by adenosine 5'-monophosphate and this provides a new method for the characterization of the alternate oxidase in mitochondria of M. tomentosa.     

The effect of calcium and inhibitors on corn mitochondrial respiration

The effects of KCN, antimycin A, malonate, rotenone, and amytal on the oxidation of malate, succinate, and extramitochondrial reduced nicotinamide adenosine dinucleotide (NADH) by corn mitochondria were studied. Potassium cyanide and antimycin A inhibited the oxidation of all three substrates. Rotenone and amytal inhibited only the oxidation of malate, and malonate inhibited only the oxidation of succinate. Rotenone, amytal, and malonate did not inhibit the oxidation of extramitochondrial NADH. The calcium stimulation of the oxidation of extramitochondrial NADH was prevented by KCN and antimycin A but not by amytal, rotenone, or malonate. It is suggested that corn mitochondria possess a flavoprotein specific for extramitochondrial NADH and that this flavoprotein is sensitive to divalent cations.     

The ultrastructural cytochemistry of lactic dehydrogenase, succinic dehydrogenase, dihydro-nicotinamide adenine dinucleotide diaphorase and cytochrome oxidase activities in hair cell mitochondria of the guinea pig cochlea.

The use of cinnamyl nitroblue tetrazolium chloride (DS-NBT) in dehydrogenase experiments (lactic dehydrogenase, succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) in cytochrome oxidase experiments indicated that mitochondrial oxidoreduction reactions from nicotinamide adenine dinucleotide to cytochrome oxidase are located on the inner mitochondrial membrane in the outer compartment and the intracristate spaces. These reactions behave according to the chemiosmotic hypothesis. The cochlear hair cell mitochondria are cytochemically indistinguishable from free liver mitochondria. The heterogeneous mitochondrial staining pattern is related to the osmolarity of the incubation media, solubility of the enzymes and pH of the medium, but not to the fixation method.     

Reduction of iron and synthesis of protoheme by Spirillum itersonii and other organisms.

Membranes from Spirillum itersonii reduce ferric iron to ferrous iron with reduced nicotinamide adenine dinucleotide or succinate as a source of reductant. Iron reduction was measured spectrophotometrically at 562 nm using ferrozine, which chelates ferrous iron specifically. Reduced nicotinamide adenine dinucleotide or succinate was also effective as a source of iron. The effects of respiratory inhibitors suggested that reduction of iron occurs at one or more sites on the respiratory chain before cytochrome c. Reduction of iron and synthesis of protoheme with the physiological reductants were also observed with crude extracts of other bacteria, including Rhodopseudomonas spheroides, Rhodopseudomonas capsulata, Paracoccus denitrificans, and Escherichia coli. The effect of oxygen upon reduction of iron and formation of protoheme was examined with membranes from S. itersonii, using succinate as a source of reductant. Both systems were inhibited by oxygen, but this effect was completely reversed by addition of antimycin A. We conclude that reduced components of the respiratory chain serve as reductants for ferric iron, but with oxygen present they are oxidized preferentially by the successive members of the chain. This could be a mechanism for regulating synthesis of heme and cytochrome by oxygen.     

Mechanism of Action of the Antifungal Antibiotic Pyrrolnitrin

Pyrrolnitrin at 10 mug/ml inhibited the growth of Saccharomyces cerevisiae, Penicillium atrovenetum, and P. oxalicum. The primary site of action of pyrrolnitrin on S. cerevisiae was the terminal electron transport system between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. At growth inhibitory concentrations, pyrrolnitrin inhibited endogenous and exogenous respiration immediately after its addition to the system. In mitochondrial preparations, the antibiotic inhibited succinate oxidase, NADH oxidase, succinate-cytochrome c reductase, NADH-cytochrome c reductase, and succinate-coenzyme Q(6) reductase. In addition, pyrrolnitrin inhibited the antimycin-insensitive reduction of dichlorophenolindophenol and of the tetrazolium dye 2,2'-di-p-nitrophenyl-(3,3'-dimethoxy-4,4'-bi-phenylene)5,5'-diphenylditetrazolium. The reduction of another tetrazolium dye, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride, that was antimycin-sensitive, was also inhibited by pyrrolnitrin. The antibiotic had no effect on the activity of cytochrome oxidase, and it did not appear to bind with flavine adenine dinucleotide, the coenzyme of succinic dehydrogenase. In whole cells of S. cerevisiae, pyrrolnitrin inhibited the incorporation of (14)C-glucose into nucleic acids and proteins. It also inhibited the incorporation of (14)C-uracil, (3)H-thymidine, and (14)C-amino acids into ribonucleic acid, deoxyribonucleic acid, and protein, respectively. The in vitro protein synthesis in Rhizoctonia solani and Escherichia coli was not affected by pyrrolnitrin. Pyrrolnitrin also inhibited the uptake of radioactive tracers, but there was no general damage to the cell membranes that would result in an increased leakage of cell metabolites. Apparently, pyrrolnitrin inhibits fungal growth by inhibiting the respiratory electron transport system.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

