Alternative Respiratory Pathway: ITS ROLE IN SEED RESPIRATION AND ITS INHIBITION BY PROPYL GALLATE

Oxygen uptake during the first hours of imbibition in intact soybean and mung bean seeds showed a marked sensitivity to potassium cyanide but was unaffected by addition of either salicylhydroxamic acid or propyl gallate. However O₂ uptake by finely ground seed particles was very sensitive to the addition of either compound. The results indicated that O₂ uptake in intact, imbibing seeds was associated with a cyanide-sensitive process, most probably mitochondrial mediated respiration, and not the result of the cyanide-insensitive lipoxygenase activity which was readily detectable in ground seed particles.     

Confounding of alternate respiration by lipoxygenase activity

The initial burst of respiratory activity (Q_o2) of imbibing soybean (Glycine max [L.] Merr. var. Wayne) seed tissue is cyanide-insensitive, and sensitive to salicylhydroxamate: presumptive evidence for the presence of alternate respiration. The initial O₂ consumption is also highly sensitive to propyl gallate. Soybean lipoxygenase exhibits similar characteristics of insensitivity to cyanide and sensitivity to salicylhydroxamate and to propyl gallate. The initial burst of respiration is enhanced by the addition of linoleic acid, a lipoxygenase substrate. These results indicate that the conventional tests for alternate respiration in plant tissues can be confounded by lipoxygenase; they also suggest that propyl gallate can be used to assess the possible participation of lipoxygenase in the O₂ uptake by plant tissues.     

The respiratory system of the aquatic phycomyceteAllomyces macrogynus wild-type and sexual mutants

A series of predominantly male mutants induced with ethidium bromide were isolated fromAllomyces macrogynus (Emerson) Emerson and Wilson strain Burma 3–35 (35°C) and crossed to the wild-type female. Gametangia and sporangia from ethidium bromide-induced mutants, predominantly male and female Mendelian mutants, and double mutants constructed from these Mendelian sexual mutants, were analyzed for their response to cyanide, propyl gallate, and CCCP. Reduced minus oxidized room temperature cytochrome difference spectra were made on homogenized mycelium from wild-type and sexual mutants. The ethidium bromide-induced male mutants showed a non-Mendelian segregation pattern when crossed to wild type. Mendelian and non-Mendelian mutants have lower respiratory rates than the wild type. Neither the mutants nor the wild type were sensitive to propyl gallate. Mutants and wild type have the same response to CCCP. The concentrations of the cytochromes from the mutants were generally the same as in the wild type, but the ratio ofa-type cytochromes toc- andb-type cytochromes was lowered in many mutants. Both higher and lower relative amounts of cyanide insensitive respiration (compared to total respiration) were observed among the sexual mutants when the sexual mutants were compared to wild type. The relative amount of cyanide-insensitive respiration of the double mutants can be related to the relative amount of cyanide-insensitive respiration of the single mutants from which the double mutants were constructed. It is concluded that the respiratory deficiency of the sexual mutants disturbs the respiratory system in a complex manner.     

Effects of Cyanide and Ethylene on the Respiration of Cyanide-sensitive and Cyanide-resistant Plant Tissues

The effects of cyanide and ethylene, respectively, were studied on the respiration of a fully cyanide-sensitive tissue-the fresh pea, a slightly cyanide-sensitive tissue-the germinating pea seedling, and a cyanide-insensitive tissue-the cherimoya fruit. Cyanide inhibition of both fresh pea and pea seedling respiration was attended by a conventional Pasteur effect where fermentation was enhanced with an accumulation of lactate and ethanol and a change in the level of glycolytic intermediates indicative of the activation of phosphofructokinase and pyruvate kinase accompanied by a sharp decline in ATP level. In these tissues, ethylene had little or no effect on the respiration rate, or on the level of glycolytic intermediates or ATP. By contrast, ethylene as well as cyanide enhanced both respiration and aerobic glycolysis in cherimoya fruits with no buildup of lactate and ethanol and with an increase in the level of ATP. The data support the proposition that for ethylene to stimulate respiration the capacity for cyanide-resistant respiration must be present.     

