Quinol oxidation in <i>Arum maculatum</i> mitochondria and its application to the assay, solubilisation and partial purification of the alternative oxidase

Rich, Peter R.

doi:10.1016/0014-5793(78)80412-8

Cited by 96 publications

(53 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Q-1 was prepared by Dr. D. Ward (Organic Chemistry, Adelaide University, Australia) and reduced according to Rich (1981). A11 other reagents were purchased from Sigma.…”

Section: A N D Methodsmentioning

confidence: 99%

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

et al. 1996

View full text Add to dashboard Cite

l h e claim that succinate and malate can directly stimulate the activity of the alternative oxidase i n plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1 042) was reinvestigated using sweet potato (Ipomoea batatas 1.) mitochondria. I n whole mitochondria, succinate (in the presence of malonate) and both i-and D-malate stimulated respiration via alternative oxidase in a pH-(and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both i-and D-malate, although the latter at only a low rate and only at acid pH. I n submitochondrial particle preparations with negligible malic enzyme activity, neither i-nor o-malate stimulated alternative oxidase activity. However, even i n the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither i-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. I n contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 p~ in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion.We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intramitochondrial generation of pyruvate via malic enzyme.

show abstract

“…Q-1 was prepared by Dr. D. Ward (Organic Chemistry, Adelaide University, Australia) and reduced according to Rich (1981). A11 other reagents were purchased from Sigma.…”

Section: A N D Methodsmentioning

confidence: 99%

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

et al. 1996

View full text Add to dashboard Cite

show abstract

“…The alternative oxidase preparation was found to be severely inhibited by heat treatment and trypsin digestion. This preparation showed inhibitor sensitivities (18). However, DOC has several disadvantages when used to purify proteins.…”

Section: Methodsmentioning

confidence: 99%

“…The thermogenic respiration of the spadices of these species is characteristically cyanide-insensitive. Mitochondria isolated from these spadices have in addition to the normal cyanide-sensitive Cyt oxidase, an alternative oxidase (8)(9)(10)18). The Cyt chain and the alternative oxidase share a common substrate, that being a reduced pool of the membrane lipid ubiquinone (10).…”

Section: Abstracimentioning

confidence: 99%

Characterization and Solubilization of the Alternative Oxidase of Sauromatum guttatum Mitochondria

Elthon

McIntosh²

1986

Plant Physiol.

View full text Add to dashboard Cite

ABSTRACIThe alternative oxidase activity of Sauromatum guttatum spadix mitochondria has been investigated as to its developmental expression and tissue localization. Mitochondria rich in alternative oxidase activity were found in a yellow cortex tissue present to varying degrees within the appendix, male floral, and sterile floral regions of the spadix. During a 5-day period just prior to anthesis, the alternative oxidase activity present in the appendix region was found to increase over 10-fold. On the following day when the appendix region becomes thermogenic, cytochrome oxidase activity was found to decrease by 92%, effectively forcing electron flow through the alternative oxidase. A procedure for efficient solubilization of the alternative oxidase from appendix region mitochondria was developed. The alternative oxidase thus solubilized was sensitive to heat inactivation and trypsin digestion. The activity showed inhibition characteristics expected of the alternative oxidase in that it was sensitive to salicylhydroxamic acid and 5-n-undecyl-6-hydroxy4,7-dioxobenzothiazole, but relatively insensitive to KCN and In this paper, we have further investigated the alternative oxidase activity ofthe voodoo lily. This activity has been partially characterized as to expression during development and tissue localization, which allowed for isolation of mitochondria with high and low levels of alternative path activity. We have solubilized the alternative oxidase from these mitochondria, and have significantly characterized the nature of this solubilized preparation. The data presented provide an essential basis for further work directed towards elucidation of the molecular nature of alternative oxidase activity. MATERIALS AND METHODSVoodoo lilies (Sauromatum guttatum Schott) were maintained during their vegetative phase in a glasshouse at 27 ± 4°C under long-d conditions. After the vegetative cycle, the corms were removed from the soil and kept dry until they resumed growth. When the resumed growth was floral in nature, the spadix and surrounding spathe developed quite rapidly over about 14 d. On, or a few days before anthesis, the spadix, enclosing spathe, and peduncle were cut from the corm and used for mitochondrial isolations. After flowering, the corms were replanted and vegetative growth rapidly ensued. The vegetative phase lasts for 4 to 6 months, followed by a I to 2 month dormant period before visible sexual growth resumes.Mitochondrial Isolation and Storage. Washed mitochondria were isolated from all tissues of the voodoo lily with the procedure of Schwitzguebel and Siegenthaler (19), using a mortar and pestle to disrupt the tissue. When purified mitochondria were desired, they were obtained with sucrose gradients according to Douce et al. (3). Isolated mitochondria were resuspended in a small volume of reaction medium consisting of 250 mm sucrose and 30 mm Tes (pH 7.4). The capacities ofthe Cyt and alternative paths were determined before storage of the mitochondria under liquid N2. After storage under liqui...

