Plant Physiol. (1973) 52, 6-12

Characterization of Plasma Membrane-associated AdenosineTriphosphatase Activity of Oat Roots'

Received for publication January , 1973

R. T. LEONARD2 AND T. K. HODGESDepartment of Botany and Plant Pathology, Purdue University, Lafayette. Intdiania 47907

ABSTRACT

ATPase activity of plasma membranes isolated from oat(Avena sativa L. cv. Goodfield) roots was activated by divalentcations (Mg2+ = Mn2+ > Zn2+ > Fe2+ > Ca+) and furtherstimulated by KCI and a variety of monovalent salts, bothinorganic and organic. The enzyme exhibited greater specific-ity for cations than anions. The presence of Mg+ was necessaryfor KCI stimulation. Ca2+ was ineffective in replacing Mg2+for activation of plasma membrane ATPase, but it did activateother membrane-bound ATPases. The pH optima for Mg2+activation and KCI stimulation of the plasma membrane ATPasewere 7.5 and 6.5, respectively.The plasma membrane ATPase showed little synergistic

effects of K+ and Na+, and it was only slightly sensitive toouahain. Oligomycin did not inhibit the ATPase, while N,N'-dicyclohexylcarbodiimide was a potent inhibitor of the enzyme.The apparent Km for Mg2+ activation (0.84 mM) of the

ATPase was about twice that of the apparent Km for ATP(0.38 mM). The effect of KCI in stimulating the enzyme was

not on the apparent Km values for ATP and Mg+ but ratheron maximum velocity. The kinetics of KCI stimulation of theplasma membrane ATPase were similar to the kinetics of 42K'influx into oat roots and neither followed the Michaelis-Menten equation but rather were best described by a singleactivity curve with continually changing kinetic parameters.These results support the concept that cation transport at theplasma membrane of root cells is coupled to a cation-activatedATPase which is functional from low (0.01 mM) to high (50.0mM) concentrations of KCI.

Based on high correlations between membrane-boundATPase activities and ion absorption rates of several plantspecies (9-11, 29), it was suggested that an ATPase mediatesenergy transfer to the ion transport system. This proposal was

strengthened by the demonstration that the monovalent ion-stimulated ATPase is associated with the plasma membrane ofoat roots (19, 20).The purified plasma membrane ATPase is specific for ATP,

requires Mg" and is further activated by monovalent ions

'This research was supported by a grant from the NationalScience Foundation (GB-31052X). Journal paper No. 5001 of thePurdue University Agricultural Experiment Station.

2Present address: Department of Plant Sciences, University ofCalifornia, Riverside, Calif. 92502.

(19, 20, 28). In this paper the enzyme is further characterizedwith respect to its requirements for ATP, Mg2+, and KCI. Thekinetics of KCl stimulation of the plasma membrane ATPase ofoat roots were virtually identical to the kinetics of K+ influxinto oat roots, both of which are best described by a singleisotherm with continually changing kinetic parameters.

MATERIALS AND METHODS

Plasma membranes were isolated from oat (Avena sativaL. cv. Goodfield) roots as previously described (19, 20). Briefly,about 50 g of 6-day-old roots were excised, washed in colddeionized water, and ground (mortar and pestle) in 200 mlof a medium consisting of 0.25 M sucrose, 0.003 M EDTA,2.5 mm dithiothreitol, and 0.025 M tris-MES, pH 7.2. Thehomogenate was strained through cheesecloth and successivelycentrifuged at 13,000g for 15 min and 80,000g for 30 min. The13,000 to 80,000g pellet was suspended in 30 ml of freshgrinding medium, pelleted. suspended in 2.5 ml of 20% (w/w)sucrose containing 1 mM MgSO4 and 1 mm tris-MES, pH 7.2,and 2 ml were layered onto a 36-ml discontinuous gradientconsisting of 28 ml of 45%4o (w/w) and 8 ml of 34% sucrosein 1 mM MgSO, and 1 mm tris-MES, pH 7.2 (Mg2+ was omittedfrom the gradient for experiments involving Mg2+ as a vari-able). The gradient was centrifuged for 2 hr at 95,000g in aSpinco SW 27 rotor. The membranes collecting at the 34 to45% sucrose interface are more than 75% plasma membranes(20).ATPase activity was measured at 38 C in a 1-ml volume

containing 3 mm ATP (tris salt) at desired pH, 33 mm tris-MESat desired pH and variable amounts of mono- and divalent ions(see tables and figure legends for exact reaction contents). Thereaction was initiated by addition of 10 to 25 ,ug of membraneprotein; Pi released was determined by the Fiske and Sub-baRow (12) procedure as previously described (19. 28). Sub-strate blanks were subtracted to calculate all enzyme activities.Proteins were estimated by the procedure of Lowry et al.(31).