Involvement of iron in the biogenesis of the cyanide-insensitive respiration in the yeast Saccharomycopsis lipolytica

The involvement of iron in the biogenesis of the cyanide-insensitive respiration in the yeast Saccharomycopsis lipolytica has been established on the following basis: (1) endogenous metal chelation by either benzyl- or salicylhydroxamic acid, EDTA or nitrilotriacetate prevented the biogenesis of the cyanide-insensitive respiratory pathway in S. lipolytica. (2) Addition of Fe(III) during the biogenesis increased both the rate of the appearance of the alternative respiratory pathway and its extent. Neither Fe(II), nor Co(II), Cu(II), Al(III), La(III), Mn(II) or Mg(II) could substitute for Fe(III). (3) The biogenesis of the alternative respiratory pathway could be dissociated into two steps: (a) a first one, slow, cycloheximide-sensitive, temperature-dependent, iron-independent, leading to cells still fully cyanide-sensitive, presumably involving the de novo biosynthesis of an inactive protein moiety and (b) a second step, fast, iron-dependent, temperature-independent, cycloheximide-insensitive, leading to cells with a cyanide-insensitive respiration, presumably the activation by iron of the inactive precursor.     

Cyanide-insensitive respiration of Candida lipolytica

Whole cells of the yeast Candida lipolytica exhibited a high, cyanide-sensitive endogenous respiration which became completely cyanide-insensitive under certain physiological circumstances namely (1) in the stationary phase of growth and (2) upon aeration in the resting state. This cannot be due to a change in permeability of the cell wall as the respiration of protoplasts showed the same (in)sensitivity to cyanide as the cells from which they were obtained.The cyanide-insensitive respiration of C. lipolytica was located in the mitochondria and coexisted with the normal respiratory chain, as the mitochondria isolated from cyanide-insensitive cells exhibited at the same time a cyanidesensitive respiration of ascorbate and N,N,N,N-tetramethyl-p-phenylenediamine and a cyanide-insensitive respiration of succinate.The alternate respiratory pathway was sensitive to benzyl- and salicylhydroxamic acids. In this respect it resembles the alternate mitochondrial pathway described in the literature for various plants.The cyanide-insensitive respiration did not appear in the resting state when the cells were aerated in the presence of cycloheximide nor at 0 C instead of at room temperature. These facts suggest some form of induction involving new protein synthesis. The induction process depends on the presence of molecular oxygen as the cyanide-insensitive endogenous respiration did not appear during agitation of yeast cells in the resting state if the gaseous atmosphere lacked oxygen.     

Evidence for target site insensitivity to cyanide in Spodoptera eridania larvae

• 1.1. Isolated mitochondria from the whole southern armyworm larvae, Spodoptera eridania, show all of the characteristics of mammalian liver mitochondria, except for target site sensitivity to cyanide.
• 2.2. The armyworm larval mitochondria are 17 times less sensitive to cyanide when compared to rat liver mitochondria and cannot be completely inhibited with extremely large doses.
• 3.3. These data suggest the presence in the southern armyworm of either a cyanide-insensitive cytochrome oxidase, or the elaboration of cyanide-insensitive oxidative pathway reminiscent of an alternative oxidative pathway that is known to coexist in plants alongside the cyanide-sensitive pathway.

     

Effect of tobacco mosaic virus infection of levels of soluble superoxide dismutase (SOD) in nicotiana tabacum and nicotiana glutinosa leaves

The activity of superoxide dismutase (SOD: E.C. 1.15.1.1) was evaluated on Nicotiana tabacum and Nicotiana glutinosa leaf tissue after Tobacco Mosaic Virus (TMV) infection. Significant increase in extracted SOD appeared to be directly related to the appearance of necrotic and systemic symptoms in hypersensitive (N. glutinosa and N. tabacum cv. Havana 425) and susceptible (N. tabacum cv. Bright BC 60) leaves, respectively. SOD activity did not change significantly during the replication of TMV in the inoculated susceptible leaves up to 4 days after inoculation. Both cyanide-insensitive (2 days after inoculation) and sensitive (3–4 days after inoculation) enzymes increased during the expression of the hypersensitivity. Only cyanide-sensitive enzyme increased in systematically infected leaves. SOD and peroxidase increased simultaneously and the enhancement of peroxidase was higher than that of SOD. The values of peroxidase greatly exceeded that of SOD only in the hypersensitive leaves during local lesion differentiation. In N. tabacum leaves 4 clear SOD bands were separated by polyacrylamide gel electrophoresis: 3 cyanide-sensitive (Cu,Zn enzyme) and 1 cyanide-insensitive, while N. glutinosa had 3 bands: 2 cyanide-sensitive and 1 cyanide-insensitive. The cyanide-insensitive band, both in N. tabacum and N. glutinosa, was sensitive to H₂O₂ and insensitive to chloroform-ethanol treatment and thus supposed to be Fe enzyme. The infection did not induce change in the electrophoretic pattern of SOD enzymes.In summary, our results indicate that the pathogenic alteration caused by TMV infection both in the compatible and in the incompatible combinations are characterized by an induction of SOD activity, particularly cyanide-sensitive Cu,Zn-SOD. The connection between the induction of SOD and a possible activation of O₂⁻ production in the hypersensitive tissue following TMV infection is discussed.     

