Improved methods for ATP analysis

Cheer, Sue; Gentile, John H.; Hegre, C. S.

doi:10.1016/0003-2697(74)90134-1

Cited by 66 publications

(18 citation statements)

References 7 publications

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“…The diluted extract can be used at once or kept frozen at -20 C for later use. In our experience, as in that of Cheer et al (6), freezing and thawing did not result in ATP losses.…”

Section: Methodssupporting

confidence: 85%

“…Background counts of each vial were measured for 0.1 min three times immediately before each assay. [5][6][7][8][9][10] ,ld sample was added to the vial, and the vial gently swirled for 10 s. Exactly 30 s after addition of the sample, counts were taken for 0.1 min consecutively three times. Two to three such assays were performed for each sample.…”

Section: Methodsmentioning

confidence: 99%

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Metabolism of Oat Leaves during Senescence

Malik¹,

Thimann²

1980

Plant Physiol.

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The ATP content of 7-day-old A vena sativa leaves during senescence in dark and in light, and after treatment with cytokinins and other reagents, has been determined by the luciferin-luciferase method. Special care was taken to avoid decomposition of the ATP, and a detailed procedure is presented for ATP analysis at the picomole level. Preliminary experiments with several inhibitors of photophosphorylation suggest, though not conclusively, that the delaying effect of light on senescence is mediated by photophosphorylation. The ATP values of the leaves senescing in darkness are found to increase in parallel with the large increase in respiratory rate, and kinetin prevents this increase just as completely as it prevents the respiratory rise. It is concluded that the respiratory increase in senescence cannot be simply due to uncoupling. In light the ATP level also rises, though more slowly, and again kinetin prevents this rise. L-Serine, which promotes dark senescence, does not significantly modify the dark ATP level, but both arginine and kinetin, which antagonize the action of serine on senescence, greatly lower the ATP level below that on serine alone. Cycloheximide has a similar effect, and the combination of cycloheximide and kinetin lowers the ATP level drastically. Fusicoccin, which opens stomata in the dark, correspondingly maintains the ATP at a low level. Thus, in general, a low level of ATP is associated with the prevention of dark senescence, ie. probably with ATP utilization, and the ATP level at any time may thus be determined more by the rate of utilization than by the efficiency of respiratory coupling.It is well understood that leaf senescence is dominated by hydrolytic processes. In the darkened oat leaf proteolysis begins within 6 h and polysaccharide breakdown soon follows (25). The increased activity of ribonuclease in leaves of several species was also an early observation (9,28,34) These and other observations could be taken to indicate some relationship between leaf senescence and the products of phosphorylation. It was not clear whether oxidative phosphorylation in the dark and photophosphorylation in the light act in the same way, nor was it at all clear just what the nature of the postulated relationship actually is. As a first step toward clarification of these difficult questions, it was felt essential to determine the levels of ATP during senescence in both darkness and light and to follow the effects on leaf ATP of some of the agents and treatments that modify the senescence process. This paper reports those determinations. A preliminary study has also been made of several inhibitors of photophosphorylation to see whether they modify senescence in light. Two of these inhibitors do in fact interfere with the light effect on senescence and thus the results give general support to the idea that phosphorylation exerts a controlling influence on senescence. MATERIALS AND METHODSA survey of the literature showed that for small samples of plant material the luciferin-luciferase meth...

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“…The diluted extract can be used at once or kept frozen at -20 C for later use. In our experience, as in that of Cheer et al (6), freezing and thawing did not result in ATP losses.…”

Section: Methodssupporting

confidence: 85%

Section: Methodsmentioning

confidence: 99%

Metabolism of Oat Leaves during Senescence

Malik¹,

Thimann²

1980

Plant Physiol.

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“…d-l. Particulate material in the experimental and control jars was analyzed for chlorophyll a (Yentsch and Menzel 1963) and adenosine triphosphate (Cheer et al 1974 …”

Section: Methodsmentioning

confidence: 99%

Containment effects in copepod grazing experiments: A plea to end the black box approach1

Roman

Rublee

1980

Limnology & Oceanography

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Containment effects from enclosing unfiltered seawater and copepods in 500-ml jars to estimate grazing rate were examined six times during 48-h incubations.Crazing rates decreased rapidly for the first 3 h (up to 50%) and then continued to decrease at slower rates. The number of bacteria increased in both control and experimental jars. Variable but significant changes were found in the size distribution of particles and concentrations of adenosine triphosphate and chlorophyll a. Ammonia concentrations were higher in jars with copepods. After 24 h, grazing rates calculated from control jars with NH4+ additions to simulate copepod excretion were about twice those calculated from unamended controls. The grazers removed 2% of the 3-5-pm particles compared to unamended controls and 17% compared to control jars plus NH4+.