RESULTS

General Properties of Plasma Membrane ATPase. ATPaseactivity of the plasma membrane fraction from oat roots wasincreased almost 3-fold bv Mg- ions (Table I). Addition of 50mM KCl with Mg2+ gave a 5-fold stimulation in activity overthat in the presence of Mg'- alone. The stimulation of enzymeactivity by KCl required Mg2+; 50 mM KCl alone has littleeffect on the ATPase (9, see also Fig. 4). The requirement forMg2+ was completely satisfied by Mn2+ and partially satisfiedby Zn2+ or Fe2+ (Table II). However, Ca2' was ineffective in re-placing Mg2' for either direct stimulation or for supporting KCIstimulation of enzyme activity (Table II). The inability of Ca2+

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PLASMA MEMBRANE-BOUND ATPase OF OAT ROOTS

Table I. Effect of Ions and Other Solutes on ATPase Activityof Plasma Membrantes from Oat Roots

Reaction mixture contained 3 mm ATP (pH 6.0), 33 mM tris-MES (pH 6.0), and other additions at concentrations indicated.

Additions ATPase

,umoles Pilmg protein hr

None 3.5350 mM KCl 4.821.5mM MgSO4 9.461.5 mM MgSO4 + 50 mM KCI 45.061.5 mM MgSO4 + 50 mM KCI + 5 g ml oli- 43.44gomycin

1.5 mM MgSO4 + 50 mM KCl + 0.01 mM 12.20DCCD

1.5 mM MgSO4 + 50 mM NaCl 40.081.5mM MgSO4 + 25 mM KCl + 25 mM NaCl 44.821.5mM MgSO4 + 25 mM KCI + 25 mM NaCl 39.46+ 0.01 mM ouabain

Table II. Divalent Ionz Specificity of Plasma Membrane ATPasefrom Oat Roots

Reaction mixture contained 3 mm ATP (pH 6.0), 33 mm tris-MES (pH 6.0), 1.5 mm divalent salt, and 50 mM KCI when added.

Divalent Ion Stimulation KCI Stimulation'

MgSO4 100 100MnSO4 97 98ZnSO4 44 78FeSO4 29 10CaSO4 6 1

1 Activity in presence of KCI and divalent salt minus activity inpresence of divalent salt alone and expressed as % of control.

0 1 2 3 0 1 2 3 0Divalent Ion (mM)

MITOCflOflOFIO

-F

--o5001-

4

CL

I w 30,2 3 E

a-

20FIG. 1. Ca2l and Mg2` stimulation of membrane-bound ATPase

of oat roots. Reaction mixture contained 3 mM ATP (pH 6.0 forplasma membrane, pH 9.0 for zone D and mitochondria), 33 mMtris-MES (pH 6.0 or 9.0) and CaSO4 or MgSO4 as indicated. Mito-chondria were isolated on sucrose gradients as described (19). ZoneD is a membrane fraction containing an unidentified membrane-bound ATPase (28).

to substitute for Mg"" in stimulating plasma membrane ATPasewas not observed for ATPases of two other membrane fractionsfrom oat roots (Fig. 1), suggesting that the plasma membranefraction was not contaminated by the other membranes.

Table III shows the effect of various K+ salts and Cl- saltson the ATPase. Thiocyanate, fluoride, and iodide are known

C- .i

0

a.

<

inhibitors of enzymes, and they did impair the activity of theATPase. The other anions, with the possible exception ofNO3-, had little effect on the ATPase activity. Cations, on theother hand, did stimulate the enzyme to varying extents. Eventhe organic cations appeared to stimulate. These data thussuggest that the ATPase is primarily a cation-activated enzymewith a definite order of specificity regarding the cations.KCI and NaCl had little synergistic effect on the plasma

membrane ATPase of oat roots (Table I and Fig. 2), and theenzyme was only slightly sensitive to ouabain (Table 1). Thus,in contrast to mammalian plasma membranes (3, 35), oat root

Table III. Monovalent Ion Specificity of Plasma MembranteA TPase from Oat Roots

Reaction mixture contained 3 mm ATP (pH 6.0), 33 mm tris-MES(pH 6.0), 1.5 mM MgSO4, and 50 mm monovalent salt.

KClKBrKNO3KNO2KHSO4K AcetateK Acid PhthalateK ThiocyanateKFKI

RbClNaClNH4ClCsClLiClTris ClCholine ClTetramethyammonium Cl

KCI 50 45 40 35 30 25 20NoCI O 5 10 15 20 25 30

Ion Concentration (mM)

Ion Stimulated ATPase

100101849310798954200

8783807842484641

I5 0 5 035 40 45 50

FIG. 2. ATPase activity of plasma membrane from oat roots atvarious KCl and NaCl concentrations. Reaction mixture contained3 mm ATP (pH 6.5), 33 mM tris-MES (pH 6.5), 3 mm MgSO4, and50 mm monovalent salt as KCl or NaCl as indicated. The verticallines connect average values obtained from two plasma membraneisolations assayed in triplicate. *: Activity in the presence of 3 mMMgSO4; 0: activities for various mixtures of KCI and NaCl.