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with α-ketoglutarate and malate as substrates. Whereas α-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.

Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD⁺, glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

     

Structural features required for inhibition of cyanide-insensitive electron transfer by propyl gallate

A series of 19 structural analogs of propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester) were tested for their ability to inhibit the cyanide-insensitive, electron transfer pathway in isolated mung bean mitochondria. The results indicate that the trihydroxy substituent, not the ester, of propyl gallate is the structural feature of the molecule required to produce inhibition. Further, only one OH group, if it is located para to the ester moiety, will bring about specific inhibition. Of the compounds which contained the appropriate hydroxyl group, the lower the pK_α of the hydroxyl group, the lower the observed inhibition constant (K_i′) for blocking the alternative pathway. Even though the observed K_i′ values varied over two orders of magnitude for the compounds tested, the calculated pH-independent, intrinsic inhibition constants (K_i) were markedly similar for all inhibitory compounds. The results indicate that a simple phenolate anion is the minimum structural feature required to observe specific inhibition of the alternative pathway and the more easily the anion can be formed, the better the observed inhibition. Similarities between the above compounds and the structural features associated with hydroxamic acids were also noted.     

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.     

Molecular approaches of a mitochondrial mutation inCandida utilis

Analysis of the cytochrome spectra of a mitochondrial mutant ofCandida utilis showed complete absence of apocytochromeb; this suggests a certain degree of damage, probably a small deletion in themit genes of mitochondrial DNA. Oxygen uptake measurements with and without cyanide of the respiratory-competentCandida utilis parent strain and its derivative mitochondrial mutant P_1,2 indicated the absence of the cyanide-sensitive or normal respiratory chain and a lowered rate of cyanide-insensitive or alternate respiration. Mitochondrial profiles and distribution of parental and mutant cells account for an altered mitochondrial DNA which affects mitochondria in the latter cell shape and function. The mutant cells ofCandida utilis were considered asmit ^– mutants from the observations reported here.     

Dibromothymoquinone inhibition of the cyanide-sensitive and -insensitive respiration in MI-1 mutant of neurospora.

Effects of dibromothymoquinone (DBMIB) on the cyanide-sensitive and -insensitive pathways of respiration in mitochondria isolated from wild type and mi-1 mutant of Neurospora crassa have been investigated. It is shown that DBMIB inhibits the overall respiration in both strains in a similar manner. However, separate measurements of the DBMIB -induced inhibition of the KCN- and salicylhydroxamic acid (SHAM)-sensitive oxidation pathways in mi-1 pointed to some differences in the pattern and the degree of inhibition of these particular pathways, as reflected by a difference in the DBMIB concentration required for half-maximal inhibition and by the finding that the KCN-sensitive pathway is resistant to low concentrations of DBMIB. These results are consistent with a regulatory function of ubiquinone (UQ) in the cyanide-insensitive pathway in addition to its known carrier function in the cyanide -sensitive pathway of oxidation.     

Binding of Butyl Gallate to Plant Mitochondria : II. Relationship to the Presence or Absence of the Alternative Pathway

[¹⁴C]butyl gallate was used in binding studies to investigate the cyanide-resistant respiratory pathway in mitochondria isolated from a variety of sources displaying varying levels of cyanide resistance. Highly cyanide-resistant mitochondria were isolated from aroid spadices, while moderately cyanide-resistant mitochondria were isolated from either mung bean (Vigna radiata L.) hypocotyls or carbon dioxide/oxygen/ethylene-treated tubers. Totally cyanide-sensitive mitochondria were isolated from untreated tubers and rat liver. With one exception, all the plant mitochondria showed a reversible butyl gallate binding site which saturated at a level of 1.0 to 2.0 nanomoles per milligram protein. The exception, freshly harvested white potato tubers (<1 month from harvest), showed little specific butyl gallate binding, and also showed no appreciable induction of the cyanide-resistant pathway following carbon dioxide/oxygen/ethylene treatment. Only a low level, linear binding, well below that seen with plant mitochondria, was observed with rat liver mitochondria. Taken together, these results suggest a model for the interaction of the alternative pathway with the cytochrome pathway. In this model, the butyl gallate binding site (alternative oxidase) is a constitutive component in those mitochondria that are capable of developing the alternative pathway, and the binding sites associated with a second, inducible component that functions to couple the oxidase to the cytochrome pathway.     

