Inhibition of Peroxidase-Catalyzed Oxidation of Chromogenic Substrates by Propyl Gallate and Its Polydisulfide

Peroxidase-catalyzed oxidation of 2,2′-azino-di-(3-ethyl-2,3-dihydrobenzthiazoline-6-sulfonate) (ABTS) was competitively inhibited by propyl gallate (PG) and its polydisulfide (PGPDS) at 20° C in 0.015 M phosphate-citrate buffer (pH 6.0). Under these conditions, the values of the inhibition constant (K_i) were equal to 62 and 5.6 μM, respectively, for PG and PGPDS. The stoichiometric inhibition factor (f; the number of radicals extinguished per molecule of an inhibitor) equaled 2.0 and 14.7, respectively, for PG and PGPDS. Peroxidase-catalyzed oxidation of o-phenylenediamine was barely affected by PG or PGPDS. PGPDS may be used as a stop-reagent of peroxidase-catalyzed ABTS oxidation, whereas PG may serve as a calibrating inhibitor in test systems for measurement of total antioxidant activity (in human biological fluids, natural preparations, juices, wines, and other objects).__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 376–382.Original Russian Text Copyright © 2005 by Naumchik, Karasyova, Metelitza.     

Effects of KCN and Salicylhydroxamic Acid on the Root Respiration of Pea Seedlings

Polarography, using cylindrical platinum electrodes, proved suitable for measuring changes in the internal apical O₂ concentration of the primary root of pea (Pisum sativum L. cv Meteor) effected by KCN and/or salicylhydroxamic acid (SHAM) in the bathing medium. An electrical rootaeration analog was used to help evaluate some of the results. Concentrations of KCN ≤0.05 millimolar had no significant effect. In response to 0.1 millimolar KCN, the O₂ concentration rose substantially for approximately 2 hours, then declined, and after 10 hours had frequently fallen below the pretreatment level. Such changes suggest an initial inhibition of cytochrome oxidase-mediated O₂ uptake followed by an induction of the alternative, cyanide-resistant respiratory pathway. These treatments proved nonlethal. Changes in O₂ concentration similar to those described for 0.1 millimolar KCN were observed in response to 1 and 10 millimolar KCN but these treatments were lethal and the root apex became soft and often appeared flooded. Roots survived and showed no significant responses when treated with SHAM at concentrations ≤5 millimolar. However, when the alternative pathway had been (apparently) induced by 0.1 millimolar KCN, the addition of 5 millimolar SHAM to the bathing medium caused a substantial and persistent rise in the root apical O₂ concentration, suggesting that this (nonlethal) concentration of SHAM could indeed inhibit O₂ uptake via the cyanide-resistant pathway.

It is concluded that while O₂ uptake normally occurs by the cytochrome pathway in the primary pea root, the alternative, cyanide-resistant pathway can be induced by 0.1 millimolar KCN.

     

Effects of KCN and Salicylhydroxamic Acid on Respiration of Soybean Leaves at Different Ages

Measurements of respiration were made on leaf discs from glasshouse-grown soybean (Glycine max [L.] Merr. cv `Corsoy') plants in the presence and absence of cyanide (KCN) and salicylhydroxamic acid (SHAM). O₂ uptake by mature leaves measured at 25°C was stimulated by 1 millimolar KCN (63%) and also by 5 millimolar azide (79%). SHAM, an inhibitor of the alternative oxidase and a selection of other enzymes, also stimulated O₂ uptake by itself at concentration of 10 millimolar. However, in combination, KCN and SHAM were inhibitory. The rate of O₂ uptake declined consistently with leaf age. The stimulation of O₂ uptake by KCN and by SHAM occurred only after a certain stage of leaf development had been reached and was more pronounced in fully expanded leaves. In young leaves, O₂ uptake was inhibited by both KCN and SHAM individually. The uncoupler, p-trifluoromethoxy carbonylcyanide phenylhydrazone, stimulated leaf respiration at all ages studied, the stimulation being more pronounced in fully expanded leaves. The uncoupled rate was inhibited by KCN and SHAM individually. The capacity of the cytochrome path declined with leaf age, paralleling the decline in total respiration. However, the capacity of the alternative path peaked at about full leaf expansion, exceeding the cytochrome capacity and remaining relatively constant. These results are consistent with the presence in soybean leaves of an alternative path capacity that seems to increase with age, and they suggest that the stimulation of O₂ uptake by KCN and NaN₃ in mature leaves was mainly by the SHAM-sensitive alternative path. The stimulation of O₂ uptake by SHAM was not expected, and the reason for it is not clear.     

