Correlations between Ion-stimulated Adenosine Triphosphatase Activities and Ion Influxes in Maize Roots

Two uptake-wash regimes were used to determine influxes of ⁸⁶Rbacross the plasmalemma and across an internal membrane of maizeroots. By using a variety of different conditions it was possibleto determine the magnitude of non-metabolic uptake at differentconcentrations and hence measure more accurately the magnitudeof the active influxes at each membrane. This data was usedto study possible correlations between ion influxes and theactivities of two different membrane-bound KCl-stimulated ATPasesisolated from maize roots. One of these ATPase activities wasassociated with a fraction enriched in plasmalemma and the otherwith a fraction containing a smooth internal membrane. Significant correlations were found between the influx of ⁸⁶Rbacross the plasmalemma and the plasmalemma-associated, KCl-stimulatedATPase activity and between the influxes and KCl-stimulatedATPase activities associated with an internal membrane. Thesecorrelations may be regarded as evidence for mediation of specificion influxes by ion-stimulated ATPases. However, a number ofsignificant cross-correlations could also be made (e.g. betweenthe influx across the plasmalemma and the inner membrane ATPaseactivity) which, together with problems of identification ofthe internal membrane and accurate flux measurement, make itdifficult to interpret the result unequivocally in terms ofthe above hypothesis.     

An Ion-stimulated Adenosine Triphosphatase from Bean Roots

A soluble ATP-ase from bean roots was discovered. The enzyme was assayed by measuring the release of inorganic P from ³²P-labelled ATP. The enzyme is strongly stimulated by hoth sodium and potassium ions, in the alkaline pH range. Its characteristics are compared to that of the animal membrnnal ATP-ase which is presumably involved in the transport of ions in animal tissues.     

Cation-stimulated Adenosine Triphosphatase Activity and Cation Transport in Corn Roots

ATPase activity of the plasma membrane fraction from primary roots of corn (Zea mays L. WF9 x M14) was activated by Mg(2+) and further stimulated by monovalent cations (K(+) > Rb(+) > Cs(+) > Na(+) > Li(+)). K(+)-stimulated activity required Mg(2+) and was substrate-specific. Maximum ATPase activity in the presence of Mg(2+) and K(+) was at pH 6.5 and 40 C. Calcium and lanthanum (<0.5 mm) were inhibitors of ATPase, but only in the presence of Mg(2+). Oligomycin was not an inhibitor of the plasma membrane ATPase, whereas N,N'-dicyclohexylcarbodiimide was. Activity showed a simple Michaelis-Menten saturation with increasing ATP.Mg. The major effect of K(+) in stimulating ATPase activity was on maximum velocity. The kinetic data of K(+) stimulation were complex, but similar to the kinetics of short term K(+) influx in corn roots. Both K(+)-ATPase and K(+) influx kinetics met all criteria for negative cooperativity. The results provided further support for the concept that cation transport in plants is energized by ATP, and mediated by a cation-ATPase on the plasma membrane.     

Adenosine Triphosphatase Activity in Cell-Wall Preparations and Excised Roots of Barley

Cell-wall preparations from barley roots contain about 20 percent of the ATP-ase activity of the whole homogenate. This activityis maximal near pH 7, activated by calcium and magnesium ionsand shows further activation when sodium and potassium chloridesare applied at alkaline pH values. High concentrations of sodiumchloride and ammonium sulphate are needed to elute the activityfrom the walls which suggests an ionic binding with the wallfraction. Excised root tips release inorganic phosphate fromATP with no lag phase, and this activity shows a response tovariation in substrate and magnesium concentration similar tothat of the cell-wall preparations, suggesting a surface locationof the enzyme. The properties of this hydrolytic activity arediscussed in relation to those described in other plant systemsand to animal transport ATP-ases.     

Membrane-bound Adenosine Triphosphatase Activities of Oat Roots

Homogenates of oat (Avena sativa 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.     

Plasma Membrane-associated Adenosine Triphosphatase Activity of Isolated Cortex and Stele from Corn Roots

The plasma membrane fractions from separated cortex and stele of primary roots of corn (Zea mays L. WF9 × M14) contained cation ATPase activity at similar levels but with somewhat different properties. ATPase activity from cortex was optimum at pH 6.5, showed a simple Michaelis-Menten saturation with increasing ATP·Mg, and showed complex kinetic data for K⁺ stimulation similar in character to the kinetic data for K⁺-ATPase and K⁺ influx in primary roots. The results for cortex indicate that homogenates of primary roots are dominated by membranes from cortical cells.

