Characteristics of potassium ion transport in pea cotyledon mitochondria isolated by a zonal technique

Hamman, Wendy; Spencer, Margaret Beale

doi:10.1139/o77-094

Can. J. Biochem.

1977

DOI: 10.1139/o77-094

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Characteristics of potassium ion transport in pea cotyledon mitochondria isolated by a zonal technique

Wendy Hamman¹,

Margaret Beale Spencer²

Abstract: Methodology was developed for measurement of K+ transport in pea cotyledon mitochondria isolated by zonal centrifugation. The valinomycin-mediated transport process required respiratory energy from Krebs cycle intermediates and was completely reversible in the absence of oxygen. The rate and extent of uptake were dependent on the presence of proton-conducting anions (with acetate plus phosphate being most effective), well-coupled mitochondria, and on the K+ concentration.

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“…The isolation procedure was basically that of Hamman and Spencer (7). Washed cotyledons (about 250 g) were ground with a mortar and pestle for 8 min in 300 ml of ice-cold extraction medium of the following composition: 0.5 M mannitol, 5 mM EDTA, 0.5% BSA, 0.05% cysteine, and 0.050 M Tes, with pH adjusted to 7.4 at 20 C with Tris.…”

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Cyanide-insensitive Respiration in Pea Cotyledons

James¹,

Spencer²

1979

Plant Physiol.

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Mitochondria isolated by a zonal procedure from the cotyledons of germinating peas possessed a cyanide-resistant respiration. This respiration was virtually absent in mitochondria isolated during the first 24 hours of germination but thereafter increased gradually until the 6th or 7th day of seedling development. At this time between 15 and 20% of the succinate oxidation was not inhibited by cyanide. The activity of the cyanide-resistant respiration was also determined in the absence of cyanide. Relationships among mitochondrial structure, cyanide-resistant respiration, and seedling development are discussed.The development of respiratory activity in the cotyledons of germinating peas has been studied extensively by several authors (2, 12, 14-17, 20, 23). It is now known that following imbibition, there is a rapid increase in several enzymic activities, as well as improvement in mitochondrial structure (2,15,20). There is no appreciable synthesis of new mitochondria but rather promitochondria, existing in the dry seeds, develop structurally and functionally.The development of respiratory activity in the cotyledons has been shown to be dependent on the presence of the developing axis (23). Experiments done by Young et al. (23) showed that if cotyledons were excised from the axis prior to 2 days of development, functional mitochondria failed to develop. Other work done to determine the relationship between the axis and cotyledons in germination has shown that there is an interdependence between the two, with regard to the development of several enzymic activities (3).The senescence of cotyledons has also been studied (2,14,17,20,23 were soaked in tap water for 6 h. Following this, damaged and nonimbibed seeds were discarded. The remaining seeds were planted in horticultural grade Vermiculite and were germinated at 27 C in the dark for various periods. Harvesting was done at 24-h intervals following the onset of imbibition.Isolation of Mitochondria. The isolation procedure was basically that of Hamman and Spencer (7). Washed cotyledons (about 250 g) were ground with a mortar and pestle for 8 min in 300 ml of ice-cold extraction medium of the following composition: 0.5 M mannitol, 5 mM EDTA, 0.5% BSA, 0.05% cysteine, and 0.050 M Tes, with pH adjusted to 7.4 at 20 C with Tris. The brei was filtered through one layer of Miracloth and the filtrate was then centrifuged at 700g for 7 min. The supernatant layer was edgeloaded into a Ti-XIV zonal rotor spinning at 1,500 rpm at 2 C in a Beckman Spinco L2-65B centrifuge. Then a sucrose-step gradient was loaded into the rotor. The gradient consisted of 100 ml each of 33, 37.5, and 43% (w/w) sucrose, and sufficient 48% (w/ w) sucrose to fill the rotor. The gradient also contained 0.1% (w/ w) BSA and 1% (w/w) Tes, with pH adjusted to 7.4 at 20 C with Tris. Centrifugation was for I h at 37,000 rpm. Following centrifugation, the rotor was center-unloaded by use of a 50% (w/w) sucrose solution. Protein peaks were monitored with a Pharmacia UV Duo Monitor (model 200). The p...

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confidence: 99%

Cyanide-insensitive Respiration in Pea Cotyledons

James¹,

Spencer²

1979

Plant Physiol.

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Characteristics of potassium ion transport in pea cotyledon mitochondria isolated by a zonal technique

Cited by 1 publication

References 1 publication

Cyanide-insensitive Respiration in Pea Cotyledons

Cyanide-insensitive Respiration in Pea Cotyledons

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