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“…At specific times the segments were suction-filtered and quickly rinsed with 20 ml of ice-cold medium of the same composition. The roots were weighed and then dropped into 10.0 ml of boiling extraction medium consisting of 4 mm MgSO4 and 20 mM Tris-HCl (pH 8.0) (10). The medium and roots were boiled I min (18) ATP content of the extracts was determined by the luciferinluciferase assay (26) using a refrigerated liquid scintillation counter in the coincidence mode of operation (10,14).…”

Section: Methodsmentioning

confidence: 99%

Comparison of Reductions in Adenosine Triphosphate Content, Plasma Membrane-associated Adenosine Triphosphatase Activity, and Potassium Absorption in Oat Roots by Diethylstilbestrol

Balke¹,

Hodges²

1979

Plant Physiol.

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The possibility was investigated that diethylstilbestrol (DES) inhibits potassium absorption in oat (Avena sativa L. cv. Goodfield) roots by inhibiting mitochondrial functions in addition to inhibiting the plasma membrane ATPase. DES at 10-6 molar stimulated the mitochondrial ATPase slightly, but higher concentrations had no effect. Oxidative phosphorylation by isolated mitochondria was inhibited 50% by 2.6 x 10-5 molar DES; concentrations of 10-4 molar or greater were completely inhibitory. After a lag of about 2 minutes, 10-4 molar DES produced a linear decrease in ATP content of excised roots. After 20 minutes, the ATP content of the tissue was about 50% of the control and remained at that level after 30 minutes in DES.Comparison of changes in ATP content, plasma membrane ATPase activity, and K+ absorption rate with time in the presence of DES showed that the rapid decrease in K' absorption rate corresponded more closely with the decrease in ATPase activity than the decrease in ATP content. Total inhibition of the ATPase was calculated by multiplying together the percentage decreases in ATPase activity and ATP content. At times greater than 10 minutes this "net" ATPase activity corresponded very closely with the K+ absorption rate.These results show that DES can inhibit potassium absorption by reducing mitochondrial ATP production in addition to inhibiting the plasma membrane ATPase. However, the rapid (less than 5 minutes) inhibition of absorption is caused by direct inhibition of the ATPase rather than a reduced ATP supply because the ATP content is lowered only slightly whereas the ATPase is inhibited dramatically in that time. The relationship between plasma membrane ATPase activity and K+ absorption rate as inhibited by DES supports the hypothesis that the ATPase is involved in cation absorption by plant roots.It has been shown that DES3 inhibits K+ absorption (5) and the plasma membrane ATPase (6) of oat roots. Because K+ absorption was inhibited more than the plasma membrane ATPase, it appeared that DES inhibited other metabolic processes involved with absorption (6 We have characterized further the effects of DES on the ATPase and oxidative phosphorylation of isolated mitochondria and on the ATP content of excised roots. DES had little effect on the mitochondrial ATPase, but it did decrease oxidative phosphorylation in vitro as well as the ATP content of excised roots. When assayed under similar conditions, the decrease in K+ absorption corresponded more closely to inhibition of the plasma membrane ATPase than to the reduction in ATP content of the tissue. MATERIALS AND METHODSOat (Avena sativa L. cv. Goodfield) plants were grown as previously described (6, 13). Isolations of mitochondria and plasma membrane vesicles were also as described (4, 6, 13) using differential and discontinuous gradient centrifugation. Because mitochondria isolated by differential centrifugation could be prepared in less than 1 hr, that preparation was assayed for oxidative phosphorylation.Oxidative phosphorylation wa...

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Improved methods for ATP analysis

Cited by 66 publications

References 7 publications

Metabolism of Oat Leaves during Senescence

Metabolism of Oat Leaves during Senescence

Containment effects in copepod grazing experiments: A plea to end the black box approach1

Comparison of Reductions in Adenosine Triphosphate Content, Plasma Membrane-associated Adenosine Triphosphatase Activity, and Potassium Absorption in Oat Roots by Diethylstilbestrol

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