Cyanide-resistant Respiration in Fresh and Aged Sweet Potato Slices

The respiration of fresh sweet potato (Ipomoea batatas) slices is resistant to, and often stimulated by, cyanide and antimycin A. m-Chlorobenzhydroxamic acid (CLAM), a selective inhibitor of the alternate path, inhibits respiration in the presence of cyanide and has a limited inhibitory effect in the presence of antimycin A. Thus, a partial bypass of the antimycinsensitive site is indicated. Respiration rises 2-fold at best with slice aging, the increment being cytochrome-mediated. The cyanide-resistant pathway contributes neither to coupled fresh slice respiration nor to the induced respiration in the absence of inhibitors of the cytochrome path. In the presence of uncoupler, however, the alternate path is engaged both in fresh and aged slices. V_cyt, the maximal capacity of the cytochrome path, remains essentially the same with slice aging, whereas V_alt decreases from 20 to 60 per cent. The induced respiration is readily accommodated by the potential cytochrome path capacity of fresh slices, which is realized on aging. Accordingly, there is no need to invoke mitochondrial proliferation in explanation of the development of the induced respiration. The engagement of the alternate path in response to uncoupler reflects substrate mobilization to a degree that substrate oxidation exceeds the electron transport capacity of the cytochrome path.

Fresh slices do not utilize exogenous substrates, whereas aged slices do so readily. Cerulenin, a specific inhibitor of fatty acid synthesis, prevents the development of the induced respiration as well as the capacity to oxidize exogenous substrates. It is suggested that lipid, and ultimately membrane, biosynthesis is central to the development of the induced respiration and the ability to use exogenous substrates, much as in potato.

     

Respiratory Contribution of the Alternate Path during Various Stages of Ripening in Avocado and Banana Fruits

The respiration of fresh slices of preclimacteric avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii var. Valery) fruits is stimulated by cyanide and antimycin. The respiration is sensitive to m-chlorobenzhydroxamic acid in the presence of cyanide but much less so in the presence of antimycin. In the absence of cyanide the contribution of the cyanide-resistant pathway to the coupled preclimacteric respiration is zero. In uncoupled slices, by contrast, the alternate path is engaged and utilized fully in avocado, and extensively in banana. Midclimacteric and peak climacteric slices are also cyanide-resistant and, in the presence of cyanide, sensitive to m-chlorobenzhydroxamic acid. In the absence of uncoupler there is no contribution by the alternate path in either tissue. In uncoupled midclimacteric avocado slices the alternate path is fully engaged. Midclimacteric banana slices, however, do not respond to uncouplers, and the alternate path is not engaged. Avocado and banana slices at the climacteric peak neither respond to uncouplers nor utilize the alternate path in the presence or absence of uncoupler.

The maximal capacities of the cytochrome and alternate paths, V_cyt and V_alt, respectively, have been estimated in slices from preclimacteric and climacteric avocado fruit and found to remain unchanged. The total respiratory capacity in preclimacteric and climacteric slices exceeds the respiratory rise which attends fruit ripening. In banana V_alt decreases slightly with ripening.

The aging of thin preclimacteric avocado slices in moist air results in ripening with an accompanying climacteric rise. In this case the alternate path is fully engaged at the climacteric peak, and the respiration represents the total potential respiratory capacity present in preclimacteric tissue. The respiratory climacteric in intact avocado and banana fruits is cytochrome path-mediated, whereas the respiratory climacteric of ripened thin avocado slices comprises the alternate as well as the cytochrome path. The ripening of intact fruits is seemingly independent of the nature of the electron transport path.

Uncouplers are thought to stimulate glycolysis to the point where the glycolytic flux exceeds the oxidative capacity of the cytochrome path, with the result that the alternate path is engaged.

     

Relative Contribution of Cytochrome-mediated and Cyanide-resistant Electron Transport in Fresh and Aged Potato Slices

The respiration of fresh potato (Solanum tuberosum, var. Russet Burbank) slices is predominantly cyanide-sensitive whether in the presence or absence of uncoupler. By contrast, the wound-induced respiration which develops in thin slices with aging is cyanide-resistant, and in the presence of cyanide, sensitive to chlorobenzhydroxamic acid, a selective inhibitor of the cyanide-resistant respiration. Titration of the alternate path in coupled slices with chlorobenzhydroxamic acid, in the presence and absence of cyanide, shows that the contribution of the cyanide-resistant pathway to the wound-induced respiration is zero. Similar titrations with uncoupled slices reveal that the alternate path is engaged and utilized extensively.

