Techniques for the application of polarography to mitochondrial respiration

Hagihara, Bunji

doi:10.1016/0006-3002(61)90656-4

Cited by 267 publications

(32 citation statements)

References 6 publications

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“…The ratio in each cycle was calculated and is shown as the first, second, or third ratio. Respiratory activity of the mitochondrial fraction was determined at 30 C by an oxygen electrode apparatus designed by Hagihara (11). The standard sequence of additions to the cuvette was as follows: 2.8 ml of reaction medium, 0.2 ml of mitochondrial fraction, 6 …”

Section: Materiais and Methodsmentioning

confidence: 99%

Rapid Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination

Nawa

Asahi

1971

Plant Physiol.

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Rapid increases in activities and components of mitochondrial partidles isolated from cotyledons of Pisum sativum var. Alaska during the early stage of germination are described. Respiratory rate of the cotyledons inceased rapidly as hydration proceeded. A simila but sightly delayed increase in respiratory activity of the isolated mitochondrial fraction was observed. The respiratory control ratio and adenosine 5'-pyrophosphate/oxygen ratio rose during imbibition. Cytochrome oxidase and malate dehydrogenase activities in the mitochondrial fraction increased during the initial phase of imbibition. The increase seemed to precede that in respiratory activity. A significant activity of cytochrome oxidase and most of the malate dehydrogenase activity in the cotyledons were present in the postmitochondrial fraction in the case of the dry seeds. Mitochondrial protein and phospholipid also increased during imbibition, and the rise in the components seemed to concur with that in respiratory activity. The mechanism of mitochondrial development during imbibition is disced.It is well known that seed germination is accompanied by a marked increase in respiratory activity of the seeds, which seems dependent on water content of seeds (14). Electron microscopic studies have revealed that the respiration rate is linked with the organization of mitochondrial structure during germination. Bain and Mercer (1-3) demonstrated that poorly differentiated mitochondria could be resolved in pea cotyledons after the seeds passed into dormancy. The mitochondrial fraction isolated from cotyledons of dry peanut seeds contains few typical mitochondria in addition to many vesicular mitochondrial particles (9). Thus, only poor mitochondrial structure was detected in dormant cotyledon cells. However, more differentiated mitochondrial structure becomes evident as germination proceeds (1-3, 9, 13 MATERIAIS AND METHODSPlant Material. Pea seeds (Pisum sativum var. Alaska) were purchased from Watanabe Seed Co., Kogota, Miyagi, Japan. The seeds were soaked in a 1% (v/v) sodium hypochlorite solution for 10 min, washed thoroughly with tap water, and then rinsed once with deionized water. The surface-sterilized seeds were germinated in Petri dishes containing deionized water in the dark at 28 C. The cotyledons were taken at the required age, washed with water, and used as the experimental material.Preparation of Mitochondrial Fraction. Mitochondrial fractions were prepared by a modification of the method of Bonner (4). The prechilled cotyledons obtained from S g of the dry seeds were ground with a pestle and mortar with 5 g of sea sand and S ml of grinding medium. The grinding medium was composed of 10 mm potassium phosphate buffer, pH 7.2; 0.7 M mannitol; 1.0 mM EDTA; 0.1% (w/v) BSA;1 and 0.05% (w/v) cysteine. The resulting slurry was diluted with 25 ml of the grinding medium. The pH was adjusted to 7.2 with KOH. The resultant homogenate was squeezed through a double layer of gauze and centrifuged at 300g for 5 min. The precipitate was suspended ...

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Section: Materiais and Methodsmentioning

confidence: 99%

Rapid Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination

Nawa

Asahi

1971

Plant Physiol.

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“…02 consumption by liver slices 02 consumption by rat liver slices was measured polarographically (Estabrook, 1967) Mitochondrial respiration and oxidative phosphorylation Mitochondria were isolated as described by Wainio (1970) by using the following isolation solution: 70mM-sucrose (calcium-free) (Schwarz/Mann, Dickinson and Co., Orangeburg, N.Y., U.S.A.), 210mM-mannitol, 1 mM-Tris-HCl buffer, pH7.4, and 1 mM-EDTA. The washed mitochondrial pellet obtained was resuspended in an incubation medium containing 62.5 mM-sucrose (calcium-free), 185.5 mMmannitol, 10mM-KCl, 10mM-Tris-HCl buffer, pH7.4, 5mM-K2HPO4, 5mM-MgCI2 and 0.2mM-EDTA (Hagihara, 1961). Mitochondrial respiration and oxidative phosphorylation were studied by the polarographic technique in a Clark 02 electrode at 25'C (Estabrook, 1967).…”

