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.     

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.

     

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.     

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.     

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.     

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.     

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.     

Correlation between Ion Fluxes and Ion-stimulated Adenosine Triphosphatase Activity of Plant Roots

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. The ratio of Rb(+) influx/Rb(+)-stimulated ATPase was approximately 0.85 and was nearly constant for all the species and Rb(+) concentrations studied. The correlation coefficient for Rb(+) influx versus Rb(+)-activated ATPase was 0.94. The results support the concept that ATP is the energy source for ion transport in roots and that an ATPase participates in the energy transduction process involved in energy-dependent ion transport.     

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.     

On the Molecular Characterization of the Sodium-Potassium Transport Adenosine Triphosphatase

The phosphorylated intermediate in the (Na + K)-activated adenosine triphosphatase (Na-K ATPase) has been characterized as an L-glutamyl-γ-phosphate residue in the enzyme. This has been accomplished by digestion of the phosphorylated and nonphosphorylated forms of the enzyme with pepsin, reaction of the pepsin digests with [2,3-³H]N-(n-propyl)hydroxylmine, further digestion of the derivatized peptides with pronase in the presence of carrier L-glutamyl-γ-N-(n-propyl)hydroxamate and carrier L-aspartyl-N-(n-propyl)hydroxamate, and chromatographic purification. An increment in radioactivity migrated with authentic L-glutamyl-γ-N-(n-propyl)hydroxamate in a total of seven electrophoretic and chromatographic systems and on gel filtration. No increment in radioactivity was associated with authentic L-aspartyl-β-N-(n-propyl)hydroxamate in five out of the seven chromatographic and electrophoretic systems. At the last stage of purification the radioactivity from the phosphorylated enzyme which migrated as L-glutamyl-γ-N-(n-propyl)hydroxamate was 2½ times that from the nonphosphorylated enzyme. On the basis of these results it is concluded that the phosphorylated intermediate in the Na-K ATPase is an L-glutamyl-γ-phosphate residue. The beef brain Na-K ATPase has been solubilized with the nonionic detergent, Lubrol, and has been purified 10 times over that in the original microsomes. The soluble enzyme remains stable in the presence of ATP and either Na⁺ or K⁺. If the partially purified enzyme is electrophoresed in 3% polyacrylamide, followed by incubation with ATP, Na⁺, K⁺, and Mg⁺⁺, a single, somewhat diffuse, ATPase band, which is ouabain-sensitive is seen. Protein impurities are also seen on the gel. Gel electrophoresis, after treatment of the partially purified enzyme with phenol-acetic acid-urea, shows about 12 discrete protein bands. Studies on the site-directed alkylation of the (Na + K)-activated adenosine triphosphatase with haloacetate derivatives of cardiotonic steroids are reviewed. Efforts are now underway to specifically alkylate the cardiotonic steroid site of the Na-K ATPase with hellebrigenin 3-[2-³H]iodoacetate and to purify the subunit of the enzyme containing the cardiotonic steroid site by following radioactivity. Finally, a working model for the role of the Na-K ATPase in the coupled transport of Na and K is presented.     

Energy-Transducing Adenosine Triphosphatase from Escherichia coli: Purification, Properties, and Inhibition by Antibody

The membrane adenosine triphosphatase (E.C. 3.6.1.3) from Escherichia coli has been solubilized with Triton X-100 and purified to near homogeneity. The purified enzyme has a sedimentation coefficient of 12.9S in a sucrose gradient, corresponding to a molecular weight of about 360,000. On electrophoresis in gels containing sodium dodecyl sulfate, it dissociates into subunits with apparent molecular weights of 60,000, 56,000, 35,000, and 13,000. The purified enzyme loses activity and breaks down into subunits when stored in the cold. Guanosine 5'-triphosphate and inosine 5'-triphosphate are alternative substrates. Ca(2+) and, to a small extent, Co(2+) or Ni(2+) will substitute for Mg(2+) in the reaction. The K(m) for Mg-adenosine triphosphate of the membrane-bound enzyme is 0.23 mM, and for the pure enzyme it is 0.29 mM. Azide is a noncompetitive inhibitor of both the membrane-bound enzyme and the pure enzyme. P(i) is a noncompetitive inhibitor of the solubilized enzyme. An antibody to the purified enzyme was obtained from rabbits. The antibody inhibits the solubilized enzyme and virtually all of the adenosine triphosphate hydrolysis by membranes from cells grown aerobically or anaerobically. The antibody also inhibits the adenosine triphosphate-stimulated pyridine nucleotide transhydrogenase (E.C. 1.6.1.1) of the E. coli membrane.     

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).     

Partial Characterization of a Potassium-stimulated Adenosine Triphosphatase from the Plasma Membrane of Meristematic and Mature Soybean Root Tissue

A K⁺-stimulated adenosine triphosphatase was partially characterized in plasma membrane from meristematic and mature soybean root tissue. The substrate concentrations required for maximum enzyme activity (3 millimolar Mg·ATP) and pH optimum (6.5) were similar for both systems. Enrichment studies, performed to ensure that the membrane vesicle preparations were comparable, indicated similar purity levels at selected steps during purification. Phospholipid and sterol analyses further substantiated their similarity.     

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.     

Salt-stimulated Adenosine Triphosphatase from Smooth Microsomes of Turnip

The turnip (Brassica rapa L.) microsome fraction contains both a Mg²⁺-inhibited acid phosphatase and a salt-stimulated Mg²⁺-activated ATPase. However, as the pH optimum of the ATPase was 8.0 to 8.5, the acid phosphatase activity could be eliminated by assaying at or above pH 7.8. The ATPase was concentrated in a fraction equivalent to the smooth microsomal membranes and was not due to fragments of mitochondria. The salt-stimulated activity showed specificity for anions rather than cations. The activity was further stimulated by carbonyl cyanide m-chloro-phenylhydrazone (CCCP), 2,4-dinitrophenol, valinomycin, nigericin, and NH₄Cl. There was a synergistic effect between CCCP and valinomycin. Activity was insensitive to oligomycin phlorizin, ouabain, and atractylate. Based on similarity to the chloroplast ATPase, it was proposed that this ATPase was situated on the outside of the vesicle.     

Characterization of a NO(3)-Sensitive H-ATPase from Corn Roots

When assayed in the presence of azide, NO₃⁻ was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 × M017) root microsomal membranes. The distribution of the NO₃⁻-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H⁺-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 μm vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO₃⁻-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H⁺ from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K⁺ > NH₄⁺ > Rb⁺ > Cs⁺ > Li⁺ > Na⁺ > choline⁺) whereas the NO₃⁻-sensitive ATPase was stimulated by anions (Cl⁻ > Br⁻ > C₂H₃O₂⁻ > SO₄²⁻ > I⁻ > HCO₃⁻ > SCN⁻). Both ATPases required divalent cations. However, the order of preference for the NO₃⁻-sensitive ATPase (Mn²⁺ > Mg²⁺ > Co²⁺ > Ca²⁺ > Zn²⁺) differed from that of the vanadate-sensitive ATPase (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO₃⁻-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO₃⁻-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO₃⁻-sensitive ATPase was stimulated by gramicidin and was associated with NO₃⁻-inhibitable H⁺ transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO₃⁻-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO₃⁻ sensitivity is a better criterion by which to identify this ATPase than either Cl⁻ stimulation or H⁺ transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization.     

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.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.