The Cytochemical Localization of Oxidative Enzymes : I. Diphosphopyridine Nucleotide Diaphorase and Triphosphopyridine Nucleotide Diaphorase

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 µ in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.     

The cytochemical localization of oxidative enzymes. I. Diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 micro in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.     

Modification of TPN-dependent isocitrate dehydrogenase by the 2', 3'-dialdehyde derivatives of TPNH and TPN

Catalytic reaction of the 2', 3'-dialdehyde analog of TPN (oTPN) with pig heart TPN-dependent isocitrate dehydrogenase in the presence of the substrate manganous isocitrate results in the formation of the dialdehyde derivative of TPNH (oTPNH). In the absence of the substrate, modification by oTPN leads to a progressive inactivation of the enzyme. The dependence of the pseudo-first order rate constants on the reagent concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification (kmax = 5.5 X 10(-2) min-1; K1 = 290 microM). Reaction of [14C]oTPN with the enzyme results in the incorporation of 2 mol of oTPN/mol of peptide chain. No appreciable protection against either inactivation or incorporation by the natural ligands TPN and TPNH was obtained, suggesting different modes of binding of the analog in the presence and absence of the substrate isocitrate. Enzymatically synthesized oTPNH has been isolated and demonstrated to act as an affinity label for a TPNH-binding site of isocitrate dehydrogenase. The inactivation process exhibits saturation kinetics (kmax = 2.67 X 10(-3) min-1; K1 = 33 microM). Protection against activity loss, as well as a decrease in incorporation from 2 to 1 eq of [14C]oTPNH bound/peptide chain was observed in the presence of 1 mM TPNH. From the TPNH concentration dependence of the inactivation rate by oTPNH, a dissociation constant of 3.4 microM is calculated for TPNH, indicating binding of the analog to a specific TPNH-binding site on the enzyme. Although dialdehyde derivatives are frequently assumed to form Schiff bases with proteins, the evidence presented suggests the formation of morpholino derivatives as the products of the covalent reaction of isocitrate dehydrogenase with the dialdehyde derivatives of TPN and TPNH. The new reagent, oTPNH, may serve as an affinity label for other dehydrogenases.     

Coenzyme binding by native and chemically modified pig heart triphosphopyridine nucleotide dependent isocitrate dehydrogenase.

The binding of TPNH to native and chemically modified pig heart TPN-dependent isocitrate dehydrogenase was studied by the techniques of ultrafiltration and fluorescence enhancement. A single site (per peptide chain) was found for TPNH with a dissociation constant (KD = 1.45 muM) that is quantitatively comparable to the Michaelis constant. The oxidized coenzyme, TPN+, weakens the binding of TPNH. The substrate manganous isocitrate also inhibits the binding of TPNH and, reciprocally, TPNH inhibits the binding of manganous isocitrate, suggesting that binding to the reduced coenzyme and substrate sites is mutually exclusive. Ultrafiltration experiments with carbonyl [14C]TPN+ revealed the existence of two sites with a dissociation constant (49 muM) more than ten times higher than the Michaelis constant. This observation excludes a random mechanism for isocitrate dehydrogenase or a sequential mechanism in which TPN+ binds first. Four chemically modified isocitrate dehydrogenases have been prepared: enzyme inactivated by reaction of a single methionyl residue with iodoacetate, by modification of a glutamyl residue by glycinamide (in the presence of a water soluble carbodiimide), by reaction of four cysteines successively with 5,5'-dithiobis(2-nitrobenzoic acid) and potassium cyanide, or by addition of two cysteine residues to N-ethylmaleimide. These enzymes were tested for their ability to bind TPN+, TPNH, and manganous isocitrate. In the cases of the cysteinyl and glutamyl-modified enzymes, inactivation appears to be due primarily to loss of the ability to bind the substrate manganous isocitrate. In constrast, the methionyl residue may participate in the coenzyme binding site or, more likely, may be involved in a step in catalysis subsequent to binding.     

Influence of substrates and coenzymes on the role of manganous ion in reactions catalyzed by pig heart triphosphopyridine nucleotide-dependent isocitrate dehydrogenase.