ATPase activity from stele was optimum at pH 6.5 and showed another maximum at pH 9. At pH 6.5, activity from stele had properties similar to that from cortex except that the kinetics of K⁺ stimulation closely approached that expected for a Michaelis-Menten enzyme. At pH 9, the enzyme activity from stele was inhibited by 5 μg/ml oligomycin, suggesting that a significant portion of the activity was of mitochondrial origin. Sucrose density gradient analysis indicated some contamination of mitochondrial membranes in the plasma membrane fraction from stele. The results for stele are consistent with the view that stelar parenchyma cells are not deficient in ion pumps.

     

Cytochemical Localization of K-stimulated Adenosine Triphosphatase Activity in Xylem Parenchyma Cells of Barley Roots

ATPase activity in xylem parenchyma cells of barley (Hordeum vulgare L.) roots was demonstrated cytochemically with a lead precipitation reaction. The methodical parameters of this cytochemical test were optimized for distinction between ATPase-specific and nonspecific precipitates. Optimum conditions were prefixation in 1% glutaraldehyde for 1 hour and incubation for 2 hours in a medium containing 2 mm each of ATP, Ca(2+), and Pb(2+) at pH 7 and 25 C. Problems of cytochemical localizations are discussed.ATPase activity occurred mainly at the plasmalemma, the endoplasmic reticulum nuclear envelope, and outer mitochondrial membranes of xylem parenchyma cells. The tonoplast of these cells showed only little ATPase activity. High K(+) concentrations stimulated ATPase activity, particularly at the plasmalemma. Diethylstilbestrol prevented the formation of ATPase-specific precipitates. The cytochemical demonstration of a K(+)-stimulated ATPase at the plasmalemma of xylem parenchyma cells is discussed in relation to the possible role of this membrane in ion transport to the vessels.     

Purification and Characterization of a Cation-stimulated Adenosine Triphosphatase from Corn Roots

A membrane-bound, monovalent cation-stimulated ATPase from Zea mays roots has been purified to a single band on sodium dodecyl sulfate gel electrophoresis. Microsomal preparations with K⁺ -stimulated ATPase activity were extracted with 1 m NaClO₄, and the solubilized enzyme was purified by chromatography on columns of n-hexyl-Sepharose, DEAE-cellulose, and Sephadex G-100 Superfine. A 500-fold purification over the activity present in the microsomes was obtained. The K⁺ -stimulated activity shows positive cooperativity with increasing KCl concentrations. The purified enzyme shows K⁺ -stimulated activity with ATP, GTP, UTP, CTP, ADP, α + β-glycerophosphate, p-nitrophenyl phosphate, and pyrophosphate as substrates. Under most conditions ATP is the best substrate. Although dicyclohexyl carbodiimide and Ca²⁺ inhibit and alkylguanidines stimulate the K⁺ -ATPase while bound to microsomes, they have no effect on the purified enzyme.     

Comparison of Reductions in Adenosine Triphosphate Content, Plasma Membrane-associated Adenosine Triphosphatase Activity, and Potassium Absorption in Oat Roots by Diethylstilbestrol

The possibility was investigated that diethylstilbestrol (DES) inhibits potassium absorption in oat (Avena sativa L. cv. Goodfield) roots by inhibiting mitochondrial functions in addition to inhibiting the plasma membrane ATPase. DES at 10⁻⁶ molar stimulated the mitochondrial ATPase slightly, but higher concentrations had no effect. Oxidative phosphorylation by isolated mitochondria was inhibited 50% by 2.6 × 10⁻⁵ molar DES; concentrations of 10⁻⁴ molar or greater were completely inhibitory. After a lag of about 2 minutes, 10⁻⁴ molar DES produced a linear decrease in ATP content of excised roots. After 20 minutes, the ATP content of the tissue was about 50% of the control and remained at that level after 30 minutes in DES.     

Further Properties of Adenosine Triphosphatase and {beta}-Glycerophosphatase from Barley Roots

Cell wall preparations from barley roots contain ATP-ase activitythat is stimulated by monovalent cations at alkaline pH values,above that obtained with calcium or magnesium ions. Sodium isthe most effective cation followed by potassium, lithium, andrubidium. Similar activation is obtained with a soluble enzymefraction and with excised root tips. ß-Glycerophosphataseshows no stimulation by calcium and sodium or potassium haveonly a small stimulatory effect. Disc electrophoresis demonstratesthe group character of ATP-ase and ß-glycerophosphataseactivities which consist of multiple forms either specific toone or other substrate or hydrolysing both.     

