Characterization and Solubilization of the Alternative Oxidase of <i>Sauromatum guttatum</i> Mitochondria

Elthon, Thomas E.; McIntosh, Lee

doi:10.1104/pp.82.1.1

Cited by 69 publications

(45 citation statements)

References 11 publications

(17 reference statements)

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“…The model plant systems that have been used to study the alternative oxidase are the Arum lilies (5,6,16), aged storage tissues (10), and ripening fruits (10). Recently, there has been interest in alternative pathway respiration in leaf tissue (18).…”

Section: Discussion Altemative Oxidase and Respiratory Changesmentioning

confidence: 99%

“…Spadix tissue of voodoo lilly (Sauromatum guttatum) has been an extremely useful source of plant material for the solubilization and identification of the proteins of the alternative oxidase and the production of monoclonal antibodies to them (5,6). The role of this activity as a thermogenic process during reproductive development has been established as well as its induction by salicylic acid (16).…”

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confidence: 99%

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Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Stewart¹,

Martin²,

Reding³

et al. 1990

Plant Physiol.

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Respiration rates of Zea mays L. seedling tissues grown at 30 and 140C were measured at 250C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 300C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 140C, the increase in altemative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 140C after growth at 300C for 2 days had the same altemative oxidase capacity as seedlings grown at 300C. Seedlings grown at 140C for 10 to 12 days, then transferred to 300C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondna were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 200C. At 41°C, very little altemative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 410C was reversible. After incubation at 410C, the alternative oxidase capacity measured at 250C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 410C as they did when incubated at 250C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.

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Section: Discussion Altemative Oxidase and Respiratory Changesmentioning

confidence: 99%

mentioning

confidence: 99%

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Stewart¹,

Martin²,

Reding³

et al. 1990

Plant Physiol.

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“…NEM is inhibitory, perhaps because of the large moiety that becomes bound to the enzyme. Similarly, the sulfhydryl reagent p-chloromercuribenzoic acid was reported to inhibit the Sauromatum guttatum alternative oxidase (31). Iodoacetate, on the other hand, was able to irreversibly stimulate the alternative oxidase when it reacted at this second site on the enzyme.…”

Section: ␣-Keto Acid Activation Of the Cyanide-resistant Oxidasementioning

confidence: 99%

The Reaction of the Soybean Cotyledon Mitochondrial Cyanide-resistant Oxidase with Sulfhydryl Reagents Suggests That α-Keto Acid Activation Involves the Formation of a Thiohemiacetal

Umbach

Siedow

1996

Journal of Biological Chemistry

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The cyanide-resistant alternative oxidase of plant mitochondria is known to be activated by ␣-keto acids, such as pyruvate, and by the reduction of a disulfide bond that bridges the two subunits of the enzyme homodimer. When the regulatory cysteines are oxidized, the inactivated enzyme is much less responsive to pyruvate than when these groups are reduced. When soybean cotyledon mitochondria were isolated in the presence of iodoacetate or N-ethylmaleimide, the intermolecular disulfide bond did not form and the alternative oxidase was present only as a noncovalently associated dimer. N-Ethylmaleimide inhibited alternative oxidase activity, but iodoacetate was found to stimulate activity much like pyruvate, including enhancing the enzyme's apparent affinity for reduced ubiquinone. The presence of pyruvate or iodoacetate blocked inhibition of the enzyme by N-ethylmaleimide, indicating that all three compounds acted at the same sulfhydryl group on the alternative oxidase protein. The site of pyruvate and iodoacetate action was shown to be a different sulfhydryl than that involved in the redox-active regulatory disulfide bond, because iodoacetate bound to the alternative oxidase at the activating site even when the redox-active regulatory sulfhydryls were oxidized. Given the nature of the covalent adduct formed by the reaction of iodoacetate with sulfhydryls, the activation of the alternative oxidase by ␣-keto acids appears to involve the formation of a thiohemiacetal.Plant mitochondria possess a terminal cyanide-resistant alternative oxidase in the electron transport system of the inner membrane (1, 2). This oxidase reduces oxygen to water with electrons derived directly from the ubiquinone pool. Because no protonmotive force is generated during this reaction, the alternative pathway appears to be energetically wasteful (1, 2). cDNA sequences have now been reported for the nuclear-encoded alternative oxidase from several plants, two fungi, and a protozoan (2-7).1 The mature protein contains 280 -290 amino acids and migrates as a 32-35-kDa protein on SDS-PAGE gels as revealed by immunoblotting. Hydropathy analysis has led to a model in which the alternative oxidase protein is anchored to the mitochondrial inner membrane by two membrane-spanning ␣-helices, with large hydrophilic domains approximately 100 amino acids in length flanking each membrane-spanning region and extending into the mitochondrial matrix (9). In the carboxyl-terminal hydrophilic domain, just beyond the second membrane-spanning region, is a set of conserved amino acid motifs found in the family of coupled binuclear iron proteins to which methane monooxygenase belongs (9, 10). Cross-linking studies have indicated that the plant alternative oxidase exists in the membrane as a homodimer (11).As a result of a search for the role of the alternative oxidase in plant metabolism, several regulators of its activity have been identified. The amounts of alternative oxidase protein in the membrane (e.g. Refs. 12-14) and the extent of ubiquinone pool reduction (...

