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B T Storey

Publications and source records attributed to B T Storey.

At least 127 records · Page 7Linked to original sources
PubMed1971

The Respiratory Chain of Plant Mitochondria: XI. Electron Transport from Succinate to Endogenous Pyridine Nucleotide in Mung Bean Mitochondria.

Energy-linked reverse electron transport from succinate to endogenous NAD in tightly coupled mung bean (Phaseolus aureus) mitochondria may be driven by ATP if the two terminal oxidases of these mitochondria are inhibited, or may be driven by the free energy of succinate oxidation. This reaction is specific to the first site of energy conservation of the respiratory chain; it does not occur in the presence of uncoupler. If mung bean mitochondria become anaerobic during oxidation of succinate, their endogenous NAD becomes reduced in the presence of uncoupler, provided that both inorganic phosphate (P(i)) and ATP are present. No reduction occurs in the absence of P(i), even in the presence of ATP added to provide a high phosphate potential. If fluorooxaloacetate is present in the uncoupled, aerobic steady state, no reduction of endogenous NAD occurs on anaerobiosis; this compound is an inhibitor of malate dehydrogenase. This result implies that endogenous NAD is reduced by malate formed from the fumarate generated during succinate oxidation. The source of free energy is most probably the endogenous energy stores in the form of acetyl CoA, or intermediates convertible to acetyl CoA, which removes the oxaloacetate formed from malate, thus driving the reaction towards reduction of NAD.In the absence of P(i) and presence of oligomycin, oxidation of succinate by the alternative cyanide-insensitive oxidase pathway, in the presence of sulfide to inhibit cytochrome oxidase, does not reduce endogenous NAD, either in the aerobic steady state or in anaerobiosis. Under these conditions, only the reversed electron transport pathway from succinate to endogenous NAD is active and ATP cannot interact with the respiratory chain. The source of energy for NAD reduction must come from the respiratory chain, and this result shows that oxidation of succinate through the alternate pathway does not provide this energy.

Journal Article↗
PubMed1970

The respiratory chain of plant mitochondria. IV. Oxidation rates of the respiratory carriers of mung bean mitochondria in the presence of cyanide.

The half-time for oxidation of cytochrome b(557) in mitochondria from etiolated mung bean (Phaseolus aureus) hypocotyls is 5.8 milliseconds at 24 Celsius in the absence or presence of 0.3 mm KCN, when the oxidation is carried out by injecting a small amount of oxygenated medium into a suspension of mitochondria made anaerobic in the presence of succinate plus malonate. Since oxygen is consumed by the alternate, cyanide-insensitive respiratory pathway of these mitochondria, cycles of oxidation and reduction can be obtained with the oxygen pulses when cyanide is present. Reduced cytochromes (a + a(3)) also become oxidized at nearly the uninhibited rate under these conditions, a(3) completely and a partially. The half-time for oxidation of c(547) is also unaffected by 0.3 mm KCN, but c(549) has a half-time equal to that of c(547) in the presence of KCN, compared to the shorter one observed in the absence of inhibitor. The maximum extent of oxidation of the cytochromes c is about 70% in the presence of 0.3 mm KCN; this oxidation is rapidly followed by an extensive reduction which is synchronous with the reduction of cytochrome a observed under the same conditions. In the presence of cyanide, it appears likely that the cytochromes c and b(557) are oxidized by cytochrome oxidase in oxygen pulse experiments, rather than by the alternate oxidase. The oxidation of cytochrome b(553) is partially inhibited by KCN, but complete oxidation is attained in the aerobic steady state with excess oxygen. If the oxygen pulse experiment is carried out in the presence of sufficient malonate so that entry of reducing equivalents into the respiratory chain occurs at a rate negligible compared to inter-carrier electron transport, the half-time for flavoprotein oxidation is unaffected by 0.3 mm KCN while that for ubiquinone oxidation is but 2-fold larger. The observed net oxidation rate of these two carriers in mung bean mitochondria is more sensitive to the entry rate of reducing equivalents, as set by succinate concentration and malonate to succinate ratio, then it is in skunk cabbage (Symplocarpus foetidus) mitochondria. These observations are interpreted in terms of a respiratory carrier Y, placed between flavoprotein plus ubiquinone and the cytochromes, which is the fork in the split respiratory pathway to the two terminal oxidases and which has lower electron transport capacity in mung bean mitochondria than in skunk cabbage mitochondria.

