The energy-dependent influx of Rb+ into excised roots of corn, wheat, and barley has been determined and compared to the Rb+-stimulated ATPase activity of membrane fractions obtained from root homogenates of these species. The external Rb+ concentrations studied were in the range of 1 to 50 mM. (17,19,20) in roots, it would seem that ATP could also serve as the actual energy source for transport. In green algae it has been concluded that anion transport is more closely linked to the photosynthetic electron transport process whereas cation transport is coupled to ATP formation and utilization (25,30,31).In animal tissues it has been shown directly that ATP could provide the necessary energy for Na+ transport (5, 18). It has also been shown that an ATPase is intimately involved in this ATP-driven reaction (33). The existence of ion-sensitive ATPases in plant tissues has been reported recently for several plant species (2,4,6,8,11,13,15,21,26,32); however, none of these reports has shown the enzyme to have any relationship to ion transport. We (8) other plant species which possess different rates of ion transport, and the results provide correlative evidence for a role of the ATPase (and therefore ATP) in the energy-dependent transport of Rb+ and K+. MATERIALS AND METHODSPlant Culture. Roots from 4-day-old, dark-grown wheat (Triticum vulgare, 59 X 844), corn (Zea mays, WF9 X M14), and barley (Hordeum vulgare var. Arivat) were used. The seeds were placed between layers of cheesecloth on stainless steel screens 2 cm above a 0.2 mm CaSO4 solution with gentle aeration. All plants were kept at 25 2 C during the 4-day growing period.Influx Experiments. Rubidium influx was determined with 88Rb. Roots were excised and washed three times in approximately 250 ml of distilled water. The terminal 6 cm of five roots were cut into 1.5-cm segments and incubated in 50 ml of 0.5 mM CaSO4 plus the desired concentration of RbCl for 30 min. Solutions were maintained at 30 C during the absorption period and gentle aeration was provided. The experiments were terminated by rapid filtration on Buchner funnels. The root segments were rinsed for 30 sec with approximately 10 ml of an ice-cold washing solution (0.5 mm CaSO4-5 mm RbCl) and then placed into ice-cold washing solution for an additional 30 min. The roots were finally rinsed with the wash solution, placed in tared stainless steel planchets and weighed. Root tissue was ashed at 500 C for 1 hr and ash was moistened with 0.25 ml of 1% Photoflo, dried, and counted for radioactivity in a gas-flow counter.ATPase Experiments. The ATPase experiments were basically as described previously (8). Roots were excised and washed three times in approximately 250 ml of deionized water. The roots were chilled in cold deionized water for 15 min prior to grinding in a mortar and pestle with 100 ml of 0.25 M sucrose, 0.003 M EDTA, 0.01 M tris, pH 7.5. The tissue was ground vigorously for 45 sec, strained through four layers of cheesecloth, and finally diluted with 200 ml of 0.25 M sucrose. ...

Homogenates of oat (Arena satira cv. Goodfield) roots contained at least five membrane-associated adenosine triphosphatase (ATPase) activities. The membrane-bound ATPases were separated on sucrose gradients and distinguished by membrane density, pH optima, sensitivity to monovalent salts, and substrate specificity.A membrane fraction sedimenting at low centrifugal force (13,000g) contained two ATPase activities at pH 9.0. One membrane ATPase was coincident with cytochrome c oxidase activity and had a density of 1.18 grams per cubic centimeter. This membrane system was identified as mitochondria. The other pH 9.0 ATPase in this fraction occurred at a density of 1.16 grams per cubic centimeter. The identity of this membrane is unknown.Three additional ATPases were in a membrane fraction sedimenting at high centrifugal forces (13,000-80,000g). One membrane ATPase coincided with NADH-cytochrome c reductase activity, had a density of about 1.09 grams per cubic centimeter, and was equally active at pH 6.0 and 9.0. A second membrane ATPase of the 13,000 to 80,000g fraction had a density of 1.13 grams per cubic centimeter and was more active at pH 9.0 than at pH 6.0. A third membrane ATPase had greater activity at pH 6.0 than at pH 9.0, and the membrane had an apparent density of 1.17 grams per cubic centimeter on the sucrose gradient. This ATPase was especially sensitive to KCI. The identity of the membranes which contain ATPases is discussed in relation to the distribution of other enzymes on the gradient.We have previously described an ATPase from roots that was associated with membranes, required Mg2+, and was stimulated by monovalent salts in general but with KCI and RbCl giving the greatest activation (8). A For the above concept to be tenable, the ATPase must be associated with either the plasma membrane or tonoplast since these membranes are the major sites of active ion transport in higher plants (20,23). Indeed, histochemical studies have shown that both the plasma membrane (11,24) and tonoplast (12,24) have ATPase activities. However, membrane systems of such structures as mitochondria, nuclei, endoplasmic reticulum, and Golgi also possess ATPase activity (12,24). Hence, in vitro assays of ATPase activity employing a crude preparation of membranes (i.e., a differentially centrifuged fraction) may measure several different ATPases. It would be desirable to know how many membrane-associated ATPases can be distinguished in cell extracts and to identify the various membrane systems containing these enzymes. This was the purpose of the investigations reported here. We show that at least five ATPase activities can be distinguished by density gradient centrifugation of membrane fractions from oat roots. One of these ATPases is associated with mitochondria, and another is associated with plasma membranes (16). The latter enzyme is especially sensitive to KCl. The membranes containing the other ATPases are unidentified. MATERIALS AND METHODSPlant Material. Oat seeds (A vena sativa cv. Goodfield) were suspend...