The maximal capacity of the cytochrome path (V_cyt) has been estimated in fresh and aged slices in the presence of the uncoupler carbonyl-cyanide m-chlorophenyl hydrazone. It has been found that V_cyt of aged slices is but 30 to 40% higher than that of fresh slices. The results suggest that the bulk of the wound-induced respiration is mediated through the cytochrome pathway which exists in fresh slices in suppressed form, and which is fully expressed by slice aging. The engagement of the alternate path by uncouplers in aged slices is attributed to an increase in substrate mobilization, with the result that the electron transport capacity of the cytochrome chain is exceeded.

     

Inhibition and stimulation of root respiration in pisum and plantago by hydroxamate : its consequences for the assessment of alternative path activity

The contribution of the alternative pathway in root respiration of Pisum sativum L. cv Rondo, Plantago lanceolata L., and Plantago major L. ssp major was determined by titration with salicylhydroxamate (SHAM) in the absence and presence of cyanide. SHAM completely inhibited the cyanide-resistant component of root respiration at 5 to 10 millimolar with an apparent K_i of 600 micromolar. In contrast, SHAM enhanced pea root respiration by 30% at most, at concentrations below 15 millimolar. An unknown oxidase appeared to be responsible for this stimulation. Its maximum activity in the presence of low SHAM concentrations (1-5 millimolar) was 40% of control respiration rate in pea roots, since 25 millimolar SHAM resulted in 10% inhibition. In plantain roots, the maximum activity was found to be 15%. This hydroxamate-activated oxidase was distinct from the cytochrome path by its resistance to antimycin. The results of titrations with cyanide and antimycin indicated that high SHAM concentrations (up to 25 millimolar) block the hydroxamate-activated oxidase, but do not affect the cytochrome path and, therefore, are a reliable tool for estimating the activity of the alternative path in vivo. A considerable fraction of root respiration was mediated by the alternative path in plantain (45%) and pea (15%), in the latter because of the saturation of the cytochrome path.     

An alternate respiratory pathway in Candida albicans

Usual concentrations of antimycin A, rotenone and EDTA, individally or in combination, reduced aerobic growth rate and cell yield of Candida albicans to about half its normal level and to about the levels of previously-described acetate-negative, cytochrome-complete and aa₃-deficient variants which were little affected by the inhibitors. Anaerobic conditions (not affected by antimycin A) reduced growth rate and cell yield of all cultures-including that of a nonrespiring aa₃, b-deficient mutant-to low, equal levels. Antimycin A but not rotenone prevented growth of the normal strain on ethanol medium. Cyanide and antimycin A blocked most of the respiration of the normal strain and cytochrome-complete variant, but did not affect that of the cytochrome aa₃-deficient mutant. Rotenone and EDTA did not affect respiration of any of the cultures. SHAM blocked cyanide- and antimycin A-insensitive respiration and prolonged the lag phases of the three respiring cultures, especially in the presence of antimycin A, but alone increased oxygen-uptake rate of the cytochromecomplete cultures while curtailing that of the cytochrome aa₃-deficient mutant. Resting cells, especially wild-type, grown in medium containing antimycin A exhibited lowered oxygen-uptake rate, which was increased upon the addition of cyanide or antimycin A. Antimycin A stimulated, but cyanide inhibited, respiration of cytochrome-complete cultures grown in the presence of rotenone but did not affect that of the cytochrome aa₃-deficient mutant. SHAM inhibited respiration of all antimycin A- or rotenone-grown cultures. The high rate of respiration of C. albicans in the presence of inhibitors for three sites of electron transport in the conventional oxidative pathway, the inhibition of this respiration by SHAM and its loss by the absence of cytochrome b, indicate an alternate oxidative pathway in this organism which crosses the conventional one at cytochrome b.This work was supported by Public Health Service Graduate Dental Training Grant DE 00144 and the Graduate School and the Department of Microbiology, Southern Illinois University.     