Inhibition by Salicylhydroxamic Acid, BW755C, Eicosatetraynoic Acid, and Disulfiram of Hypersensitive Resistance Elicited by Arachidonic Acid or Poly-l-Lysine in Potato Tuber

The hypothesis that arachidonic acid (AA) induction of sesquiterpene accumulation and browning in potato (Solanum tuberosum) is mediated by a lipoxygenase metabolite of AA was tested using lipoxygenase inhibitors. Salicylhydroxamic acid (SHAM) and 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C) delayed the response to AA. Inhibition by eicosatetraynoic acid (ETYA) was more persistent. These results are consistent with previous reports that SHAM and BW755C are reversible inhibitors of lipoxygenase and easily oxidized by potato while ETYA acts as an irreversible inhibitor. Disulfiram (tetraethylthiuram disulfide) also inhibited AA elicitor activity. SHAM was most effective if applied at the time of AA treatment, having no effect if applied 6 hours afterward. SHAM was effective in the presence of MES or MOPS buffers but not in acetate-buffered or unbuffered solutions; neither BW755C nor ETYA exhibited this restriction. However, SHAM, BW755C, and ETYA also were inhibitors of browning and sesquiterpene accumulation elicited in potato by poly-l-lysine, which, unlike AA, is not a lipoxygenase substrate. SHAM effectiveness also was restricted to 6 hours after treatment with poly-l-lysine. While the results with AA support a role for lipoxygenase, those with poly-l-lysine may be evidence that these compounds are having other effects in potato tissue.     

Uptake of the glucose analogue 2-deoxyglucose by germinating mitospores of Allomyces macrogynus.

Mitospores or cysts of Allomyces macrogynus do not take up the glucose analogue 2-deoxyglucose. Uptake of 2-deoxyglucose by germlings begins at 25 min into germination, the start of the rhizoid stage, and increases in rate by approximately 50-fold until 100 min into germination. The rate remains constant from 100 to 200 min, at which time germination is completed and hyphal formation begins. The presence of glucose in the germination medium blocks the uptake of 2-deoxyglucose. Of the other sugars tested, only galactose had any effect on 2-deoxyglucose uptake. Actinomycin D treatment during germination in a glucose-containing medium prevented the appearance of the uptake system, but actinomycin D was not effective after the transfer to a glucose-free medium. Cycloheximide treatment prevented the appearance of the uptake system if it was added at the time of the transfer to the glucose-free medium; it inhibited uptake only partially if the germlings were starved of glucose before its addition. It appears, therefore, that both ribonucleic acid synthesis during germination and protein synthesis after the removal of glucose are required for the uptake of 2-deoxyglucose.     

Functional necessity of the cytoskeleton during cleavage membrane development and zoosporogenesis in Allomyces macrogynus

Cleavage membrane development and cytokinesis were examined in zoosporangia of Allomyces macrogynus treated with cytoskeletal inhibitors and compared to zoosporogenesis under control conditions. Developing membranes were visualized in living zoosporangia with laser-scanning confocal microscopy using the lipophilic membrane dye FM4-64. Under control conditions, cleavage membranes developed in four discrete stages, ultimately interconnecting to delimit the cytoplasm into polygonal uninucleate domains of near uniform size. Disruption of microtubules did not impede the normal four-stage development of cleavage membranes, and cytokinesis occurred with only minor detectable anomalies, although zoospores lacked flagella. Disruption of actin microfilaments did not inhibit membrane formation but blocked nuclear migration and significantly disrupted membrane alignment and cytoplasmic delimitation. This resulted in masses of membrane that remained primarily in cortical regions of the zoosporangia, as did nuclei, throughout zoosporogenesis. Zoospores formed in the absence of microtubules had only a slightly larger mean diameter than control zoospores, although nearly 50% of spores contained two or more nuclei. Microfilament inhibitor treatments produced spores with substantially larger mean diameters and correspondingly larger numbers of nuclei per spore, with greater than 85% containing three or more nuclei. These results showed that a functional actin microfilament cytoskeleton was required for proper alignment of cleavage elements and cytokinesis in Allomyces zoosporangia while microtubules played a less significant role.     

The effect of monensin on gametogenesis and zoosporogenesis in the aquatic fungus,Allomyces macrogynus

Summary Cytoplasmic cleavage in the gametangia and zoosporangia ofA. macrogynus was studied using monensin, an ionophore known to disrupt several endomembrane functions in plant and animal cells. Monensin interfered with normal gamete and zoospore formation in a dose dependent manner such that at a 20 M concentration very abnormal cells were released from the reproductive structures. It was evident that monensin's effect was most pronounced during the first 25 minutes of gametogenesis and parallels in time the onset and continuation of the cytoplasmic cleavage events. Observations using fluorescence and differential interference contrast microscopy indicated that the ionophore inhibited normal cytoplasmic cleavage resulting in the production of multinucleate cells, many of which had either no flagella or multiple flagella. Transmission electron microscopy showed that the monensin-treated gametangia had many large vacuoles which contained amorphous electron-opaque material. X-ray microprobe analysis demonstrated that the elemental composition of the large vacuoles was identical to that of the dense globular inclusions seen in untreated gametangia, and morphological analysis confirmed the relationship between these endomembrane structures. Thus this swollen endomembrane component probably is not involved in the cleavage process. Single endomembrane cisternae which were very common in untreated gametangia were seldom seen in monensin-treated preparations. Instead, many smaller electron-transparent vacuoles were observed. These swollen cisternae may both represent monensin-modified Golgi apparatus equivalents and/or play a critical role during the process of gametogenesis and zoosporogenesis inA. macrogynus.     

