Assay of nicotinamide deamidase. Determination of ammonia by the indophenol reaction

The indophenol reaction for the determination of ammonia has been adopted for the assay of nicotinamide deamidase. Nicotinamide interferes with the development of the blue color characteristic of indophenol by competing with ammonia for the assay reagents. This difficulty is circumvented by varying the concentration of nitroprusside used in the determination of ammonia with the nicotinamide contents of individual samples. It is not possible to adopt a unique concentration of nitroprusside for all nicotinamide concentrations because both insufficient and excessive amounts of the reagent lead to low color yields. The method permits the determination of 0.02–1.4 μmoles ammonia and as little as 1 μg rabbit liver nicotinamide deamidase. The reduction of the scale of the procedure to one-tenth is possible and would permit a ten-fold increase in sensitivity. The inhibition of the indophenol reaction by amino acids has also been found to be reversed by increased nitroprusside levels in assay mixtures.Since a number of substances can interfere with the indophenol method, attention must be given to the control of their effects on the color reaction. Ammonia and other nitrogenous compounds may have to be removed if their concentrations in assay samples are high. The effects of low levels of such compounds ordinarily can be controlled through the appropriate use of reagent blanks, internal standards, and adjustment of the nitroprusside concentration employed in the ammonia assay mixtures.     

Carbon dioxide and the reduction of indophenol and ferricyanide by chloroplasts

Pea chloroplasts isolated in salt media show decreased rates of 2:6 dichlorophenolindophenol (DCPIP) and ferricyanide reduction when depleted of CO₂ at pH values below 7.5. The greatest effect of CO₂ was on uncoupled systems. The incorporation of 10⁻², 2 × 10⁻² and 4 × 10⁻² m sodium acetate into the reaction mixtures progressively increased the bicarbonate concentration required for half maximal rates of reduction of DCPIP. The reaction was saturated by bicarbonate concentrations of 1 to 4 × 10⁻² m. With both DCPIP and ferricyanide, the addition of bicarbonate to illuminated chloroplast systems depleted of CO₂ gave very rapid increases in the rates of reduction. Bicarbonate also stimulated oxygen uptake by the illuminated chloroplasts when added hydrogen acceptors had been reduced. There was no effect of bicarbonate on ferricyanide reduction at low light intensities, but with DCPIP reduction, the apparent magnitude of the effect was independent of light intensity. This suggests that DCPIP reacts with the chloroplast electron transport chain at a site nearer to a photochemical stage than does ferricyanide. It also suggests that CO₂ has at least 2 sites of action.     

Correction for creatine interference with the direct indophenol measurement of NH3 in steady-state nitrogenase assays.

Creatine was identified as a major source of interference with the direct phenol/hypochlorite colorimetric determination of ammonia in nitrogenase reaction mixtures. A method is described for removing other compounds which inhibit color development and for compensating for the interference produced by creatine. This method avoids time-consuming microdiffusion and also routinely makes available the efficiency of ATP hydrolysis coupled to substrate reduction (ATP/2e ratio) with N2 as a reducible substrate. Using this method we determined values for this ratio at 30 degrees C of 4.87 +/- 0.03 during the reduction of protons to H2 and 7.16 +/- 0.14 during the reduction of N2 by the vanadium-containing nitrogenase of Azotobacter chroococcum.     

Design of novel electron transfer mediators based on indophenol derivatives for lactate sensor

Novel indophenol derivatives which have limited solubility in water solution functioned as excellent electron-transfer mediators for lactate oxidase and a lactate sensor, showing good sensor performance, including a high sensitivity and good durability. These less water-soluble mediators were prepared by the O-alkylation or acylation of indophenols in order to prevent the mediator being leached from the sensor. Consequently the characteristic substituent effects of indophenol derivatives were found to be follows, (1) modification of the phenolic OH group improved sensor durability; (2) the presence of chloride groups at the 2,6-positions of the phenol ring improved the sensitivity of the sensor; (3) the introduction of substituents at the quinoide moiety led to a deterioration in durability. A mediator based lactate sensor using these derivatives was found to be nearly independent of oxygen concentration, showed a low level of interference effect, and a quite long durability.     

In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction

Two methods for measurement of urease activity are described and demonstrated on extracts from several crop plants. With an in-gel staining method based on the principle of Fishbein's noninhibitory stain for urease as little as 25 microU of jackbean urease can be detected within 2 h following electrophoresis. A comparison with published in-gel staining methods shows that the sensitivity is improved by at least two orders of magnitude. The second method allows quantification of urease activity from small amounts of plant material without the need for special laboratory equipment. It employs the detection of ammonium by the indophenol reaction. To eliminate reducing agents which are often necessary to maintain urease activity during extraction, but which interfere with ammonium detection, a simple spin-column procedure is used. The quantification of less than 5 mU/ml extract is possible.     