Effect of Age on Adenosine Triphosphatase Activity from Tobacco Chloroplasts

Adenosine triphosphatase activity of tobacco leaf chloroplasts in the dark was measured, using leaves of different age as determined by the position of the leaves along the stem. The activity of the chloroplast preparations strongly decreased with age, regardless of the addition of Mg²⁺ or Ca²⁺. Opposite effects of Mg²⁺ and Ca²⁺ on the activity of the chloroplasts were noted in experiments where different ratios of Mg²⁺/Ca²⁺ were applied. They were related to the age of the leaves, Ca²⁺ strongly stimulated the activity of the preparations from old leaves but was practically without effect in young, just expanded leaves. Mg²⁺ slightly stimulated the activity from old leaves while it invariably inhibited the hydrolytic activity of preparations from young leaves.     

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200–400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40–60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 µM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5–10 µM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.     

Dependence of Delayed Luminescence upon Adenosine Triphosphatase Activity in Chlorella

Delayed luminescence and fluorescence yield after illumination by a short flash were measured in Chlorella pyrenoidosa in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Addition of tri-N-butyl-tin (TNBT), a specific inhibitor of ATPase, drastically increases the life-time of the reduced photosystem II primary acceptor Q⁻ and decreases the intensity of delayed luminescence. This indicates a slowing of the charge recombination between the oxidized donor and reduced acceptor of photosystem II centers. No inhibition is observed in isolated chloroplasts when the membrane is permeable to ions, i.e. in the presence of Gramicidin D and KCl.     

Presence of Two Different Membrane-bound, KCl-stimulated Adenosine Triphosphatase Activities in Maize Roots

Recent publications have indicated that a KCl-stimulated ATPase from cereal roots is specifically associated with plasmalemma-enriched membrane fractions. However, in previous work we found that relatively high specific activities of this enzyme were also associated with a membrane fraction which did not contain plasmalemma. In an attempt to clarify this discrepancy, we have investigated the effect of density gradient composition upon the association of the enzyme with different membrane fractions isolated from the roots of Zea mays L. (WF9 × M14).     

Adenosine Triphosphatase Activity at the External Surface of Chicken Brain Synaptosomes

An ATP-hydrolysing activity on the external surface of intact synaptosomes from chicken forebrain has been investigated. The observed ATPase activity was not due to leakage of the intracellular ATPase activities, of artefacts resulting from breakage of the nerve endings during the incubation and isolation periods, or to possible contamination by other subcellular particles. Disruption of the synaptosomes resulted in an approximately 2.5-fold increase of the basal, Mg2+-dependent ATPase activity, suggesting that the plasma membrane was acting as permeability barrier to the substrate. ATP hydrolysis was maximal (0.8 mumol Pi/min/mg protein) at pH 8.2 in a medium containing either Mg2+ or Ca2+ ions. Ouabain (0.2 mM) and oligomycin (2 micrograms/mg protein) had no appreciable effect on this ATPase activity. Kinetic studies of the enzyme revealed an apparent Km value of ATP of approximately 4 x 10(-5) M. These data are consistent with the view that the observed ATP hydrolysis was being catalysed by an ectoenzyme, i.e., an enzyme in the plasma membrane of the nerve endings with its active site facing the external medium. The rapid hydrolysis of the released ATP is a suspected function for this ecto-ATPase.     

Analysis of the Distribution of Potassium-stimulated Adenosine Triphosphatase Activity in Soybean Root

The distribution of activity of a potassium-stimulated ATPase associated with the plasma membrane was determined in 4-day-old soybean roots. Changes in protein-based specific activity of the enzyme coincided with developmental changes in the root. Activity was low in the region of the root cap, increased to a maximum in the meristematic region, decreased to a minimum as cell elongation proceeded, and then increased as lateral root development began. Analysis of the data indicated that fluctuations in enzyme activity can be described by a three-phase system which can be approximated by a linear-linear-linear piece-wise regression curve. The need for constructing a biological model to describe plasma membrane development is suggested.     

Purification and Properties of the Plasma Membrane H-Translocating Adenosine Triphosphatase of Phaseolus mungo L. Roots

The plasma membrane ATPase of mung bean (Phaseolus mungo L.) roots has been solubilized with a two-step procedure using the anionic detergent, deoxycholate (DOC) and the zwitterionic detergent, zwittergent 3-14 as follows: (a) loosely bound membrane proteins are removed by treatment with 0.1% DOC; (b) The ATPase is solubilized with 0.1% zwittergent in the presence of 1% DOC; (c) the solubilized material is further purified by centrifugation through a glycerol gradient (45-70%). Typically, about 10% of the ATPase activity is recovered, and the specific activity increases about 11-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the peak fraction from the glycerol gradient contains three major polypeptides of M_r = 105,000, 67,000, and 57,000 daltons. The properties of the purified ATPase are essentially the same as those of membrane-bound ATPase, with respect to pH optimum, substrate specificity, inhibitor sensitivity, and ion stimulation.