Section: Formate Oxidation In Vivomentioning

confidence: 99%

“…described by Hagihara (1961) except that phosphate was omitted and 1 ,umol ofATP/ml was added; duplicate sets were tested in the absence and in the presence of 1 mM-dinitrophenol. Pi was measured as described by Martin & Doty (1949).…”

Section: Mg2+ and Dinitrophenol-stimulated Mitochondrial Atpase* Actimentioning

confidence: 99%

Metabolic alterations produced in the liver by chronic ethanol administration. Increased oxidative capacity

Videla

Bernstein

Israel

1973

Biochemical Journal

180

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1. Administration of ethanol (14g/day per kg) for 21-26 days to rats increases the ability of the animals to metabolize ethanol, without concomitant changes in the activities of liver alcohol dehydrogenase or catalase. 2. Liver slices from rats chronically treated with ethanol showed a significant increase (40-60%/) in the rate of 02 consumption over that of slices from control animals. The effect of uncoupling agents such as dinitrophenol and arsenate was completely lost after chronic treatment with ethanol. 3. Isolated mitochondria prepared from animals chronically treated with ethanol showed no changes in state 3 or state 4 respiration, ADP/O ratio, respiratory control ratio or in the dinitrophenol effect when succinate was used as substrate. With f-hydroxybutyrate as substrate a small but statistically significant decrease was found in the ADP/O ratio but not in the other parameters or in the dinitrophenol effect. Further, no changes in mitochondrial Mg2+-activated adenosine triphosphatase, dinitrophenol-activated adenosine triphosphatase or in the dinitrophenol-activated adenosine triphosphatase/Mg2+-activated adenosine triphosphatase ratio were found as a result of the chronic ethanol treatment. 4. Liver microsomal NADPH oxidase activity, a H202-producing system, was increased by 80-100 % by chronic ethanol treatment. Oxidation offormate to CO2 in vivo was also increased in these animals. The increase in formate metabolism could theoretically be accounted for by an increased production of H202 by the NADPH oxidase system plus formate peroxidation by catalase. However, an increased production of H202 and oxidation of ethanol by the catalase system could not account for more than 10-20% of the increased ethanol metabolism in the animals chronically treated with ethanol. 5. Results presented indicate that chronic ethanol ingestion results in a faster mitochondrial 02 consumption in situ suggesting a faster NADH reoxidation. Although only a minor change in mitochondrial coupling was observed with isolated mitochondria, the possibility of an uncoupling in the intact cell cannot be completely discarded. Regardless of the mechanism, these changes could lead to an increased metabolism of ethanol and of other endogenous substrates.Previous studies by our group (Videla & Israel, 1970) indicate that the rate of ethanol metabolism by the liver depends on the rate of mitochondrial reoxidation of the cytoplasmic NADH produced in the oxidation of ethanol. Uncoupling agents such as 2,4-dinitrophenol or arsenate were shown to increase markedly the rate of ethanol metabolism by normal rat liver slices. Dinitrophenol was also shown to be effective in increasing the rate of ethanol metabolism in vivo . However, although dinitrophenol increased the ethanol metabolism in liver slices from control animals, it was not effective in those from animals chronically treated with ethanol, in which the rate of ethanol metabolism had already been increased. This suggested that the rate ofethanol metabolism in these animals was no longer ...

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“…Mitochondria were prepared from rat liver according to common methods [9]. Mitochondrial protein was determined by the Biuret-method.…”

Section: Methodsmentioning

confidence: 99%

Properties of ADP‐ and ATP‐1‐N‐oxide in the adenine nucleotide translocation in rat liver mitochondria

Schlimme¹,

Schäfer²

1972

FEBS Letters

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Techniques for the application of polarography to mitochondrial respiration

Cited by 267 publications

References 6 publications

Rapid Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination

Rapid Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination

Metabolic alterations produced in the liver by chronic ethanol administration. Increased oxidative capacity

Properties of ADP‐ and ATP‐1‐N‐oxide in the adenine nucleotide translocation in rat liver mitochondria

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