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

Abstract

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...

Solubilization and Partial Purification of the Adenosine Triphosphatase from a Corn Root Plasma Membrane Fraction

“…Both the basal, Mg2+ ATPase activity and the K+-stimulated ATPase activity were affected by the detergents. Maximal activation of Triton X-100 was at 0.1% (v/v), by octyl-glucoside at 20 mm and by sodium cholate between 2 and 3% (w/w) (Figs. 1 and 2).…”

“…Measurements of membrane potentials and ion fluxes suggest that the plasma membrane contains an electrogenic, proton-pumping ATPase (26,28). K+ transport in higher plants is closely linked to ATP hydrolysis (2,26), but K+ may be directly pumped by an ATPase or indirectly driven by the electrical component of the proton motive force. It has been difficult to distinguish between these possibilities.…”

“…It has been difficult to distinguish between these possibilities. No specific inhibitors of the plasma membrane ATPase or of K+ transport in plants are known (2,19) and ATP dependent ion transport has not been demonstrated in plasma membrane vesicles isolated from higher plants (31). It appears that it will be necessary to reconstitute the K+-stimulated ATPase into impermeable lipid vesicles to determine its transport properties.…”

Effect of Vanadate, Molybdate, and Azide on Membrane-Associated ATPase and Soluble Phosphatase Activities of Corn Roots

“…DES appears to be a very effective inhibitor (1), but specificity ofDES for plasma membrane-ATPase has not been demonstrated. Also, DES inhibits oxidative phosphorylation which limits its usefulness in studies on ion transport in intact cells (1). The other inhibitors vary in effectiveness and are not selective for plasma membrane ATPase.…”

Effect of Diethylstilbestrol on Ion Fluxes in Oat Roots

“…Because the inhibition of absorption by DES is very rapid, indicating effects at the plasma membrane, DES inhibition of the plasma membrane ATPase that is associated with ion absorption across the plasma membrane of oat roots (13) has been studied in the following paper (5). The effects of DES on mitochondrial functions have been addressed in a subsequent paper (4).…”