Flavensomycin, an inhibitor of enzyme reactions involving hydrogen transfer

The antifungal antibiotic flavensomycin inhibited the oxidation of amino acids and of glucose by Penicillium oxalicum. The compound inhibited l-amino acid oxidase (EC 1.4.3.2) activity for l-leucine and l-phenylalanine, and also d-amino acid oxidase (EC 1.4.3.3) in the oxidation for dl-alanine. The addition of flavin adenine dinucleotide, which is a cofactor for this enzyme, antagonized the action of the antibiotic. Glucose oxidase (EC 1.1.3.4) was also inhibited. The antibiotic inhibited the reduced nicotinamide adenine dinucleotide (NADH(2)) cytochrome c reductase (EC 1.6.2.1) as well as the much slower nonenzymatic reduction of this cytochrome by the nucleotide. Reduced cytochrome c was also oxidized nonenzymatically by flavensomycin. The antibiotic completely inhibited the action of rabbit muscle lactic dehydrogenase (EC 1.1.1.27) in promoting the reduction of pyruvate by NADH(2) but only slightly affected the reverse reaction. Alcohol dehydrogenase (EC 1.1.1.1) was also similarly inhibited. Flavensomycin prevented the reduction of nicotinamide adenine dinucleotide phosphate by isocitrate in the presence of isocitrate dehydrogenase (EC 1.1.1.42). The hexokinase (EC 2.7.1.1)-catalyzed phosphorylation of glucose, in which the adenosine triphosphate acts as a phosphate donor, was only slightly affected. Flavensomycin also inhibited the action of yeast lactate dehydrogenase (EC 1.1.2.3) on the reduction of cytochrome c. High concentrations of cytochrome c were antagonistic to this reaction. The results point to an interference with enzymatically controlled hydrogen or electron transfer as the mechanism of the antifungal activity of flavensomycin.     

Thiosulfate- and Sulfide-Dependent Pyridine Nucleotide Reduction and Gluconeogenesis in Intact Thiobacillus neapolitanus

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.     

Respiratory Chain of a Pathogenic Fungus, Microsporum gypseum: Effect of the Antifungal Agent Pyrrolnitrin

Pyrrolnitrin has been reported to inhibit Bacillus megaterium primarily by forming complexes with phospholipids and to block electron transfer of Saccharomyces cerevisiae between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. We found that pyrrolnitrin inhibited respiration of conidia of Microsporum gypseum. In mitochondrial preparations, pyrrolnitrin strongly inhibited respiration and the rotenone-sensitive NADH-cytochrome c reductase. The rotenone-insensitive NADH-cytochrome c reductase, the succinate-cytochrome c reductase, and the reduction of dichlorophenolindophenol by either NADH or succinate were inhibited to a lesser extent. However, the activity of cytochrome oxidase was not affected by pyrrolnitrin. The extent of reduction of flavoproteins by NADH and succinate, measured at 465 - 510 nm, was unaltered; however, the reduction of cytochrome b, measured at 560 - 575 nm, was partially inhibited by pyrrolnitrin. The level of totally reduced cytochrome b was restored with antimycin A. We, therefore, concluded that the primary site of action of this antifungal antibiotic is to block electron transfer between the flavoprotein of the NADH-dehydrogenase and cytochrome b segment of the respiratory chain of M. gypseum.     

A model for NADH-tetrazolium reductase

Summary In the presence of light, reduced nicotinamide adenine dinucleotide (NADH) and riboflavin formed a complex which was able to reduce certain tetrazolium salts. Neither NADH (10^–3 M) nor riboflavin (10^–4 M) alone was able to induce tetrazolium reduction in the presence of oxygen, but in a nitrogen atmosphere photoreduction of riboflavin induced reduction of tetrazolium salts. Only electrophilic nitro and thiazolyl substituted tetrazolium salts with more positive redox potentials were reduced by the NADH-riboflavin complex, and only monoformazans were produced from the ditetrazolium salts. The reduction kinetics of these tetrazolium salts are given, and the spectral area capable for induction of electron transfer in the NADH-riboflavin complex is screened. It is concluded that the electron transfer in flavin nucleotide dependent dehydrogenase systems will probably proceed without direct interference with the apoenzyme. This may have practical implications for the histochemistry of tetrazolium reductases especially as regards fixation. The catalytic action of light on tetrazolium reduction should also be taken into consideration when tetrazolium salts are used as electron acceptors in a histochemical reaction.     