show abstract

“…The current model of the AOX, supported by mutagenesis studies, predicts a monotopic integral membrane protein (2,(13)(14)(15) associating with one leaflet of the lipid bilayer. Although analyses of yeast and trypanosomal enzymes have established that iron is required for activity (16,17), early investigations of either mitochondria or partially purified protein failed to reveal spectroscopic signatures of its active site (18,19). The first spectroscopic evidence for iron involvement was provided by Berthold et al (20) (20) were not successful.…”

mentioning

confidence: 99%

Three Redox States of Trypanosoma brucei Alternative Oxidase Identified by Infrared Spectroscopy and Electrochemistry

Maréchal

Kido

Kita

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Electrochemistry coupled with Fourier transform infrared (IR) spectroscopy was used to investigate the redox properties of recombinant alternative ubiquinol oxidase from Trypanosoma brucei, the organism responsible for African sleeping sickness.Stepwise reduction of the fully oxidized resting state of recombinant alternative ubiquinol oxidase revealed two distinct IR redox difference spectra. The first of these, signal 1, titrates in the reductive direction as an n ‫؍‬ 2 Nernstian component with an apparent midpoint potential of 80 mV at pH 7.0. However, reoxidation of signal 1 in the same potential range under anaerobic conditions did not occur and only began with potentials in excess of 500 mV. Reoxidation by introduction of oxygen was also unsuccessful. Signal 1 contained clear features that can be assigned to protonation of at least one carboxylate group, further perturbations of carboxylic and histidine residues, bound ubiquinone, and a negative band at 1554 cm ؊1 that might arise from a radical in the fully oxidized protein. A second distinct IR redox difference spectrum, signal 2, appeared more slowly once signal 1 had been reduced. This component could be reoxidized with potentials above 100 mV. In addition, when both signals 1 and 2 were reduced, introduction of oxygen caused rapid oxidation of both components. These data are interpreted in terms of the possible active site structure and mechanism of oxygen reduction to water.Mitochondria from many higher plants possess, in addition to the conventional cytochrome c oxidase, a second terminal oxidase that oxidizes ubiquinol (1-3). In thermogenic plants this alternative oxidase (AOX) 6 plays a key role in the release of heat for pollination purposes or for maintaining a warm environment within the flower at low ambient temperatures. In nonthermogenic plants its function is still under debate; proposed roles include maintaining tricarboxylic acid cycle turnover under high cytosolic phosphorylation potentials, defense against oxidative stress, and growth rate and energy charge homeostasis (4). AOX is also found in species of fungi, green algae, bacteria, and protozoa (5) and, more recently, in mollusks, nematodes, and chordates (6). Of particular importance, however, is its presence in pathogenic protozoa such as the blood parasite Trypanosoma brucei (7) and the intestinal parasite Cryptosporidium parvum (8, 9). T. brucei is a parasite that causes African sleeping sickness in humans and Nagana in livestock and is transmitted by the tsetse fly (7). The bloodstream forms of T. brucei appear to depend solely on its alternative oxidase (TAO) for respiration. Because the protein is absent from the mammalian host, TAO is an attractive and important chemotherapeutic target for African trypanosomiasis (7-10). In this respect it is interesting to note that ascofuranone, isolated from the pathogenic fungus Ascochyta visiae, specifically and potently inhibits the quinol oxidase activity of TAO (11) and rapidly kills the parasites. In addition, the chemotherapeutic eff...

show abstract

Quinol oxidation in Arum maculatum mitochondria and its application to the assay, solubilisation and partial purification of the alternative oxidase

Cited by 96 publications

References 14 publications

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

Characterization and Solubilization of the Alternative Oxidase of Sauromatum guttatum Mitochondria

Three Redox States of Trypanosoma brucei Alternative Oxidase Identified by Infrared Spectroscopy and Electrochemistry

Contact Info

Product

Resources

About