Stimulation of Plasmalemma Adenosine Triphosphatase from Oat Roots by Inorganic and Organic Cations: Concentration-Dependence, Selectivity, and Sites

Abstract

K-stimulated ATPase activity of a plasmalemma-enriched fraction from excised roots of oat was triphasic in the range 5 to 80 m;limolar KCI. The phases obeyed MlchaeLs-Menten kinetics and were separated from each other by jumps or sharp breaks at about 10 and 20 nillilar. Stimulation by alkali cations was in the order K+ > Rb+ > Na+ > Cs' > Li' or in a closely related sequence. The specificity reflected differences in ,,.,, not in affinity (K.'). Stimulation by the organic cations ethanolamine and choline in the interval 11 to 80 mllmolar appeared monophasic rather than biphasic. Substitution on the quatemary nitrogen of the amino alcohols decreased their effectiveness, as did extension and brhing of the chain. Stimulation was maximal at about pH 7 both for K' and choline.The kinetics of K' stimulation are multiphasic, not cooperative, as was also found for uptake. The ATPase is also stimulated by organic cations, but the difference in kinetics indicates the existence of separate sites for stimulation and transition.Membrane-bound ATPases have been implicated in the transport of ions in plants (17,18,30). Particularly close correlations exist between a K+-stimulated ATPase and uptake of K+ across the plasmalemma (13,27,28). The role of this enzyme in ion transport remains unclear, however (25, 43).The present paper examines some unresolved questions concerning this relationship. The complex kinetics of ion uptake and ATPase stimulation have been described variously as cooperative (17,27,28) MATERIALS AND METHODS Seedlings of oat (Avena sativa L. cv. Titus) were grown in 0.5 mM CaC12 for 6 to 8 days at 16 to 22 C in the dark (40).A plasmalemma-enriched membrane fraction was isolated as described by Hodges and Leonard (19) with some minor modifications (32). The excised roots were ground with a mortar and pestle in a solution of 250 mm sucrose, 3 mm EDTA, 2.5 mm DTT, and 25 mm Tris-Mes buffer (pH 7.2). The filtered homogenate was centrifuged successively at 13,000g for 15 min and 40,000g for 30 min. The 40,000g pellet was suspended in 34% sucrose (w/w) and layered onto a discontinuous gradient consisting of 20 ml of 45% sucrose (w/w), 8 ml of 40%o sucrose, and 8 ml of 34% sucrose. All sucrose solutions contained 1 mm DTT and 1 mM Tris-Mes (pH 7.2). The gradient was centrifuged for 2 h at 80,000g in a Spinco SW 27 rotor, and the membrane fraction was collected at the 34/ 40% sucrose interface. For most experiments the membrane fraction was frozen at -18 C and used the following day (cf. Figs. 1 and 2).ATPase activity was measured in a l-ml volume containing 3 mM ATP (Tris salt, pH 6.5), 30 mm Tris-Mes (pH 6.5), 3 mM MgSO4, varying concentrations of monovalent inorganic or organic cations (as chlorides), and 10 to 25 ,tg of membrane protein.Reactions, initiated by the addition of ATP, were carried out at 38 C in a shaking water bath for 30 to 45 min, depending on the amount of protein. (ATPase activity was linear for at least 1 h, and the ATP concentrations remained essentially constant.) Pi released was de...

Multiphasic uptake of phosphate by corn roots

“…Pronounced and reproducible jutnps have also been observed in other isotherms for uptake (e.g. Bowen & Nissen 1977) ATPase stimulation by potassium (Havarstein & Nissen, 1981) and long-tertn accutnulation of nutrients (Nissen et al, 1980). The fmding of .separate uptake and transition sites (see above) can be used experitnentally to produce jutnps in a reproducible tnanner (Vange et ai, 1974a).…”

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membrane after treatment of intact tissues and cell free extracts with triacontanol

“…Conquest) is Ca-*-and Mg^*dependent ATPase activity which has been suggested to represent multiple, non-interacting catalytic proteins capable of using these substrates (Caldwell 1981, Caldwell andHaug 1980). Although the specific function of this ATPase activity has not been well established, similar ATPases from maize (Zea mays L.) (Gallagher and Leonard 1982) and oat (Avena sativa L., Havarstein and Nissen 1981) have been suggested to play a roie in transmembrane transport or maintenance of membrane potential. Extensive research using electron paramagnetic resonance spectrometry linked the biochemical activity of these ATPases to the physico-chemical state of membrane lipids.…”

Uptake of Benzyladenine by Tuber Slices of Jerusalem Artichoke (Helianthus tuberosus L.) over a Wide Concentration Range

“…Each slice was solubilized separately in 0.5 ml of BTS solubilizer (Beckman) for 3 h at 500C. Radioactivity was assayed by liquid scintillation counting.Details of the statistical analysis of uptake data have been described previously (11,22,24). …”

“…A preliminary report of these findings has appeared (17). ' Details of the statistical analysis of uptake data have been described previously (11,22,24).…”