Ozone‐induced Membrane Permeability Changes

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

mentioning

confidence: 99%

“…ATPase activity of the plasma membrane fractions was determined on 1.0-ml volumes of assay medium (30 mm buffer, 3 (9). However, preliminary experiments indicated that when 3 mm DTE was present in the isolation solutions, ozone had no effect on ATPase activity.…”

mentioning

confidence: 99%

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

Dominy¹,

Heath²

1985

“…The culture solution contained 10 mm KCI, 1.0 mM MgSO4, 1.0 mm KH2PO4, 0.5 mM CaCl2, 5.0 mm NaNO3, and 5.0 mm Na-glutamate as well as sucrose, micronutrients, vitamins, and hormones. Cells were filtered and washed three times at 30-min intervals with a wash solution containing 1 mM CaCl2 and 0.1 mM KCl.…”

Section: Cultured Rose Cellsmentioning

confidence: 99%

“…Ion leakage as a stress response has also been shown to be caused by other factors such as herbicides (14), chilling (7), ozone (5), heavy metals (8), and certain plant pathogens (2). However, while most of these stress factors cause a generalized leakage of a variety of solutes, UV at low fluences causes a specific K+ leakage from cultured rose cells (12).…”

mentioning

confidence: 99%

Effects of Extracellular pH on UV-Induced K⁺ Efflux from Cultured Rose Cells

Huerta

Murphy²

1989

Ultraviolet (UV) light causes a specific leakage of K+ from cultured rose cells (Rosa damascena). During K efflux, there is also an increase in extracellular HC03-and acidification of the cell interior. We hypothesized that the HC03-originated from intracellular hydration of respiratory CO2 and served as a charge balancing mechanism during K+ efflux, the K+ and HC03-being cotransported out of the cell through specific channels. An alternative hypothesis which would yield similar results would be the countertransport of K+ and H+. To test these hypotheses, we studied the effect of a range of extemal pH values (pH 5-9), regulated by various methods (pH-stat, 100 millimolar Tris-Mes buffer, or CO2 partial pressure), on the UV-induced K efflux. Both UV-C (<290 nanometers) and UV-B (290-310 nanometers) induced K+ efflux with a minimum at about pH 6 to 7, and greater efflux at pH values of 5, 8, and 9. Since pH values of 8 and 9 increased instead of reduced the efflux of K+, these data are not consistent with the notion that the efflux of K+ is dependent on an influx of H+, a process that would be sensitive to extemal H+ concentration. We suggest that the effect of pH on K+ efflux may be mediated through the titration of specific K -transporting proteins or channels in the plasma membrane. Since we could not detect the presence of carbonic anhydrase activity in cell extracts, we could not use the location of this enzyme to aid in our interpretation regarding the site of hydration of CO2. Solar UV radiation can stimulate a leakage of ions from plant cells. Ion leakage as a stress response has also been shown to be caused by other factors such as herbicides (14), chilling (7), ozone (5), heavy metals (8), and certain plant pathogens (2). However, while most of these stress factors cause a generalized leakage of a variety of solutes, UV at low fluences causes a specific K+ leakage from cultured rose cells (12). Treatment of suspension-cultured tobacco cells with Pseudomonas syringae, a bacterial plant pathogen, can also cause a specific leakage of K+ (2).The mechanism by which UV induces K+ leakage is not known. At present, it is thought to involve active metabolism, since the leakage is inhibited by anaerobiosis (10), as well as by chemical inhibitors of respiration, protein synthesis, and other biochemical processes (9,12 cellular HC03-as well as an increase in pH (about 0.2-0.3 pH units). There were no comparable net fluxes of other ions. They proposed a mechanism by which the electrical charge of the K+ efflux was balanced by a concomitant efflux of HCO3-, the HCO3-presumably originating from intracellular hydration of respiratory CO2 (Fig. lA). An alternative mechanism involves a counterflux of H+ from the outside to the inside of the cell (Fig. 1 B): in respiring cells, the H+ on the outside would be supplied to a great extent by the hydration of CO2 and dissociation of H2CO3 outside the cell. The second mechanism would be consistent with the conclusions of Atkinson et al. (2) regarding the K+/H+ counterfl...

“…Previously, ozone treatment of plant material has been shown to induce changes in the transport kinetics of many substances, including water (9), sugars (21,22), and ions (6,10). Accordingly, the loss of K+ has been shown to be one of the first indications of membrane permeability changes under a variety of stresses (5,17,18,20).…”

mentioning

confidence: 99%

Ozone Alteration of Membrane Permeability in Chlorella

Heath

Frederick²

1979

(21,22), and ions (6, 10). Accordingly, the loss of K+ has been shown to be one of the first indications of membrane permeability changes under a variety of stresses (5,17,18,20).The net loss of K+ from ozone-treated Chlorella cells suspended in a Tris buffer has been measured using a cation-specific electrode (6). The electrode method measured only the net loss of K+ at a low external K+ concentration. Thus, the effect of ozone upon the K+ influx and efflux could not be studied separately under physiological conditions. The use of Tris as a buffer induces K+ loss itself, thus making the interpretation of K+ loss difficult (7). For influx measurements, I09 cells were added to a total volume of 5 ml of medium in a stirred and temperature-regulated cuvette (38 C). Label ("6RbCl, New England Nuclear) was added prior to cell addition and subsequent gassing. Either oxygen or ozone in oxygen, produced and measured as previously described (6, 11), was introduced into the agitated solution through a 50-,ul micropipette. At specific times, a 200-jul aliquot of the cell suspension was withdrawn, filtered on a Millipore filter (0.45 ,um), and washed with 5 ml of unlabeled medium. The cell sample on the filter was removed to a drying stand, bleached with I drop of Purex, and placed in a toluene-Triton cocktail for scintillation counting (14).Uptake rates were calculated from the amount of 8'Rb present in the cells at various time intervals, based upon the external specific radioactivity of Rb+ and K+.In experiments during which the initial uptake kinetics were examined, l-ml aliquots from a batch of cells (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)