Inhibition of the K⁺-Stimulated ATPase of the Plasmalemma of Pinto Bean Leaves by Ozone

Abstract

MATERIALS AND METHODSThree varieties of Phaseolus vulgaris which differ in their sensitivity to ozone were examined for changes in some physiological and structural plasma membrane characteristics. Plasma membrane vesicles were prepared from control and ozone-treated (0.2 to 0.5 microliters per liter ozone for 5 hours) leaf tissue, and the (K' + Mg2')-ATPase activity determined and compared. No major changes were observed in the resistant varieties. The sensitive variety showed a severe inhibition of ATPase activity which was largely due to a decrease in the K-stimulated component. This inhibition was completely reversed by the addition of sulfhydryl compounds.Ozone-induced plasma membrane permeability changes may be effected by damage to membrane proteins, perhaps by oxidation of amino acid sulfhydryl groups to disulfide and sulfenic moieties. (15). Ozonation was carried out as described previously (13).Preparation of Cell Fractions. Plasma membrane fractions were prepared from leaf tissue according to slightly modified procedures described elsewhere for root tissue (5,10). Briefly, 10 g of leaf tissue was homogenized for 5 s in 150 ml of isolation medium (50 mM Tris-Mes (pH 6.8), 0.3 M sucrose, 3 mM EDTA, 3 mM DTE, and 1% BSA) in a Polytron. The resulting brei was centrifuged at 3,000g (3 min) and then at 13,000g (15 min) and the pellets discarded. The 13,000g supernatant was then centrifuged at 80,000g (45 min) and the resultant pellet resuspended in 1 ml of isolation medium and placed on top of a 20-33-38% discontinuous sucrose gradient and centrifuged at 80,000g for 2 h. The milky band which appeared at the 33% to 38% sucrose interface was removed. Previous studies have shown this fraction to be enriched in plasma membrane vesicles (5). At all stages of the isolation procedure, the cell fractions were kept at 2°C. All fractions were kept in liquid nitrogen until required.To show that we had a good ATPase preparation from the plasmalemma, we examined the effect of pH and inhibitors on the ATPase activity of our fraction (Fig. 1). This fraction showed maximal stimulation at pH 6.0 to 6.5; increasing or decreasing pH produced a severe inhibition of activity. Mitochondrial and acid phosphatase inhibitors, azide and molybdate, respectively, had little or no effect upon the ATPase activity. However, orthovanadate, a plasma membrane ATPase inhibitor (5), completely abolished the stimulation at pH 6.0, producing a relatively flat pH profile with an activity of approximately 25% of the maximum value. These results are similar to those reported elsewhere for corn plasma membranes (5,13

Effect of SO2 and O3 on Production of Antioxidants in Conifers

“…Exposure of fir and spruce to a combination of both gases gave a higher response of the antioxidants than exposure to each pollutant alone. But, because of the low potency of 03, the pollutant seems not to be the dominator in 03 + S02 action as recently proposed by Olszyk and Tingey (12 (3). In our studies, increase of antioxidants was a general response to air pollutant action.…”

Effects of low ozone exposure of spring wheat on net CO2 uptake, Rubisco, leaf senescence and grain filling

“…Tingey & Taylor (1982) reported that SH concentration of crude foliar extracts of spinach (Spinacia oleracea), bean (Phaseolus vulgaris) and tobacco (Nicotiana tabacum) became smaller after exposure to O $ by the formation of disulphide bonds. In a sensitive variety of common bean, Dominy & Heath (1985) observed severe ozone-induced inhibition of plasmalemma ATPase activity, which could be reversed by treatment with sulphydryl compounds. They concluded that the inactivation was caused by the oxidation of a protein sulphydryl to a disulphide bridge and that sulphydryls can repair ozone-damaged plasmalemma proteins.…”

Ozone inhibits guard cell K + channels implicated in stomatal opening

“…There is indirect evidence that O 3 affects the plant plasma membrane because altered membrane permeability and ion leakage are frequently detected in leaves and in the unicellular alga Chlorella sorokiniana after treatment with the gas (11). Little research has been performed on the underlying mechanisms, although two studies have demonstrated that membrane vesicles isolated from leaves of O 3 -treated pinto bean (Phaseolus vulgaris L. var Pinto) plants display altered Ca 2ϩ transport (12) and a decrease in K ϩ -stimulated ATPase activity (13). However, all of these studies lack specificity with regard to cell type and involvement of molecularly defined transport molecules.…”