The interaction of manganous ions with pig heart triphosphopyridine nucleotide (TPN) specific isocitrate dehydrogenase has been studied by kinetic experiments and by direct ultrafiltration measurements of manganous ion binding. At low metal ion concentrations, a lag is observed in the time-dependent production of reduced triphosphopyridine nucleotide (TPNH) that can be eliminated by adding 20 muM TPNH to the initial reaction mixture. A plot of 1/upsilon vs. 1/ (Mn2+) obtained at relatively high TPNH concentrations (20 muM) is linear and yields of Km value of 2 muM for metal ion, which is comparable to the direct binding constant measured in the presence of isocitrate. A similar plot at low TPNH concentrations (2 muM) reveals a biphasic relationship: at high metal concentrations the points are collinear with those obtained at high levels of TPNH, but at low metal concentrations that line is characterized by a Km of 19 muM for Mn2+. A difference in the deuterium oxide solvent isotope effect on Vmax observed with 20 muM TPNH as compared with 2 muM TPNH suggests that at high TPNH concentrations or high manganous ion concentrations the rate-limiting step is the dehydrogenation of isocitrate, while at low manganous ion concentrations and low TPNH concentrations, the slow step is the decarboxylation of enzyme-bound oxalosuccinate. Evidence to support this hypothesis is provided by the sensitivity to isocitrate concentration of the Km for total manganese measured in the presence of 20 muM TPNH that contrasts with the relative insensitivity to isocitrate of the Km measured at 2 muM TPNH and low manganous ion concentration. Direct measurements of oxalosuccinate decarboxylation reveal that the Vmax and the Km for manganous ion are influenced by the presence of oxidized or reduced TPN with the Km being lowest (5-7 muM) in the presence of TPNH. The dependence of the Km for manganous ion on the presence of substrate, TPN, and TPNH, is responsible for the variation with conditions in the rate-determining step. The enzyme binds only 1 mol of metal ion and 1 mol of isocitrate/mol of protein under all conditions. The pH dependence of the binding of free manganous ion, free isocitrate, and manganous-isocitrate complex indicates differences in the interaction of these species with isocitrate dehydrogenase. These results can be described in terms of two functions for manganous ion in the reactions catalyzed by isocitrate dehydrogenase, each of which requires a distinct binding site for metal ion: in the dehydrogenation step, Mn2+ facilitates the binding of the substrate isocitrate, and in the decarboxylation step it may stabilize the enolate of alpha-ketoglutarate which is generated.     

Variazioni di attività della clorogenico ossidasi e di altri enzimi interessati all'ossidoriduzione del TPN durante l'attivazione di fettine di tubero di patata

Abstract

CHANGES IN THE ACTIVITY OF CHLOROGENIC ACID OXIDASE AND OTHER ENZYMES INVOLVED IN OXIDATION AND REDUCTION OF TPN IN AGEING POTATO TUBER SLICES. — The activation of respiration, and in particular of the pentose phosphate pathway, during incubation of potato tuber slices could depend on the increase of activity of oxidative enzymes mediating electron transfer from Gl. 6-P to oxygen.

The present report deals with the activity changes, in the first period of incubation, of the following enzymes: Gl. 6-P-dehydrogenase, TPNH-glutathione reductase, gluta-thione-dehydroascorbate reductase, chlorogenic acid oxidase and a TPNH diaphorase utilizing tetrazolium salts as electron acceptors.

The activity of all of these enzymes, with the exception of TPNH diaphorase, was found to bs, at all stages of incubation, in large excess respect that required to account for the estimated contribution of the pentose phosphate pathway to respiration.

Gl. 6-P dehydrogenase, glutathione reductase and chlorogenic oxidase activities markedly incresed during incubation; but their increase appeared to be clearly delayed (of some hours) respect that of oxygen uptake. This seems to indicate that the increase in activity of these anzymes is rather a consequence than a cause of the respiratory activation.

TPNH diaphorase showed a very low activity in the fresh slices, and it increased quite significantly already in the very first period (5 hours) of incubation. This behaviour suggests the possibility that this enzyme could limit TPNH oxidation, and thus the pentose phosphate pathway activity, and that its activation could be correlated with that of oxidative metabolism in the ageing slices. Further investigation of this hypothesis requires the identification of the natural electron acceptor of this enzyme.     

Fat Metabolism in Higher Plants XXXVI: Long Chain Fatty Acid Synthesis in Germinating Peas

A low lipid, high starch containing tissue, namely cotyledons of germinating pea seedlings was examined for its capacity to synthesize fatty acid. Intact tissue slices readily incorporate acetate-¹⁴C into fatty acids from C₁₆ to C₂₄. Although crude homogenates synthesize primarily 16:0 and 18:0 from malonyl CoA, subsequent fractionation into a 10,000g pellet, a 10⁵g pellet and supernatant (soluble synthetase) revealed that the 10⁵g pellet readily synthesizes C₁₆ to C₂₈ fatty acids whereas the 10,000g and the supernatant synthesize primarily C₁₆ and C₁₈. All systems require acyl carrier protein (ACP), TPNH, DPNH if malonyl CoA is the substrate and ACP, Mg²⁺, CO₂, ATP, TPNH, and DPNH if acetyl CoA is the substrate. The cotyledons of germinating pea seedlings appear to have a soluble synthetase and 10,000g particles for the synthesis of C₁₆ and C₁₈ fatty acid, and 10⁵g particles which specifically synthesize the very long chain fatty acid from malonyl CoA, presumably via malonyl ACP.     