Molecular phylogeny of Allomyces macrogynus: Congruency between nuclear ribosomal RNA- and mitochondrial protein-based trees

We have sequenced the nuclear and mitochondrial small subunit rRNA genes (rns) and the mitochondrial genes coding for subunits 1 and 3 of the cytochrome oxidase (cox1 and cox3, respectively) of the chytridiomycete Allomyces macrogynus. Phylogenetic trees inferred from the derived COX1 and COX3 proteins and the nuclear rns sequences show with good bootstrap support that A. macrogynus is an early diverging fungus. The trees inferred from mitochondrial rns sequences do not yield a topology that is supported by bootstrap analysis. The similarity and the relative robustness of the nuclear rns and the mitochondrial protein-derived phylogenetic trees suggest that protein sequences are of higher value than rRNA sequences for reconstructing mitochondrial evolution. In addition, our trees support a monophyletic origin of mitochondria for the range of analyzed eukaryotes. Correspondence to: B. Franz Lang     

Structural Features Required for Inhibition of Soybean Lipoxygenase-2 by Propyl Gallate : Evidence that Lipoxygenase Activity Is Distinct from the Alternative Pathway

The ability of 19 structural analogs of propyl gallate to inhibit purified soybean seed (Glycine max [L.] Merr. var. Ransom) lipoxygenase-2 (EC 1.13.11.12) was determined. The results indicate that the o-dihydroxy and not the ester function of propyl gallate is essential for inhibition of lipoxygenase. Catechol thus represents the minimum inhibitory structure. Among those compounds possessing an o-dihydroxy function, the K_i′ for inhibition of lipoxygenase is directly related to the lipophilicity of the inhibitor as measured by the octanol-water partition coefficient. The structural features of propyl gallate necessary for inhibition of lipoxygenase were found to differ from those required for inhibition of the plant mitochondrial alternative pathway. This further supports the concept that the alternative oxidase and lipoxygenase are functionally distinct species.     

Effect of Salicylhydroxamic Acid on Respiration, Photosynthesis, and Peroxidase Activity in Various Plant Tissues

Earlier reports from our laboratory described salicylhydroxamicacid (SHAM) stimulation of O₂ uptake by expanded soybean leavesor older green cotyledons. This stimulation could not be interpretedin terms of engagement or capacity of the cytochrome and alternativerespiratory pathways. In this report, we tested the possibilitythat a soluble peroxidase, which can be easily eluted from soybeanleaves and cotyledons, might be responsible for SHAM stimulationin whole tissue. The peroxidase catalyzes oxidation of NADHby O₂, is strongly stimulated by SHAM and benzhydroxamic acid(BHAM) and inhibited by KCN, propyl gallate and gentisic acid.This peroxidase, however, does not seem to be responsible forSHAM-stimulated O₂ uptake in whole, green tissue. In our earlier work reporting SHAM-stimulated respiration ingreen tissue, the samples had not been shielded from room light(10–20 µmol photons m^–2.s^–1). In thisreport, we show that O²-uptake rates of controls measured indarkness were always greater than those measured in room light.SHAM stimulation was not observed in the dark or in tissue withoutchlorophyll. We also found that CO₂ uptake of whole leafletsin saturating light was completely inhibited by SHAM fed throughthe transpiration stream. SHAM, therefore, is a potent inhibitorof photosynthesis. We conclude that the SHAM-stimulated respirationof green tissues we reported earlier likely was due to verylow rates of photosynthesis occurring under room light. ³Present address: SANDOZ Ltd., Agrobiological Research Station,4108 Witterswil, Switzerland⁴Present address: WTC 1A3, Weyerhaeuser Co., Tacoma, WA 98477,U.S.A. (Received June 23, 1989; Accepted October 20, 1989)     

Carbon Source and Micronutrient Requirements of the Aquatic Phycomycete, Catenaria anguillulae Sorokin

The requirements of the aquatic Phycomycete, Catenaria anguillulaewere analysed in liquid, shake cultures using a standardizedzoospore inoculum. Growth was determined by measuring mycelialdry weight and rate of production of titratable acid. D-glucose was the best carbon source and had an optimum concentrationof 166 mM of carbon for the medium used. When other carbon sourceswere supplied, only those related to glucose (fructose and mannose)or composed of glucose units with an alpha-linkage (maltose,glycogen, and starch) were readily utilized. Lactic acid wasdetermined qualitatively as an end-product of carbon metabolism. The optimum level of phosphate was 1.0 mM. The optimum concentrationof EDTA was 0.032 mM. Of the chelated cations included in themedium only the omission of iron, zinc, calcium, or magnesiumreduced growth. Concentrations of calcium below 0.4 mM and ofmagnesium below 0.2 mM were limiting; whereas, concentrationsof both ions up to 1 mM were non-toxic.     

