A K +‐stimulated ATPase from suspension‐cultured rose cells was isolated and subjected to UV radiation. The characteristics of the ATPase resembled those of a plasma‐membrane associated enzyme and not those of the mitochondrial enzyme. The ATPase required Mg2+ and was further stimulated up to 100% by K+. K+ stimulation was specific for ATP. The order of stimulation by monovalent cations was K+ > Na+ > Li+. The enzyme had a pH optimum of 6.5 in the presence of 50 mM K+. It was almost completely inhibited by diethylstilbestrol and partially inhibited by vanadate. but was not affected by azide or oligomycin. The inhibition of ATPase activity by various fluences of UV indicated that one fraction of the K+‐stimulated activity was very sensitive to radiation, while another fraction was relatively insensitive. It is possible that UV distinguished between two enzymes. The action spectra for inhibition of both fractions showed maxima at 290 nm and significant but much lower action throughout the near‐UV region, resembling spectra in the literature for the inhibition of transport processes in bacteria.

The KV-stimulated ATPase was partially purified from a plasma membrane fraction of suspension cultured cells of rose (Rosa damascena) by two different solubilization procedures. Solubilization with 30 mm octyl-,6-n-glucopyranoside followed by precipitation with ammonium sulfate increased the specific activity of the enzyme about 6-fold. Solubilization with 1% cholate removed all but 1% of the phospholipids and resulted in an almost total loss of ATPase activity. The subsequent addition of polar lipids restored >90% of the ATPase activity with a doubling in specific activity. Fractionation of the cholate-solubilized ATPase activity on a Sephadex G-150 column resulted in 88% of the ATPase activity being recovered in two discrete, approximately equal peaks. Both ATPase activities were similar to plasma membrane ATPase activities in pH optimum, substrate specificity, ion stimulation, and inhibitor sensitivity. Assays of marker enzymes for Golgi apparatus, endoplasmic reticulum, and mitochondria revealed only a low contamination (<7%) from other membranes in the plasma membrane-enriched preparations. Lacking an unequivocal marker for the tonoplast, intact vacuoles were isolated, and their membrane density and ATPase activity were characterized and shown not to correspond to those of the putative plasma membrane preparation. These results suggest that there are two forms of ATPase separable by size in the plasma membrane of rose.The probable role of ATPases in a variety of energy transducing processes has long been recognized. It is now widely accepted that certain membrane-associated ATPases, such as the Na+,K+-stimulated, ouabain-sensitive Mg-ATPase in a variety of animal cells and the Ca2+-dependent Mg-ATPase in the sarcoplasmic reticulum are directly involved in the active transport of cations. In bacteria, membrane-bound ATPases are considered to be biological energy transducers which can utilize the energy ofATP hydrolysis to generate proton gradients and/or membrane potentials by effecting the net separation of H+ and OH-across the membrane in which they reside.Monovalent cation-stimulated ATPases are associated with membranes from many species of higher plants (10), and it is probable that they function in the transport of ions across the plasma membrane (10,11,24,25). However, the precise transport properties of the plasma membrane ATPase have not been clearly determined.If the ATPase were solubilized with detergents, it might be possible to purify it, reconstitute it, and determine both its structure and its transport properties. Despite rapid advances in purification and characterization of membrane transport pro- teins from biological membranes (9,14,20), there is no example of a purified, well characterized transport protein from the plasma membrane of higher plants. However, there are several reports of the purification of a supposed transport ATPase (1, 23) and a variety of solubilization techniques (5, 6, 22).Here, we describe the development of a procedure for obtaining two distinct, partially...