Studies on the Wound-Induced CN-Resistant Respiration in Potato Tuber Slices

The respiration of fresh potato slices was sensitive to CN-, and was not inhibited by m-CLAM in the presence or absence of CN-. By contrast, the wound-induced respiration of slices, incubated in air for 24 hours, was not only relatively resistant to CN-, but also markedly inhibited by m-CLAM in the presence or absence of CN-. When m-CLAM and CN- were added togather, the inhibitory effect was higher than that of them when used separately. The observations indicated that the alternate path is operative in aged potato slices. The data determined by the method of m-CLAM titration showed that the actual contribution of alternate path and cytochrome path in aged potato slices was approximately 28% and 54% of the total respiration respectively in the absence of CN-. When the cytochrome path was inhibited by CN-, the maximal capacity of alternate path (Valt was higher than the actual contribution of them (ρ·Valt). The increased contribution of alternate path in presence of CN- might be thought to indicate that there is a diversion of electron flux from the cytochrome path to the alternate path. When the respiratory flux of aged slices was reduced by treatment with iodoacetate and malonate, the proportions of respiration inhibited by CN- and m-CLAM respectively were not changed.     

Cyanide-Resistant Respiration in Neurospora crassa

Cell respiration in wild type and poky was studied as part of a long-term investigation of cyanide-resistant respiration in Neurospora. Respiration in wild type proceeds via a cytochrome chain which is similar to that of higher organisms; it is sensitive to antimycin A or cyanide. Poky, on the other hand, respires by means of two alternative oxidase systems. One of these is analogous to the wild-type cytochrome chain in that it can be inhibited by antimycin A or cyanide; this system accounts for as much as 15% of the respiration of poky f(-) and 34% of the respiration of poky f(+). The second oxidase system is unaffected by antimycin A or cyanide at concentrations which inhibit the cytochrome chain maximally. It can, however, be specifically inhibited by salicyl hydroxamic acid. The cyanide-resistant oxidase is not exclusive to poky, but is also present in small quantities in wild type grown under ordinary circumstances. These quantities may be greatly increased (as much as 20-fold) by growing wild type in the presence of antimycin A, cyanide, or chloramphenicol.     

Contribution of a Cyanide-insensitive Alternate Respiratory System to Increases in Formamide Hydro-lyase Activity and to Growth in Stemphylium loti in Vitro

Stemphylium loti, a pathogen of a cyanogenic plant, possesses a cyanide-insensitive alternate respiratory pathway. In the absence of cytochrome inhibitors, the alternate system had only a minor role in respiration. When S. loti was grown in medium amended with antimycin to block the cytochrome chain, the alternate system accounted for the total oxygen consumption associated with respiration.     

Appearance of an Alternate Pathway Cyanide-resistant during Germination of Seeds of Cicer arietinum

The combined action of the inhibitors antimycin A and cyanide with benzohydroxamic acid indicates the presence of a cyanide-resistant pathway of respiration in chick pea (Cicer arietinum L.) seeds. The appearance of this pathway takes place during germination. During the first 12 hours of germination, the respiration is predominantly cyanide-sensitive, showing after this time a shift to an “alternate” respiration which is sensitive to benzohydroxamic acid, reaching the maximal cyanide resistance between 72 and 96 hours of germination. The appearance of the alternate pathway is initiated by high O₂ concentrations and depends on cytoplasmic protein synthesis, since its appearance is inhibited by cycloheximide but not by chloramphenicol. Actinomycin D has no effect on the appearance of the alternate pathway. Our results indicate, in agreement with other authors, that the branching point is located between the flavoproteins and cytochromes b, probably at the level of ubiquinone, but the possibility of more than one branching point of the electron flow is also considered.     

Investigation of respiratory and ion transport properties of aging bean stem slices

Ion and oxygen uptake were studied on aging bean stem slices. Oxygen uptake was high immediately after slicing, decreased to a minimum at 100 minutes, and then increased again. Ion uptake per unit of O₂ uptake data suggested that metabolic energy was utilized almost exclusively for sodium transport in fresh tissue but was diverted to potassium transport as the slices aged. Oxygen and ion uptake in fresh slices was less sensitive to 2,4-dinitrophenol as compared to the aged slices, indicating major metabolic and physiological changes occurred during aging. This was further substantiated by the tissue response to cyanide and antimycin A. Oxygen uptake was decreased by cyanide (22% by 1 mm) and antimycin A (14% by 1 microgram per milliliter) in fresh slices but not in aged slices. Potassium uptake that developed during aging was sensitive to cyanide and antimycin A. The results are pertinent to understanding the role of the stem in regulating ion transport in plants.     