Respiration of Senecio Shoots: Inhibition during Photosynthesis, Resistance to Cyanide and Relation to Growth and Maintenance

Respiration and dry matter producation were measured in shoots of senecia aquaticus Hill, which is flood tolerant and in shoots of S. jacobaea L., which is flood- sensitive. Both species were grown in culture solutions of high and of low oxygen concentration Growth of food of S. jacobaca was unaffected by a low oxygen supply bur growth of S. jacobaca was severly hampered by a low oxygen concentration in the root medium. Kinetic data about the rate of apparent photosynthesis at low oxygen conetration and different carbon dioxide concentrations indicated that at light saturation respiration was strongly repressed during photosynthesis. Shoot growth respiration, i.e. the amount of carbon dioxide produced for synthesis of shoot dry, matter appeared to be absent on S. jacobaea and to be very low (13.mg CO₂/g dry shoots) in S. aquaticus. In comparison with values prepiration rate was 2.8. 2.0. 1.5 and 1.3mg CO₂/h.g dry shoots in aerobically and anaerobically growth S. jacobaea and in aerobically and anaerobically growth S. aquabaea respectively. These values were also low in comprision with values previously found for roots of the same species. Shoot dark respiration on S. aquaticus was inbihitedd by a com bination on CN and salicylhydroxamic acid (SHAM), but not by application on one of these inhibitors alone. It was therefore concluded that an alternative oxidative pathway was present but not active in shoots of S. aquaticus. In the absence of inhibited of the cylochorome pathway. The low value of growth respiration and maintenance respiration rate in the shoots as compared with those in the roots of the investigated Sencio species are discussed in relation to the activity of the alternative oxidative pathway and to the possibilbity of a direct supply of ATP by photosynthesis intead of respiratory meta bolism.     

The Effect of Cyanide on the Respiration and the Oxidative Assimilation of Glucose by Chlorella vulgaris

It is shown that low concentrations of cyanide stimulate theendogenous respiration of Chlorella vulgaris. When glucose isadded the respiration rate is much increased but is now reversiblyinhibited by cyanide. Some 30–60 per cent. of the totalrespiration remains uninhibited. One-eighth to one-ninth ofthe glucose added is completely oxidized. Most of the remainderis assimilated to di- or polysaccharide. Low concentrationsof cyanide which inhibit the rate of glucose oxidation alsoinhibit the assimilation of glucose. Two possible interpretationsof this fact are discussed. It is suggested that the assimilationof glucose is coupled with the oxidation of glucose by a cyanide-sensitiverespiratory system. The mathematical consequences of this theoryare considered and shown to agree with the experimental results.The effect of cyanide on the respiratory quotient is also discussed.     

Inhibition of the Development of Induced Respiration and Cyanide-insensitive Respiration in Potato Tuber Slices by Cerulenin and Dimethylaminoethanol

The interdependence of the development of wound-induced respiration and membrane-related phospholipid biosynthesis in potato tuber (Solanum tuberosum var. Russet) slices was established by the use of agents which selectively affect lipid and phospholipid synthesis. Cerulenin, a specific inhibitor of de novo fatty acid synthesis, inhibited the ultimate development of wound-induced respiration and of cyanide resistance only when given in the critical first 10 to 12 hours of slice aging. Similarly, when slices were exposed to the choline analogue dimethylaminoethanol within the first 10 hours, the phospholipid composition of the membrane lipids was drastically altered, the wound-induced respiration in a 24-hr period was substantially curtailed, and the development of cyanide insensitivity was sharply inhibited. These observations indicate that time-restricted membrane-related phospholipid synthesis is prerequisite to the development of wound-induced respiration and concurrent cyanide insensitivity.     

Changes in Respiration and Cyanide Sensitivity of the Barley Floret during Development and Maturation

The 6-week period of development and maturation of the barley (Hordeum vulgare L.) floret from anthesis to harvest is characterized by two phases: an early phase of rapid increase in respiration rate and dry weight, and a late phase during which respiration decreased rapidly whereas dry weight remained unchanged. Consumption of O₂ by the embryo changed little during the entire developmental period, whereas O₂ uptake by the endosperm and the lemma and palea decreased significantly during the late phase.