Cyanide-insensitive respiration in relation to growth of a low-temperature basidiomycete

The cyanogenic low-temperature basidiomycete (Coprinus psychromorbidus Redhead and Traquair), unlike other cyanide-tolerant fungi, does not detoxify cyanide via formamide hydro-lyase. Instead, tolerance apparently depends on cyanide-insensitive respiration involving activity of the mitochondrial alternative oxidase. Respiration and growth of young mycelium that lacks alternative oxidase activity are blocked both by cyanide and 1 mum antimycin. When activity of the alternative oxidase is elicited in young mycelium by 0.05 mm cyanide, subsequent treatment with antimycin stimulates respiration and fails to halt growth. Older mycelium becomes tolerant coincidentally with the release of cyanide by the mycelium. Tolerant older mycelium in medium containing 0.05 to 1.0 mum antimycin grows at 30 to 45% of the control rate. Cyanide- and antimycin-tolerant growth and respiration are blocked by salicyl hydroxamic acid, an inhibitor of the alternative oxidase, and by rotenone, which inhibits ATP synthesis at site I.     

m-Chlorobenzhydroxamic Acid–an Inhibitor of Cyanide-Insensitive Respiration in Trypanosoma brucei*

Cyanide-insensitive respiration of bloodstream and culture forms of T. brucei was inhibited by m-chlorobenz-hydroxamic acid (m-CLAM). The cyanide-sensitive respiration of culture forms of this organism was not affected by m-CLAM. This compound is the 1st really effective inhibitor to be described that acts specifically on the cyanide-insensitive respiration of the T. brucei group of trypanosomes; as such it may be of some importance as a trypanocidal drug. Evidence is adduced which suggests that a branched electron transport chain is present in culture forms of T. brucei and that the cyanide-insensitive terminal oxidase found in these stages is the same as that found in bloodstream forms.     

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.     

Allotopic expression of a mitochondrial alternative oxidase confers cyanide resistance to human cell respiration

Human mitochondrial respiration is distinct from that of most plants, microorganisms and even some metazoans in that it reduces molecular oxygen only through the highly cyanide-sensitive enzyme cytochrome c oxidase. Here we show that expression of the cyanide-insensitive alternative oxidase (AOX), recently identified in the ascidian Ciona intestinalis, is well tolerated by cultured human cells and confers spectacular cyanide resistance to mitochondrial substrate oxidation. The expressed AOX seems to be confined to mitochondria. AOX involvement in electron flow is triggered by a highly reduced redox status of the respiratory chain (RC) and enhanced by pyruvate; otherwise, the enzyme remains essentially inactive. AOX expression promises to be a valuable tool to limit the deleterious consequences of RC deficiency in human cells and whole animals.     

Nippostrongylus brasiliensis and Ascaridia galli: mitochondrial respiration in free-living and parasitic stages

Aerobic respiratory pathways have been delineated and respiratory efficiency has been assessed in mitochondria isolated from embryonated eggs, infective larvae, and adult Nippostrongylus brasiliensis and Ascaridia galli. Mitochondrial respiration in free-living stages of N. brasiliensis is mediated mainly by a mammalian-like antimycin A- and cyanide-sensitive pathway; specific respiratory activity is high and oxidative phosphorylation efficient. In mitochondria of adult N. brasiliensis, antimycin A- and cyanide-sensitive respiration is decreased relative to respiration though an alternative pathway, and specific respiratory activity and mitochondrial efficiency are lower. Respiration in mitochondria from embryonated eggs and tissues of adult A. galli is comparable, and apparently mediated by an antimycin A- and cyanide-insensitive alternative respiratory pathway; no evidence for the presence of a mammalian-like respiratory pathway in embryonated eggs of A. galli was found. The results of this study are compared to mitochondrial respiration in eggs, larvae, and adult body wall muscle of Ascaris suum.