Recovery of loss in chlorophyll and 2,6-dichlorophenol indophenol Hill reaction of isolated chloroplasts during dark induced aging of intact maize seedlings

Loss in the content of pigments and decline in the efficiency of thylakoid membranes to reduce 2,6-dichlorophenol indophenol (DCPIP) have been investigated during dark induced senescence of attached leaves of maize seedlings. The chlorophyll degradation during senescence is differentially inhibited by indoleacetic acid (IAA), gibberellic acid (GA) and kinetin. IAA and GA behave as mild senescence inhibitors in comparison to kinetin. However, in comparison to light, kinetin is relatively less efficient in counteracting senescence. Dark-induced loss in chlorophyll content is fully recovered by light when the dark incubation period is relatively short. The pattern of light recovery of loss in photoelectron transport during dark-aging is similar to the recovery kinetics of chlorophyll. Continuous kinetin treatment of dark-incubated seedlings inhibits the chlorophyll degradation but with decreased duration of kinetin treatment, the efficiency of the hormone to inhibit chlorophyll loss is reduced. The kinetin-induced inhibition of pigment loss is small in comparison with the effect of light.     

2,6-Dichloro-phenol indophenol prevents switch-over of electrons between the cyanide-sensitive and -insensitive pathway of the mitochondrial electron transport chain in the presence of inhibitors

To evolve a simple oxygen electrode-based method to estimate alternative respiration, one needs to develop a procedure to prevent switch-over of electrons to either pathway upon inhibition by cyanide or salicylhydroxamic acid. It was hypothesized that the inclusion of appropriate electron acceptor, possessing redox potential close to one of the electron transport carriers in between ubiquinone (branch point) and cytochrome a-a3, should be able to stop switch-over of electrons to either pathway by working as an electron sink. To test the hypothesis, 2,6-dichloro-phenol indophenol (DCPIP; redox potential +0.217 V), an artificial electron acceptor having a redox potential quite similar to the site near cytochrome c1 (redox potential +0.22 V) on the cyanide-sensitive pathway, was used with isolated mitochondria and leaf discs in the absence and presence of inhibitors (potassium cyanide, antimycin A, and salicylhydroxamic acid). Polarographic data confirmed electron acceptance by DCPIP only from the inhibited (by cyanide or salicylhydroxamic acid) mitochondrial electron transport chain, hence preventing switch-over of electrons between the cyanide-sensitive and cyanide-insensitive pathway of respiration. Results with antimycin A and reduction status of DCPIP further confirmed electron acceptance by DCPIP from the mitochondrial electron transport chain. Possible implications of the results have been discussed.     

Chromosome assignments of genes in man using mouse-human somatic cell hybrids: Mitochondrial superoxide dismutase (indophenol oxidase-B,tetrameric) to chromosome 6

Summary Evidence from mouse/human somatic cell hybrids is presented for the synteny of the genes for indophenol oxidase-B (tetrameric) and cytoplasmic malic enzyme (EC 1.1.1.40). Assignment of these two genes to chromosome 6 is further confirmed. The identification of indophenol oxidase-B (tetrameric) as mitochondrial superoxide dismutase is discussed.

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Zusammenfassung Befunde an somatischen Zellhybriden Maus/Mensch werden dargestellt, die für die Syntenie der Gene für Indophenol-Oxidase-B (tetramer) sowie für cytoplasmatisches malic enzyme (EC 1.1.1.40) sprechen. Außerdem wird die Zuordnung dieser beiden Gene zu Chromosom 6 bestätigt. Die Identifizierung der Indophenol-Oxidase-B (tetramer) als mitochondriale Superoxid-Dismutase wird diskutiert.

     

Reduction of exogenous quinones and 2,6-dichlorophenol indophenol in cytochrome b-deficient yeast mitochondria: a differential effect on center i and center o of the cytochrome b-c1 complex

The reduction of duroquinone (DQ), 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), and dichlorophenol indophenol (DCIP) by succinate and NADH was investigated in yeast mitochondria which have no spectrally detectable cytochrome b. Succinate reduces DB in the cytochrome b-deficient mitochondria at rates comparable to that observed in wild-type mitochondria, suggesting that succinate:ubiquinone oxidoreductase is unaffected by the lack of cytochrome b. In the mutant mitochondria, succinate does not reduce DQ or DCIP at significant rates; however, NADH reduces both DQ and DCIP at rates similar to that of the wild-type mitochondria in a myxothiazol, but not antimycin, sensitive reaction. The Ki for myxothiazol in this reaction is close to that for electron transfer through the cytochrome b-c1 complex. In addition, myxothiazol does not inhibit NADH:ubiquinone oxidoreductase. These results confirm our previous suggestion that the cytochrome b-c1 complex is involved in electron transfer from the primary dehydrogenases to DQ and DCIP and suggest that cytochrome b is not the binding site for myxothiazol.