Oligomycin effect on ion absorption by excised barley roots and on their ATP content

Jacoby, B.; Plessner, Ora E.

doi:10.1007/bf00387173

Cited by 19 publications

(13 citation statements)

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“…The inhibition increases with increasing oligomycin concentration, and the oligomycin concentration giving 50%o inhibition is about 6 (6) show that oligomycin inhibits Cl-influx more than K+ influx in excised barley roots. Lin and Hanson (7) showed that oligomycin inhibits phosphate influx in corn roots to a greater extent than can be accounted for by its effect on ATP levels, and Malone et al (8) …”

Section: Resultsmentioning

confidence: 90%

“…Disks about 0.8 mm thick and 4 mm diameter were cut from storage tissue of red beet (Beta vulgaris L.), and washed in 0.1 mm aerated CaSO4, changed daily, at 22-24 C. The washing was continued for [5][6][7] days, except where otherwise indicated, in order to allow development of Cl-transport (12 15,000g. Eight-ml samples of supernatant + 10 ml Aquasol (New England Nuclear) were counted in a scintillation counter, and then recounted for 36C1 after decay of the 42K.…”

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confidence: 99%

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ATP Levels and their Effects on Plasmalemma Influxes of Potassium Chloride in Red Beet

Petraglia¹,

Poole²

1980

Plant Physiol.

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Tissue ATP concentrations in slices of red beet increase progressively with time for up to 7 days after cutting the root. ATP The manner in which metabolic energy is supplied to ion transport processes in plant cells is not well established. It is generally agreed that cation influx can be coupled to ATP hydrolysis (13), although the manner of coupling, and the relationship of cation influx to electrogenic H+ efflux, remains in doubt. In the present study, the dependence of K+ influx on ATP concentration has been investigated in more detail. We show in another report (14) that the present relationship between influx and ATP concentration is different from that observed for the electrogenic pump activity.With regard to anion influx (13), there is some evidence for its dependence on ATP but contrary claims have also been made, especially for storage tissues (2, 11). The present study shows that in storage tissue of red beet Cl-influx shows a dependence on ATP concentration similar to that observed for K+ influx. Evidence is also presented that, in addition to its effect on ATP levels, oligomycin inhibits the Cl-pump at the plasmalemma. (1) with modifications as indicated below. One g beet disks frozen in liquid N2 were disrupted in 40 ml ice-cold 0.4 N HC104 + 1 mm EDTA disodium salt, in a Virtis homogenizer for 1 min. One-half of the homogenate was taken for isotope counting. This was decolorized with activated charcoal (2.5 ml of a 10%7b suspension in water), and centrifuged 10 min at 15,000g. Eight-ml samples of supernatant + 10 ml Aquasol (New England Nuclear) were counted in a scintillation counter, and then recounted for 36C1 after decay of the 42K. MATERIALS AND METHODSThe remainder of the tissue homogenate was taken for ATP assays after centrifugation at 10,000g for 15 min to sediment debris. Immediately before the ATP assays, 5 ml ofthe supernatant were added to 5 ml 40 mm glycylglycine buffer (pH 7.4) and the mixture was adjusted to pH 7.4 with 1 M KOH + 0.1 M KHCO3. The extract was then centrifuged at 5,000g for 5 min to remove KC104 and ATP assays were performed without delay. The luciferin-luciferase ATP assays were performed without delay. The luciferin-luciferase ATP assays were performed as described by Schram (15) with a few modifications. The content of one vial of firefly extract (Sigma FLE-50) was dissolved in 5 ml cold distilled H20. This reconstitution of the firefly extract gives a final concentration of 50 mm potassium arsenate and 20 mm MgSO4 (pH 7.4). This enzyme solution was agitated occasionally and incubated on ice for 2-5 h. When ready for use, the enzyme solution was centrifuged 10 min at 5,000g. The supernatant was either used immediately in the ATP assay or stored frozen for future use.To scintillation vials containing 700 Id glycylglycine buffer were added 100 tA of the enzyme solution prepared as described above.

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Section: Resultsmentioning

confidence: 90%

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confidence: 99%

ATP Levels and their Effects on Plasmalemma Influxes of Potassium Chloride in Red Beet

Petraglia¹,

Poole²

1980

Plant Physiol.

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“…Histochemical techniques have been used to localize ATPases at cell membranes (13,14,26,28,32), and correlations between salt-stimulated ATPase activity and ion absorption have been established (8)(9)(10). Support for the view that ATP levels govern ion absorption by roots is found in several reports (3,6,(18)(19)(20)24 (Tables III and V).…”

Section: Resultsmentioning

confidence: 92%

“…The final reaction volume was 3.0 ml and consisted of 25 [LM NADH, 25 ltM cytochrome c, 0.167 mm KCN, and 50 mm KH2PO4, pH 7.6. The reaction was run at room temperature, about 24 C. Enzyme concentration was chosen to give an optical density change of less than 0.1 unit/min (about 20 pg protein). The reaction was initiated by the addition of substrate.…”

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confidence: 99%

Increased Membrane-bound Adenosine Triphosphatase Activity Accompanying Development of Enhanced Solute Uptake in Washed Corn Root Tissue

Leonard

Hanson

1972

Plant Physiol.