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“…The general techniques used were as reported by Elthon and McIntosh (1986). Two-dimensional gels were run using pH 3 to 10 ampholytes according to the method of Barent and Elthon (1992).…”

Section: Sds-page Antibody Production Immunoblotting and Lmmunoprementioning

confidence: 99%

Purification, Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

et al. 1995

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An NADH dehydrogenase activity from red beet (Befa vulgaris 1.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays 1. 873) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein crosslinking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis(P-aminoethyl ether)-N,N'-tetraacetic acid or CaCI,, was stimulated somewhat (21 %) by flavin mononucleotide, was inhibited by pchloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containhg plataneth (61 %).In contrast to their mammalian counterparts, plant mitochondria are capable of oxidizing cytoplasmic NAD(P)H directly, coupling this oxidation to the electron transport chain (Moller and Lin, 1986). This occurs via exogenous NAD(P)H DHs located on the cytosolic face of the inner mitochondrial membrane (Palmer and Moller, 1982;Moller, 1986;Moller and Lin, 1986; Douce and Neuberger, 1989). The oxidation of endogenous mitochondrial matrix substrates (NADH and succinate) has been shown to take precedence over the oxidation of exogenous NAD(P)H (Dry et al., 1983;Day et al., 1985). Thus, it has been suggested that the exogenous NAD(P)H DHs may function in balancing the redox levels between NAD(P)H pools in the cytosol and the mitochondrion (Moller and Lin, 1986; Douce and Neuberger, 1989). As a result, the exogenous NAD(P)H DH could coordinate glycolytic flwc with flow through the Krebs cycle. If this is true, then regulation of exogenous NAD(P)H DH activity could greatly influence plant metabolism. Consistent with this hypothesis, Kromer and Heldt (1991) have shown that the oxidation by mitochondria of reducing equivalents generated during photosynthesis is vital for obtaining maximum photosynthetic rates. Exogenous NAD(P)H DH activity can be distinguished from the other mitochondrial NAD(P)H DH activities by its insensitivity to rotenone (Wilson and Hanson, 1969), stimulation by Ca2' and inhibition by EGTA (Coleman and Palmer, 1971), and sensitivity to platanetin (Ravanel et al., 1986). Although these characteristics ca...

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Characterization and Solubilization of the Alternative Oxidase of Sauromatum guttatum Mitochondria

Cited by 69 publications

References 11 publications

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

The Reaction of the Soybean Cotyledon Mitochondrial Cyanide-resistant Oxidase with Sulfhydryl Reagents Suggests That α-Keto Acid Activation Involves the Formation of a Thiohemiacetal

Purification, Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

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