Alternative path mediated chloride absorption in cyanide-resistant tissues

The ability of two cyanide-resistant tissues—aged potato slices and fresh preclimacteric banana slices—to take up chloride in the presence of cyanide has been established. Extensive inhibition of chloride uptake by cyanide and chlorbenzhydroxamate together indicates that chloride absorption in the presence of cyanide is supported by respiration mediated by the alternative path. The partial inhibition of respiration-dependent chloride uptake by chlorbenzhydroxamate alone is independent of its effect on the alternative oxidase, and points to inhibition of the transport process per se.     

Respiration of the Roots of Flood-Tolerant and Flood-Intolerant Senecio Species: Affinity for Oxygen and Resistance to Cyanide

The affinity of respiration for oxygen in the roots of six Senecio species studied was low compared with the affinity of cytochrome oxidase for oxygen. Half saturation values of approximately 22 μM oxygen were measured. Root respiration was to a large extent insensitive to cyanide in flood-tolerant as well as in flood-sensitive species. The evidence presented suggests that high activity of salicylhydroxamic acid (SHAM)-sensitive oxidase in Senecio roots was the basis for the low oxygen affinity and for the high cyanide-insensitivity of root respiration in the Senecio species. Methods are described to determine the in vivo activity of the SHAM-sensitive oxidase. It was estimated that it contributed 70% to the total root respiration. The presence of SHAM-sensitive oxidase activity could explain a higher efficiency of root growth respiration under a low oxygen tension if this alternate oxidase was inhibited at a low oxygen concentration in the root medium. However, the SHAM-sensitive oxidase was not specifically involved in either growth respiration or maintenance respiration. Its significance in regulation of the redox state of the cells is discussed.     

The respiratory system of Rhodotorula glutinis. II. Mechanism of inhibitor tolerant respiration

The mechanism of inhibitor-tolerant respiration in Rhodotorula glutinis was studied. This inhibitor-tolerant respiratory pathway was not due to the presence of an excess of cytochrome c oxidase, nor to the operation of an inhibitor-resistant cytochrome c oxidase. Carotenoids do not appear to be involved in this respiratory chain pathway; data are also presented which show that the inhibitors penetrate into the cell. Although the initial rate of oxygen uptake by intact cells was not inhibited in the presence of cyanide or antimycin A, in the presence of these inhibitors the rate of oxygen uptake decreased significantly when the oxygen concentration fell below 100 mum. This change in rate of oxygen uptake as a function of pO(2), suggests that a respiratory chain with a low affinity for oxygen operates in the presence of inhibitors. The characteristics of this alternate pathway are described.     

The alternate respiratory pathway of Candida albicans

Candida albicans contains a cryptic cyanide and antimycin A insensitive respiratory system. This alternate oxidase was found (i) at all growth rates from =0.05 to 0.26 in a chemostat culture and (ii) in both mycelial and yeast forms of the organism. Neither chloramphenicol nor cycloheximide prevented the expression of the alternate oxidase. Salicyl-hydroxamic acid was a potent inhibitor of the cyanide insensitive respiration. The respiration of mitochondria grown in the presence of antimycin A was not inhibited by cyanide or antimycin A but was inhibited by salicylhydroxamic acid.Abbreviations KCN potassium cyanide - SHAM salicyl hydroxamic acid     

Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b.

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reoxidation is observed in the wild type in the present of low concentrations of antimycin. 2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steady-state reduction; reduction in the presence of substrate, cyanide and oxygen; the 'red shift' and lowering of E'-o of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable. 3. The red shift in the mutant is more extensive than in the wild type. 4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes. 5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant. 6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH-2-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycin-binding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.     