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Washing of excised corn (Zea mays L., variety WF9xM14) root tissue is accompanied by an increase in (Mg2+ + K+)-stimulated adenosine triphosphatase. This is the adenosine triphosphatase described by Fisher, Hansen, and Hodges as positively correlated with ion accumulation rates. The increase in activity is confined to the microsomal fraction. A close parallel exists between increases in adenosine triphosphatase and phosphate absorption, and they respond similarly to inhibitors of RNA and protein synthesis. However, the amplitude of change is much smaller in adenosine triphosphatase. Possible reasons for this discrepancy are discussed.In the preceding paper (25) we describe the increase in absorption rates of phosphate, and of some other ions and solutes, which accompanies the washing of roots from seedlings grown under highly aerobic conditions. After an inductive lag period of about 30 min, there is a rapid development of phosphate absorption rate without significant change in respiration rate.Inhibitor studies indicate that RNA and protein synthesis are involved. However, no increase in membranes with washing could be found by electron microscopy or phospholipid analysis. Therefore, "transport" enzymes synthesized must be added to existing membranes. Since the apparent Km values for '2P and '6Rb absorption do not change, it would appear that existing carrier systems must be augmented or activated.One enzymatic system which is being explored for a possible role in ion transport is membrane-bound ATPase (1, 4, 5, 7-10, 12, 15, 17, 22, 29, 33 Incorporation and Distribution of "4C-Leucine. Root segments were incubated in 500-mg lots for 15 min in 10 ml of 0.2 mM CaCi2 containing 0.1 ,uc/ml of uniformly labeled 4C-leucine (316 mc/mmole). After being rinsed for 30 sec in ice-cold water and for 20 min in ice-cold unlabeled 1 mM leucine, the tissue was ground with a conical glass homogenizer in 0.2 M sucrose. The homogenate was successively centrifuged at 2,000g for 10 min (cell wall and nuclei), 12,000g for 20 min (mitochondria), and 80,000g for 60 min.The pellets were washed by resuspension and recentrifugation and were suspended in 0.2 M sucrose. Protein was precipitated from the supernatant, first in 50% saturated (NH4),-SO4 and finally in 8% trichloroacetic acid. Each fraction was washed by resuspension and centrifugation and finally was suspended in 0.2 M sucrose. Aliquots of the pellets, precipitates, and final supernatant were assayed for radioactivity.Protein content was determined colorimetrically (27

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“…The external Rb+ concentrations studied were in the range of 1 to 50 mM. (17,19,20) in roots, it would seem that ATP could also serve as the actual energy source for transport. In green algae it has been concluded that anion transport is more closely linked to the photosynthetic electron transport process whereas cation transport is coupled to ATP formation and utilization (25,30,31).…”

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Correlation between Ion Fluxes and Ion-stimulated Adenosine Triphosphatase Activity of Plant Roots

1970

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The energy-dependent influx of Rb+ into excised roots of corn, wheat, and barley has been determined and compared to the Rb+-stimulated ATPase activity of membrane fractions obtained from root homogenates of these species. The external Rb+ concentrations studied were in the range of 1 to 50 mM. (17,19,20) in roots, it would seem that ATP could also serve as the actual energy source for transport. In green algae it has been concluded that anion transport is more closely linked to the photosynthetic electron transport process whereas cation transport is coupled to ATP formation and utilization (25,30,31).In animal tissues it has been shown directly that ATP could provide the necessary energy for Na+ transport (5, 18). It has also been shown that an ATPase is intimately involved in this ATP-driven reaction (33). The existence of ion-sensitive ATPases in plant tissues has been reported recently for several plant species (2,4,6,8,11,13,15,21,26,32); however, none of these reports has shown the enzyme to have any relationship to ion transport. We (8) other plant species which possess different rates of ion transport, and the results provide correlative evidence for a role of the ATPase (and therefore ATP) in the energy-dependent transport of Rb+ and K+. MATERIALS AND METHODSPlant Culture. Roots from 4-day-old, dark-grown wheat (Triticum vulgare, 59 X 844), corn (Zea mays, WF9 X M14), and barley (Hordeum vulgare var. Arivat) were used. The seeds were placed between layers of cheesecloth on stainless steel screens 2 cm above a 0.2 mm CaSO4 solution with gentle aeration. All plants were kept at 25 2 C during the 4-day growing period.Influx Experiments. Rubidium influx was determined with 88Rb. Roots were excised and washed three times in approximately 250 ml of distilled water. The terminal 6 cm of five roots were cut into 1.5-cm segments and incubated in 50 ml of 0.5 mM CaSO4 plus the desired concentration of RbCl for 30 min. Solutions were maintained at 30 C during the absorption period and gentle aeration was provided. The experiments were terminated by rapid filtration on Buchner funnels. The root segments were rinsed for 30 sec with approximately 10 ml of an ice-cold washing solution (0.5 mm CaSO4-5 mm RbCl) and then placed into ice-cold washing solution for an additional 30 min. The roots were finally rinsed with the wash solution, placed in tared stainless steel planchets and weighed. Root tissue was ashed at 500 C for 1 hr and ash was moistened with 0.25 ml of 1% Photoflo, dried, and counted for radioactivity in a gas-flow counter.ATPase Experiments. The ATPase experiments were basically as described previously (8). Roots were excised and washed three times in approximately 250 ml of deionized water. The roots were chilled in cold deionized water for 15 min prior to grinding in a mortar and pestle with 100 ml of 0.25 M sucrose, 0.003 M EDTA, 0.01 M tris, pH 7.5. The tissue was ground vigorously for 45 sec, strained through four layers of cheesecloth, and finally diluted with 200 ml of 0.25 M sucrose. ...

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Oligomycin effect on ion absorption by excised barley roots and on their ATP content

Cited by 19 publications

References 20 publications

ATP Levels and their Effects on Plasmalemma Influxes of Potassium Chloride in Red Beet

ATP Levels and their Effects on Plasmalemma Influxes of Potassium Chloride in Red Beet

Increased Membrane-bound Adenosine Triphosphatase Activity Accompanying Development of Enhanced Solute Uptake in Washed Corn Root Tissue

Correlation between Ion Fluxes and Ion-stimulated Adenosine Triphosphatase Activity of Plant Roots

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