Redox involvement in acid secretion in the amphibian gastric mucosa

Summary Gastric fundic metabolism was studied by spectroscopic observation in frog mucosa during transitions of secretory status in vitro and by direct measurement of pyridine nucleotides and associated metabolites in biopsies of dog fundic mucosa also during secretory oxidation of the redox components from flavin adenine dinucleotide (FAD) to cytochromea ₃. Addition of histamine resulted in reduction of these components with onset of secretion by about 50%. In contrast, the effect of apparently, burimamide and subsequently histamine on the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced (NAD⁺/NADH) was relatively slight. Further, the presence of burimamide substantially reduces the effect of amytal on the pyridine nucleotide spectrum and abolishes the effect of amytal on FAD and the cytochromes. Measurements of lactate, pyruvate, -ketoglutarate, NH₃ and glutamate in the dog showed that whereas the calculated NAD⁺/NADH ratio in the cytoplasm declined with onset of secretion, the calculated mitochondrial ratio rose. No change was noted in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate, reduced (NADP⁺/NADPH) ratio. It is concluded that (1) H₂ antagonists act by blocking substrate flow into the mitochondrial respiratory chain, (2) conversely, histamine stimulation acts at the level of substrate mobilization, and (3) there may be a cross-over in the mitochondrial chain between NAD⁺ and FAD.     

Respiratory Chain of Antimycin A-producing Streptomyces antibioticus

An active respiratory chain system was demonstrated in sonically treated mycelium of Streptomyces antibioticus, the producer of antimycin A. The respiratory electron transfer from substrate to oxygen proceeded successively through flavoprotein(s), b-, c-, and a-type cytochromes, and terminated with the cyanide-sensitive cytochrome oxidase. The cytochrome composition of the culture was not affected by the age of the mycelium, the intensity of antimycin A production, or differences in the media. Slater factor, coenzyme Q, and vitamin K were not interposed as hydrogen carriers in the respiratory chain between flavoproteins and cytochromes. The oxidation of reduced nicotinamide adenine dinucleotide and succinate was unaffected by antimycin A. Evidence is presented in support of the absence of the antimycin A-sensitive site from the electron transport system of S. antibioticus.     

Respiratory System of Rhodotorula glutinis I. Inhibitor Tolerance and Cytochrome Components

Oxygen uptake by the carotenoid-containing yeast, Rhodotorula glutinis was not affected by concentrations of cyanide and antimycin A which completely inhibit the respiration of Saccharomyces cerevisiae. The tolerance of R. glutinis to these inhibitors was somewhat dependent on the age of the cultures. Reduced minus aerated difference spectra of cells revealed spectral changes presumably due to cytochromes and carotenoids. The kinetics of these spectral changes induced by oxygen were followed. Carotenoid deficient cells were prepared by growth in the presence of diphenylamine. Difference spectra of these cells revealed the presence of flavoprotein, and a, b, and c type cytochromes. Growth of R. glutinis was completely inhibited by concentrations of cyanide which did not affect respiration. Oxidation of reduced nicotinamide adenine dinucleotide by sub-cellular fractions was sensitive to cyanide and antimycin A. Although respiration of intact cells is tolerant to these inhibitors, studies with cell-free extracts suggest the presence of a cyanide and antimycin A-sensitive, cytochrome-linked, respiratory chain.     

Oxidation of ethane by an Acremonium species.

Ethane oxidation was studied in ethane-grown resting cells (mycelia) of an Acremonium sp. and in cell-free preparations of such mycelia. From resting cell experiments evidence was found for a pathway of ethane oxidation via ethanol, acetaldehyde, and acetic acid. In vitro studies indicated that ethane-oxidizing activity in such mycelia occurred predominantly in the microsomal fraction of crude homogenates. Microsomal preparations were inactive in the absence of added coenzyme. Marked stimulation of activity was obtained in such preparations with reduced nicotinamide adenine dinucleotide phosphate and to a much lesser degree with nicotinamide adenine dinucleotide phosphate. Ethane oxidation was inhibited by sodium azide and carbon monoxide.     

Oxidation of ethane by an Acremonium species.

Ethane oxidation was studied in ethane-grown resting cells (mycelia) of an Acremonium sp. and in cell-free preparations of such mycelia. From resting cell experiments evidence was found for a pathway of ethane oxidation via ethanol, acetaldehyde, and acetic acid. In vitro studies indicated that ethane-oxidizing activity in such mycelia occurred predominantly in the microsomal fraction of crude homogenates. Microsomal preparations were inactive in the absence of added coenzyme. Marked stimulation of activity was obtained in such preparations with reduced nicotinamide adenine dinucleotide phosphate and to a much lesser degree with nicotinamide adenine dinucleotide phosphate. Ethane oxidation was inhibited by sodium azide and carbon monoxide.