Cyanides↗
PubMed1970

The respiratory chain of plant mitochondria. V. Reaction of reduced cytochromes a and a3 in mung bean mitochondria with oxygen in the presence of cyanide.

The half-times of oxidation by oxygen pulses of reduced cytochromes a and a(3) in mung bean mitochondria made anaerobic with succinate have been measured by means of a rapid mixing flow apparatus coupled to a dual wave length spectrophotometer in the presence and absence of cyanide. The absorbance changes at 438 to 455 millimicrons and 603 to 620 millimicrons are suitable for recording the time course of cytochrome a oxidation; the half-time is 2.0 milliseconds at 24 Celsius. This half-time does not change over the range 0 to 300 mum KCN, but the fraction of cytochrome a oxidized falls to a limiting value of 0.3 at the higher cyanide concentrations. The absorbance changes at 445 to 455 millimicrons record the time course of both cytochrome a and cytochrome a(3) oxidation; the former contributes 60% of the absorbance change and the latter 40%. The half-time for a(3) oxidation is calculated as 0.9 milliseconds at 24 Celsius. This half-time increases slightly to 1.3 milliseconds at 300 mum KCN. Reduced cytochrome a(3), whether uncomplexed or complexed with cyanide, becomes fully oxidized. The dissociation constant for the reduced cytochrome a(3)-cyanide complex is estimated to be 30 mum, whereas that for the oxidized a(3)-cyanide complex which inhibits electron transport is estimated to be 2 mum. This suggests two different binding sites for cyanide on the reduced and oxidized forms of cytochrome a(3). The fact that a limiting fraction of reduced cytochrome a can be oxidized at high cyanide concentrations implies that there is no interference by cyanide with electron transport from a to a(3), if cyanide remains bound to the site it occupies on reduced a(3) after this carrier becomes oxidized on reaction with molecular oxygen. Rearrangement of cyanide from this noninhibitory site to the inhibitory site occurs rapidly enough to compete with cytochrome a oxidation. The half-time for the rearrangement is calculated to be 0.9 milliseconds.

Cyanides↗
PubMed1970

The Respiratory Chain of Plant Mitochondria: VI. Flavoprotein Components of the Respiratory Chain of Mung Bean Mitochondria.

Redox changes of the flavoproteins of mung bean (Phaseolus aureus) mitochondria were measured by differential absorbance at 468 to 493 nanometers and by fluorescence emission above 500 nanometers excited at 436 nanometers. Four flavoproteins are distinguishable by the ratio of their fluorescence to absorbance changes, and by their requirement, or lack of it, for energy-linked reverse electron transport for reduction by succinate. Two flavoproteins are reduced by succinate in fully depleted mitochondria which lack the capacity for reverse electron transport. These are designated Fp(ha) and Fp(hf) and have fluorescence to absorbance ratios of 0 and 1.4, respectively. The two flavoproteins have the same half-time for oxidation, but Fp(hf) is reduced more slowly than Fp(ha) by substrate in the presence of cyanide. One flavoprotein with a fluorescence to absorbance ratio of 0 is not reduced by succinate in anaerobic, fully depleted mitochondria, but is rapidly reduced on subsequent addition of malate; it is designated Fp(m). The fourth distinguishable flavoprotein component is reducible by succinate in an energy-linked reaction, even in partially depleted mitochondria. This component has a fluorescence to absorbance ratio of 3.8 and is designated Fp(1f). In addition to these four flavoproteins reducible by substrates, there is a highly fluorescent flavin-containing component in or associated with these mitochondria, which is rapidly reduced by dithionite.

Journal Article↗
PubMed1970

The Respiratory Chain of Plant Mitochondria: VII. Kinetics of Flavoprotein Oxidation in Skunk Cabbage Mitochondria.