Circular dichroic titration of dihydrofolate reductase with TPNH

Dihydrofolate reductase from Streptococcus faecium shows a marked aromatic side chain Cotton effect in the 260–310 nm region of its circular dichroic spectrum. This effect consists of three distinct ellipticity bands with maxima centered at 305 nm, 295 nm and 270 nm. Titration of the enzyme with TPNH to a 1:1 stoichiometry results in the generation of an extrinsic Cotton effect at ca. 340 nm and a decrease in the magnitude of the side chain Cotton effect. This is the first such example of a TPNH-generated extrinsic Cotton effect. The data suggest the involvement of tryptophyl residues in coenzyme binding.     

Antimycin inhibition as a probe of mitochondrial function in isolated rat hepatocytes Effects of chronic ethanol consumption

Previous studies have established that hepatic mitochondria and submitochondrial particles from rats, fed ethanol chronically, display diminished respiratory activities and alterations in the contents of specific electron transfer chain components. The latter include a decrease of about 50% in cytochrome b content. Titrations of respiratory activity in submitochondrial particles with antimycin, a stoichiometric inhibitor of electron flow through the cytochrome b-c₁ region of the respiratory chain, indicated a comparable decrease (35%) in the amount of antimycin required to elicit maximal inhibition (‘titer’) after chronic ethanol treatment. Measurements of antimycin binding to submitochondrial particles by fluorescence quenching demonstrated a similar diminution in the number of tight binding sites per mg protein. By contrast, hepatocytes isolated from control and ethanol-fed rats exhibited nearly identical rates of oxygen utilization under a variety of conditions. However, antimycin titrations of respiratory activity in isolated hepatocytes revealed a 60% decrease in the antimycin titer, but no change in the maximal extent of inhibition after chronic ethanol treatment. Direct measurements of cytochrome b which could be reduced in the presence of antimycin in hepatocytes confirmed a comparable decrease (42%) after chronic ethanol treatment. The results demonstrate that molecular alterations in the cytochrome b region of the respiratory chain caused by ethanol feeding are present in intact liver cells, but suggest that substrate accessibility, rather than the respiratory chain, limits the rate of oxygen utilization in isolated hepatocytes. The data also suggest that mitochondria account for at least 80% of total oxygen utilization by liver cells from both control and ethanol-fed rats.     

Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus: Cofactor Requirements

The rates of oxidation of ent-kaur-16-ene to ent-kaur-16-en-19-ol, ent-kaur-16-en-19-al, ent-kaur-16-en-19-oic acid, and ent-kaur-16-en-7alpha-ol-19-oic acid are maximal in microsomes prepared from the endosperm of immature Marah macrocarpus seeds in which the cotyledons are approximately one-half the overall length of the seed. The supernatant fraction remaining from the preparation of the microsomes contains factors which stimulate the rates of oxidation catalyzed by the microsomes. Added TPNH is more effective than added DPNH in meeting the requirement for reduced pyridine nucleotide. A mixture of DPNH, ATP, and TPN(+) is much more effective than DPNH alone. Experiments with 2,4-dinitrophenol as a selective inhibitor indicate that the ATP-stimulated synthesis of TPNH which occurs in these microsomes in the presence of this mixture of coenzymes provide TPNH for use in the mixed function oxidations. Relatively low concentrations of DPNH and TPNH together are much more effective than either alone at equivalent concentration. This is consistent with the involvement of two pathways of electron transfer associated with the mixed function oxidations, one of which preferentially utilizes TPNH and the other favoring DPNH. FAD added to microsomes at an optimal concentration of about 10 mum in the presence of TPNH stimulates the rate of the oxidations; higher concentrations are inhibitory. FMN by itself does not produce this stimulation. However, FMN and FAD added together at low concentrations (0.5 mum each) have approximately the same effectiveness as FAD alone at 10 mum. This suggests a role for both flavin nucleotides in the normal electron transfer pathways associated with these oxidations. Some of the stimulatory properties of the supernatant fraction may be accounted for by its content of reduced pyridine nucleotides, FAD, and FMN; the concentrations of FAD and FMN were determined to be 1.1 mum and 0.4 mum, respectively. However, the effects of the supernatant fraction are not completely explained by its content of these coenzymes since other experiments indicate the presence of a heat-labile, nondialyzable stimulatory factor(s) in the supernatant fraction in addition to heat-stable, dialyzable fractors.     