Cyanide-Resistant Respiration in Relation to Fruit Respiratory Climacteric

Changes in respiratory rate and the effects of respiratory inhibitorson respiration were determined in apple (Malus sylvestris cv. Delicious) and red pepper (Capsicum fructescens) fruits dusting different stages of development and ripening.The results showed that there was an abrupt rise in respiration daring ripening inapple fruit, but the respiration of the red pepper declined continuously throughout theripening period. Thus the apple is climacteric and the red pepper is non-climacteric fruit. The respiration of apple fruit was sensitive to KCN (1 mM) during the period ofdevelopment but changed to CLAM-sensitive and CN-resistant during preclimactericand climacteric phases, indicating that a diversion of respiratory pathways from the cy-tochrome path to the alternative path has occurred. The respiration of the red pepperfruit was CN-sensitive thoughout the whole period of fruit ripening, suggesting thatthe operation of the CN-resistant path was insignificant. Slices from climacteric apple fruits developed induced .respiration after aging, bothKCN and CLAM (1 mM) inhibited the induced respiratic considerably. However, slices from red pepper fruits showed no evidence of induced respiration after aging. Slices from climacteric apple fruits infiltrated with 3 mM CLAM before aging, reducedthe peak of the induced respiration by about 30%, indicating that the development ofinduced respiration was suppressed by the presence of CLAM. The above results indicated that the: climacteric fruits were characterized by diversion of traffic from the cytochrome path to the alternative path during ripening andby the development of induced respiration after slicing and aging. While in nonclimacteric fruits no .diversion of electron transport path was observed during ripening andno induced respiration occurred after aging. Although both the eytochrome and alternative pathways were present in the tissue of red pepper fruits, the alternative pathwas not operating except when the cytochrome path was blocked or was saturated by electron flow.     

Terminal oxidases of Crithidia oncopelti

Abstract Washed cell suspensions of Crithidia oncopelti oxidizing a variety of substrates gave complex plots for the inhibition of respiration by potassium cyanide or azide. The data indicated the presence of at least two and possibly three terminal oxidases on the basis of their differential sensitivity to these inhibitors. The oxidase most sensitive to cyanide, azide and CO accounted for approx. 65–70% of whole cell respiration and is probably cytochrome oxidase a/a₃. A second oxidase exhibiting low affinity for CO required high concentrations of KCN or azide for inhibition. This haemoprotein had the spectral characteristics of cytochrome o and accounted for 15–20% of cell respiration. Incomplete inhibition of respiration by high concentrations of KCN or azide suggested the presence of a third oxidase which was CO-unreactive.     

Energy coupling in lysolecithin-treated submitochondrial particles.

Mitochondria isolated from mung bean hypocotyls, possessing a significant level of cyanide and antimycin A — resistant respiration $\text{via}$ an alternate pathway, were assayed for hydrogen peroxide production by yeast cytochrome $\text{c}$ peroxidase compound II formation. Rates of antimycin A — insensitive hydrogen peroxide production of 0.7–3 nmol/mg/min were observed which were too low to account for the observed oxygen consumption $\text{via}$ the alternate pathway. However, further investigations revealed the presence of significant levels of catalase, peroxidase and hydrogen donor to peroxidase, even in gradient purified mitochondria and these could easily utilize any hydrogen peroxide produced by the alternate pathway. Similar experiments performed upon submitochondrial particles demonstrated a rate of H₂O₂ production which could easily account for the net electron flux through the alternate pathway. From these results, we postulate that the alternate pathway reduces oxygen only partially to hydrogen peroxide, and that the peroxidase and catalase activities of the mitochondria prevent its accumulation.     

Respiration of barley roots: Assessment of activity of the alternative path using SHAM

The activity of the alternative path of O₂ consumption in detached and intact roots of barley [ Hordeum distichum (L.) Lam. cv. Maris Mink] was determined by titration with salicylhydroxamic acid (SHAM) in the presence and absence of cyanide. In the absence of cyanide, only high concentrations were inhibititory (> 5 m M ). whilst in its presence low SHAM concentrations (2.5–5.0 m M ) gave maximum inhibition: the resulting ϱ V_alt plots were non-linear. A SHAM-stimulated peroxidase could readily be washed from these roots, but non-linearity cannot be explained in terms of SHAM-stimulation of this peroxidase as it is not active in the absence of an exogenous supply of NADH. In detached roots the degree of inhibition of respiration with 25 m M SHAM was nearly double the capacity of the alternative path (measured as the degree of inhibition by SHAM in the presence of cyanide), suggesting non-specific inhibition. Effects of SHAM on cytochrome path activity in intact roots were examined by reverse titration with cyanide in the presence and absence of SHAM. At 5 m M SHAM had no effect on the cytochrome path, but at 25 m M it inhibited. We conclude that the only factor causing non-linearity of ϱV_alt plots in barley roots is non-specific inhibition of the cytochrome path by high concentrations of SHAM; consequently only low concentrations of SHAM (2.5–5.0 m M ) are suitable for estimating alternative path activity in barley roots.