The oxidation kinetics of the two high potential flavo-proteins, one (Fp(hf)) fluorescent and the other (Fp(ha)) nonfluorescent, in mitochondria from skunk cabbage (Symplocarpus foetidus) spadices have been measured by combined spectrophotometry and fluorimetry. In the absence of respiratory inhibitors, both flavoproteins are oxidized at nearly the same rate with half-times between 120 and 160 milliseconds at 24 C. When slight differences in rate are observed, it is Fp(ha) which consistently has the shorter half-time. The presence of 0.3 millimolar KCN has no perceptible effect on the oxidation rate of either component. Antimycin A (2 nanomoles per milligram of protein) increases the oxidation half-time of Fp(ha) about 3-fold, but it has no effect on the oxidation half-time of Fp(hf). In contrast to these two inhibitors, m-chlorobenzhydroxamic acid-an inhibitor specific to the cyanide insensitive, alternate oxidase pathway in these mitochondria-increases the oxidation half-time of Fp(hf) 10-fold to about 2 seconds, while increasing that of Fp(ha) only some 20%. This result implies that the flavoprotein Fp(hf) mediates electron transport to the alternate oxidase from the region of the mitochondrial respiratory chain encompassing Fp(ha), ubiquinone, and the cytochromes b. The oxidation rate of cytochrome b(557) is unaffected by either m-chlorobenzhydroxamic acid or cyanide but is strongly inhibited by antimycin A. This result implies that cytochrome b(557) plays no direct role in the respiratory pathway to the alternate oxidase and is different from cytochrome b(7) found in mitochondria from the spadices of Arum maculatum.

Journal Article↗
PubMed1970

The Respiratory Chain of Plant Mitochondria: VIII. Reduction Kinetics of the Respiratory Chain Carriers of Mung Bean Mitochondria with Reduced Nicotinamide Adenine Dinucleotide.

Addition of 90 micromolar reduced nicotinamide adenine dinucleotide (NADH) in the presence of cyanide to a suspension of aerobic mung bean (Phaseolus aureus) mitochondria depleted with ADP and uncoupler gives a cycle of reduction of electron transport carriers followed by reoxidation, as NADH is oxidized to NAD(+) through the cyanide-insensitive, alternate oxidase by excess oxygen in the reaction medium. Under these conditions, cytochrome b(553) and the nonfluorescent, high potential flavoprotein Fp(ha) of the plant respiratory chain become completely reduced with half-times of 2.5 to 2.8 seconds for both components. Reoxidation of flavoprotein Fp(ha) on exhaustion of NADH is more rapid than that of cytochrome b(553). There is a lag of 1.5 seconds after NADH addition before any reduction of ubiquinone can be observed, whereas there is no lag perceptible in the reduction of flavoprotein Fp(ha) and cytochrome b(553). The half-time for ubiquinone reduction is 4.5 seconds, and the extent of reduction is 90% or greater. About 30% of cytochrome b(557) is reduced under these conditions with a half-time of 10 seconds; both cytochrome b(562) and the fluorescent, high potential flavoprotein Fp(hf) show little, if any, reduction. The two cytochromes c in these mitochondria, c(547) and c(549), are reduced in synchrony with a half-time of 0.8 second. These two components are already 60% reduced in the presence of cyanide but absence of substrate, and they become completely reduced on addition of NADH. These results indicated that reducing equivalents enter the respiratory chain from exogenous NADH at flavoprotein Fp(ha) and are rapidly transported through cytochrome b(553) to the cytochromes c; once the latter are completely reduced, reduction of ubiquinone begins. Ubiquinone appears to act as a storage pool for reducing equivalents entering the respiratory chain on the substrate side of coupling site 2. It is suggested that flavoprotein Fp(ha) and cytochrome b(553) together may act as the branching point in the plant respiratory chain from which forward electron transport can take place to oxygen through the cytochrome chain via cytochrome oxidase, or to oxygen through the alternate, cyanide-insensitive oxidase via the fluorescent, high potential flavoprotein Fp(hf).

Journal Article↗
PubMed1969

The respiratory chain of plant mitochondria. I. Electron transport between succinate and oxygen in skunk cabbage mitochondria.