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.     

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.     

The interaction of rubroskyrin, a modified bis-anthraquinone pigment fromPenicillium islandicum Sopp, with respiratory chain of liver mitochondria

Summary  Rubroskyrin, a modified bisanthraquinone pigment from an yellow rice moldPenicillium islandicum Sopp, was examined for its redox-interaction with the mitochondrial respiratory chain by using rat liver submitochondrial particles (SMP) and was compared with luteoskyrin and rugulosin. Rubroskyrin showed a redox interaction with the NAD-linked respiratory chain of SMP, promoting NADH oxidase in the presence of rotenone, a specific inhibitor to coupling site I of the respiratory chain. Rubroskyrin-mediated NADH oxidase was not inhibited by antimycin A and cyanide, inhibitors to coupling sites II and III, respectively, indicating a generation of an electron transport shunt from a rotenone-insensitive site of NADH dehydrogenase (complex I) to dissolved oxygen. An electrontransport shunt to cytochromec oxidase from complex I was also observed in the experiment with cytochromec and antimycin A. Rubroskyrin did not interact with succinate-linked respiratory chain. Such enzymatic redox response which generates electron transport shunt was not detected for luteoskyrin and rugulosin in the present study.     

Microsomal electron transport reactions. II. The use of reduced triphosphopyridine nucleotide and-or reduced diphosphopyridine nucleotide for the oxidative N-demethylation of aminopyrine and other drug substrates

The ratio of formaldehyde formed to TPNH oxidized during aminopyrine oxidative demethylation as catalyzed by rabbit liver microsomes was found to be about 0.5. This is less than the expected 1:1 ratio for a mixed function oxidase reaction and may reflect the oxidation of TPNH by other reactions. Similar results were obtained when measuring the oxidative demethylation of codeine and ethylmorphine. In all cases the addition of DPNH significantly increased the yield of formaldehyde formed in the presence of TPNH. The stimulatory effect of DPNH was a linear function of the DPNH concentration added until the initial concentrations of DPNH and TPNH were equal. Increasing the DPNH concentration above a DPNH:TPNH ratio of 1:1 had no further effect upon the final concentration of formaldehyde formed. This observation, as well as the inhibition of DPNH-supported aminopyrine metabolism by TPN⁺, argue against the role of a transhydrogenase mechanism for the DPNH effect. The rate of DPNH oxidation catalyzed by liver microsome was also observed to increase markedly in the presence of TPNH.     

A new approach to understanding kinetic cooperativity when the hill coefficients are less than 2

Dihydrofolate reductase from chicken liver has a single sulfhydryl group which reacts stoichiometrically and specifically with a wide variety of organic mercury compounds to yield an enzyme derivative which exhibits up to 10-fold the activity of the unmodified form when measured at pH 6.5, the optimum for the modified enzyme. The sulfhydryl group is apparently not at the active site since a 25-fold excess of either major cosubstrate, dihydrofolate or TPNH, affects neither the rate nor extent of the modification reaction. The reaction is essentially instantaneous and yields an enzyme with altered kinetic properties for all the substrate pairs examined (TPNH/dihydrofolate, TPNH/ folate, and DPNH/dihydrofolate) when tested near their pH optima. V values increased 3- to 10-fold when TPNH was cofactor; K_m values increased 10- to 15-fold for the TPNH/dihydrofolate pair. The mercurial-activated enzyme, unlike the native form, exhibits a markedly increased sensitivity to heat, proteolysis, and the ionic environment, losing approximately 50% of its activity under conditions where there is no loss of activity in the native form. However, substrates can afford protection, the order of effectiveness being identical with the relative affinities of the substrates for the native enzyme (Subramanian, S., and Kaufman, B. T. (1978) Proc. Nat. Acad. Sci. USA75, 3201). Thus, dihydrofolate, with the largest binding constant is the most efficient, protecting completely against trypsin digestion when present at a 1:1 ratio with enzyme. Heating the mercury enzyme in the absence of substrates gives rise to a stable but altered conformation characterized by a time course which shows marked hysteresis. The striking similarity of the properties of the mercurial-activated dihydrofolate reductase to the reductase activated by 4 m urea, a reagent known to affect the tertiary structure of proteins, suggests that covalent binding of organic mercurials to the sulfhydryl group results in a similar conformational change characterized by a marked facilitation of the dihydrofolate reductase reaction.     