The sensitivity of Rosa damascena cultured cells to chlorate was measured by plating samples of suspensions in agar containing NaCIO3. This sensitivity depended on the age of the cultures that were plated. Chlorateresistant colonies isolated from 5-to 7-day cultures retained their resistance through many generations of growth in medium lacking NaCIO3; they also retained resistance when mixed with sensitive cells. Treating cell aggregates with ultraviolet (UV) light (254 nanometers), or UV light (360 nanometers) in the presence of 4'-methoxymethyltrioxsalen, increased the proportion that was resistant to NaCIO3. However, the amount of increase was low (three times) and required very specific doses of UV light. The UV treatments did not select for chlorate-resistant cells over chlorate-sensitive cells. The data suggested that UV had induced mutations leading to chlorate resistance. Approximately 15% of the resistant strains did not grow on medium containing nitrate as the sole nitrogen source. These strains lacked ability to reduce chlorate to chlorite. This observation supports the current idea that chlorate toxicity depends on the activity of nitrate reductase. Approximately 85% of the resistant strains grew on medium containing nitrate as the sole nitrogen source. These strains lost catalase activity following chlorate treatment, indicating that they took up and reduced chlorate. These strains have a mechanism for tolerating chlorate and its reduction products, rather than avoiding them.In tobacco, Muller and Grafe (11) isolated nine strains on the basis ofchlorate resistance. Seven ofthese strains lacked detectable nitrate reductase and did not grow on medium containing nitrate as the sole nitrogen source. Two other strains had very little nitrate reductase and grew very poorly on nitrate medium. It seems that the presence of nitrate reductase, which presumably reduces chlorate to chlorite, is necessary for the full toxic effect of chlorate to be expressed.If strains of plant cells that possess both nitrate reductase and chlorate resistance were located, they might provide clues to further steps in the mechanism by which chlorate kills normal cells. Cove (3) discovered a group of chlorate-resistant mutants of A. nidulans that could utilize NO3; at least three separate genes were involved, but these mutants were very rare. One group of chlorate-resistant mutants of N. muscorum (17) retained nitrate reductase activity (inferred, not measured) and lacked nitrogenase activity. Here we report that when strains of Rosa damascena cultured cells are selected for chlorate resistance, only a minor fraction lacks the ability to grow on medium containing nitrate as the sole nitrogen source and probably lacks the ability to reduce chlorate to chlorite; the major fraction retains the ability to grow on nitrate medium and possesses the ability to transform chlorate to toxic products. MATERIALS AND METHODSThe lethal effect of NaClO3 on higher plants has long been recognized, but the mechanism of chlorate toxicity rema...

The photochemistry of vesicular and detergent-solubilized preparations of plasma membrane-associated ATPase was investigated in Rosa damascena. The cholate-solubilized ATPase activity fractionated into two peaks on a Sephadex G-150 column with simple, but different ultraviolet (UV) sensitivities. The larger enzyme was UV sensitive; the smaller enzyme was relatively insensitive. The activity of both ATPase fractions depended on environment: both were inactive in cholate, relatively inactive in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and active in phosphatidylglycerol and phosphatidylserine. The UV sensitivities of both fractions also depended on their environment. For the UV sensitive fraction, the action spectrum differed in the 300 to 400 nanometers range when the fraction was irradiated with and without lipids. For the resistant fraction, UV sensitivity at 290 nanometers differed (up to 6-fold) in different lipids. The resistant fraction solubilized in octylglucoside had an action spectrum very different from that in cholate or in lipid vesicles. The absorption spectra of the different preparations reflected the action spectra. For both UV sensitive and insensitive fractions, the action spectra for photoinactivation had peaks at 290 nanometers, suggesting that the chromophores were tryptophanyl residues. The loss of ATPase activity was strictly correlated with the loss of fluorescence from tryptophan in the partially purified enzymes. Cs' protected the UV sensitive activity but not the insensitive one. We propose a model which explains the difference in UV sensitivities based on the positions of the tryptophan residues in the two proteins.The inactivation of a Mg2"-requiring, K+- MATERIALS AND METHODSPlant materials used and procedures for detergent solubilization and gel filtration, as well as ATPase and protein assays used, were described previously (9). Polar Lipid Dispersion. PC,2 PE, and PI from soybean, PG from egg lecithin, and PS from bovine brain, all 99.9% purity, were obtained from Sigma Chemical Company. A mixture of lipids (approximately 50% PC from soybean) was also obtained from Sigma. The lipids were dispersed as described before (9)

UV radiation at 290 and 365 nm inactivates two forms of the K+‐stimulated ATPase associated with the plasma membrane of suspension‐cultured cells of Rosa damascena. One form is 15 and 36 times more sensitive than the other to 290 and 365 nm, respectively. For both forms, the inactivation requires oxygen, is inhibited by azide and diazobicyclo(2.2.2.2)octane, but not glycerol, and is enhanced up to 7.5 times in deuterium oxide solvent. Inactivation occurs concomitantly with loss of absorbance at 290 nm. Cs+ and NO−3, quenchers of tryptophan fluorescence, inhibit inactivation. The results suggest that inactivation involves singlet‐oxygen mediated destruction of tryptophans in the ATPases.