The kinetics of oxidation of ubiquinone, flavoprotein, cytochrome c, and the cytochrome b complex in skunk cabbage (Symplocarpus foetidus) mitochondria made anaerobic with succinate have been measured spectrophotometrically and fluorimetrically in the absence of respiratory inhibitor and in the presence of cyanide or antimycin A. No component identifiable by these means was oxidized rapidly enough in the presence of one or the other inhibitor to qualify for the role of alternate oxidase. Cycles of oxidation and rereduction of flavoprotein and ubiquinone obtained by injecting 12 mum oxygen into the anaerobic mitochondrial suspension were kinetically indistinguishable in the presence of cyanide or antimycin A, implying that these 2 components are part of a respiratory pathway between succinate and oxygen which does not involve the cytochromes and does involve a cyanide-insensitive alternate oxidase. The cytochrome b complex shows biphasic oxidation kinetics with half times of 0.018 sec and 0.4 sec in the absence of inhibitor, which increase to 0.2 sec and 1 sec in the presence of cyanide. In the presence of antimycin A, the oxidation of the cytochrome b complex shows an induction period of 1 sec and a half-time of 3.5 sec. A split respiratory chain with 2 terminal oxidases and a branch point between the cytochromes and flavoprotein and ubiquinone is proposed for these mitochondria.

Electron Transport↗
PubMed1969

The respiratory chain of plant mitochondria. II. Oxidative phosphorylation in skunk cabbage mitochondria.

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3. Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.

Mitochondria↗
PubMed1969

The Respiratory Chain of Plant Mitochondria. III. Oxidation Rates of the Cytochromes c and b in Mung Bean Mitochondria Reduced With Succinate.

Oxidation rates of the 2 cytochromes c and the 3 cytochromes b in mitochondria from the hypocotyls of etiolated mung beans (Phaseolus aureus) have been measured with a rapid mixing flow apparatus coupled to a dual wavelength spectrophotometer. The mitochondria were depleted with uncoupler and ADP prior to starting the experiments to minimize the complications caused by energy-linked processes on electron transport. The half-time for oxidation and absorbance change at a given wavelength for the rapidly oxidized cytochromes was plotted as a function of wavelength, with 570 mmu as reference, to yield a spectrum in which absorbance maxima and half-times were correlated. The 2 cytochrome c components have oxidation half-times of 2 milliseconds for c(549) and 3 milliseconds for c(547). (The subscripts refer to the difference absorbance maxima observed for these cytochromes in reduced-minus-oxidized difference spectra obtained at -196 degrees . The maxima observed at room temperature are 550 mmu for c(547) and 552 mmu for c(549).) Cytochrome b(557) (room temperature maximum 560 mmu) was oxidized with a half-time of 8 milliseconds, while b(553) (room temperature maximum 556 mmu) was oxidized relatively slowly with a half-time of 0.5 sec. Cytochrome b(562) (room temperature maximum 565 mmu) was oxidized with half-times in the range 15 to 35 milliseconds, depending on mitochondrial preparation. It was shown that b(562) is only partially reduced by succinate in anaerobic mitochondria previously depleted with uncoupler and ADP; this cytochrome can be fully reduced by succinate in coupled mitochondria under conditions where endogenous pyridine nucleotide is also reduced. Both b(553) and b(557) in depleted mitochondria are reduced in anaerobiosis by succinate. With ascorbate plus N,N'-tetramethylphenylenediamine as substrate, b(553) is only partially reduced in anaerobiosis, while b(557) is completely reduced in depleted mitochondria. This observed difference in ease of reducibility provides another method for differentiating the 3 cytochromes b of mitochondria isolated from plant tissues.

Journal Article↗
PubMed1967

Peroxidase associated with higher plant mitochondria.

The presence of peroxidase in mitochondria from etiolated mung bean hypocotyls and skunk cabbage spadices, suggested by carbon monoxide difference spectra and the spectral response to methyl hydroperoxide, was shown to result from contamination of the isolated mitochondria by a denser particle fraction with high peroxidase activity. The mitochondria themselves have no peroxidatic activity. Analysis of the homogenate of mung bean hypocotyls revealed that over 90% of the peroxidase was soluble, as expected. Sonication of the isolated mitochondria, however, did not remove all the peroxidase from the particulate fraction, indicating that some of this enzyme is tightly bound to a membrane. These results suggest that the peroxidase in plant cells is localized in lysosomes or microbodies, most of which are ruptured on tissue homogenization to give soluble peroxidase, but some of which survive and appear as membrane bound peroxidase.

Lysosomes↗