A kinetic assay of TPNH-dependent microsomal lipid peroxidation by changes in difference spectra

In the presence of TPNH, O₂ and ADP-Fe⁺³ rat liver microsomes yield difference spectral changes at 237 nm and 267–270 nm that correlate with the kinetics of lipid peroxidation as measured by the rate of malonaldehyde formation and O₂ and TPNH consumption. Mn⁺² EDTA, aniline, and reduced glutathione were inhibitory. It is suggested that the difference spectral changes at 237 nm and 267–270 nm are essentially due to conjugated diene and malonaldehyde formation, respectively.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

NADH- and NADPH-dependent lipid peroxidation in bovine heart submitochondrial particles. Dependence on the rate of electron flow in the respiratory chain and an antioxidant role of ubiquinol.

Malondialdehyde formations by bovine heart submitochondrial particles supported by NADH or NADPH in the presence of ADP and FeCl3 was studied. The NADH-dependent reaction was maximal at very low rate of electron input from NADH to the respiratory chain and it decreased when the rate became high. The reaction was stimulated by rotenone and inhibited by antimycin A when the input was fast, whereas it was not affected by the inhibitors when the input was slow. The input rate of the electrons from NADPH was also so low that the reaction supported by NADPH was not affected by the inhibitors. Most of the endogenous ubiquinone in the particles treated with antimycin A was reduced by NADH even in the presence of ADP-Fe3+ chelate, but uniquinone was not reduced by NADPH when ADP-Fe3+ was present. Succinate strongly inhibited both NADH- and NADPH-dependent lipid peroxidation. The inhibition was abolished when uniquinone was removed from the particles, and it appeared again when uniquinone was reincorporated into the particles. Reduced uniquinone-2 also inhibited the peroxidation, but duroquinol, which reduces cytochrome b without reducing endogenous uniquinone, did not. Thus the malondialdehyde formation appeared to be inversely related to the extent of the reduction of endogenous uniquinone. These observations suggest that both NADH- and NADPH-dependent liquid-peroxidation reactions are closely related to the respiratory chain and that the peroxidation is controlled by the concentration of reduced ubiquinone.     

Electron transport particles from beef-heart mitochondria as a test system for inhibitors of the respiratory chain]

Respiratory inhibitors exert a potentially biocidic action on all aerobic organisms. Therefore the systematic search for them and the elucidation of their mechanism of action is of great importance. The non-phosphorylating electron transfer particles (ETP) according to Crane et al. are a suitable test object for this aim. A standardized preparation of ETP from beef heart is described and characterized enzymatically. The ETP show a variety of advantages in comparison with other systems; their field of application is broad. Owing to the absence of bypasses of the respiration, particles from animals are more suitable than those from plants for the search for fungicides and herbicides which inhibit the respiratory chain. With relatively little expense, the two standard assay systems proposed (NADH oxidase and succinate-cytochrome c oxidoreductase) pick up with certainty all inhibitors of the main pathway of the respiratory chain; they are useful for screening studies. The system proposed is also suitable for the analysis of the exact site and the mechanism of action of respiratory inhibitors. Furthermore, ETP can be used to obtain parameters of the hydrophobicity of drugs as well as for toxicological studies.     

Physiology and metabolism of pathogenic Neisseria: partial characterization of the respiratory chain of Neisseria gonorrhoeae.

The cell membrane-associated respiratory electron transport chain of Neisseria gonorrhoeae was examined using electron paramagnetic spectroscopy (EPR) at liquid helium temperatures and optical spectroscopy at liquid nitrogen and room temperatures. EPR spectra of dithionite-reduced particles indicated the presence of centers N-1 and N-3 in the site I region of the respiratory chain, whereas reduction with succinate revealed the existence of center S-1 from the succinate cytochrome c reductase segment. Free radical(s) resembling that due to falvin semiquinone were observed with both reductants. Low temperature (77 K) optical difference spectra indicated the presence of cytochromes with alpha band maxima at 549, 557, and 562. Bands at 567, 535, and 417 nm, characteristic of the CO compound of cytochrome o, were also identified. Cytochromes a1 and a3 were not detected; however, a broad but weak absorbance with an alpha band maximun at 600 nm and a Soret shoulder at 440 nm was observed. Hence the respiratory chain of N. gonorrhoeae appears to contain several nonheme iron centers, cytochrome c, two b cytochromes, with cytochrome o which probably serves as the terminal oxidase.