- 3 mM MgSO4

I I:

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LEONARD AND HODGES

50-

4000E.1 300In0

0-4 0

E io

Cn

0Ifr-nI( _

5 6 7 8 9pH

FIG. 3. Effect of pH on ion-stimulated ATPase activity ofplasma membrane fraction from oat roots. Assay mixture con-tained 3 mM ATP, 33 mM tris-MES (proportions varied dependingon pH) and, when added, 1.5 mM MgSO4 and 50 mM KCl. Mg2+-stimulated activity was determined by difference in activities inpresence and absence of MgSO4. KCl-stimulated activity wascalculated by subtracting activity in presence of MgSO4 from thatin presence of MgSO4 plus KCl.

shifts reported for membrane-bound ATPases. In all sub-sequent work, pH 6.5 was employed. At this pH the KClstimulation at 50 mm is about 2-fold greater than the Mg2+-stimulated activity (see Figs. 5 and 7) which compares to the5-fold KCl stimulation observed at pH 6.0 (Table I).

Kinetic Constants for ATP and Mg2+ for Plasma MembraneATPase. To determine the apparent Michaelis constant torATP and Mg2+ and maximum velocity (Vm,1) of the plasmamembrane ATPase, enzyme activities were determined atseveral ATP and Mg2+ concentrations in the presence or ab-sence of 50 mm KCI (Fig. 4). With or without KCl, typicalsaturation kinetics for ATP were observed at 3 mm MgSO,.There was little effect of KCl on ATPase activity in the ab-sence of Mg2+ (Fig. 4).Enzyme kinetic constants for ATP were determined at a

Mg` concentration of 3 mM (the basal activity in the absenceof Mg2+ for each ATP concentration was subtracted) (Fig. 5).Lineweaver-Burk (30) plots of these data showed that the effectof KCl was not on apparent Km for ATP but rather on Vi3ax(Fig. 6). Michaelis constants were also determined at saturatingconcentrations of Mg2+ (30 mm MgSO4) and were similar tothose at 3 mm MgSO, (unreported).

Mg2+ kinetic data at 3 mm ATPase are shown in Figures7 and 8. Again, KCl had little effect on apparent Kmn for Mg2+but increased Vmax (Fig. 8). Similar results were obtained at

ATP ( mM)

FIG. 4. Effect of ATP and Mg2+ concentrations on plasma mem-brane ATPase activity of oat roots. Assay mixture contained 33mM tris-MES (pH 6.5), 50 mM KCl when added, and variable ATP(pH 6.5) and MgSO4.

plasma membranes contain virtually no (Na+ + K+)-ATPaseactivity.

Oligomycin, at a concentration that inhibited mitochondrialATPase of oat roots by 89% (unpublished results), had littleeffect on the ATPase of the plasma membrane fraction (TableI). DCCD3 strongly inhibited the plasma membrane ATPaseof oat roots (Table I) which is similar to its effect on theplasma membrane ATPase of bacterial cells (1, 15).The pH optimum for Mg'-enhancement is 7.5, whereas the

optimum in KCl stimulation is broad, lying between pH 6.0and 7.0 (Fig. 3). A high pH optimum for Mg2+-ATPase, ascompared with monovalent ion-stimulated ATPase, is a gen-eral phenomenon for both animal and bacterial cells (3). Ionicstrength-induced shifts in pH optimum occur for polyelec-trolyte-supported enzymes (13, 26) and may account for the

I Abbreviations: DCCD: N,N'-dicyclohexylcarbodiimide.

ATP (mM)

FIG. 5. Effect of ATP concentrations on ATPase activity ofplasma membranes from oat roots. Data taken from Figure 4 at 3mM MgSO,, and with activity at zero MgSO4 subtracted.

ATP (mM)

FIG. 6. Lineweaver-Burk plot of data in Figure 5. Kinetic con-stants were calculated by linear regression analysis.

8 Plant Physiol. Vol. 52, 1973

vII

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PLASMA MEMBRANE-BOUND ATPase OF OAT ROOTS

3 4Mg (mM)

FIG. 7. Effect of Mg2+ concentrations on ATPase activity ofplasma membrane fraction from oat roots. Reaction mixture con-tained 3 mM ATP (pH 6.5), 50 mM KCI when added and MgSO4as indicated.

5 mm ATP (unreported). The apparent Km for Mg` activationof the enzyme was about twice that of the apparent Km forATP (Fig. 6 and 8).

Kinetics of KCI Stimulation of Plasma Membrane ATPase.Figure 9 shows the KCl stimulation of the plasma membraneATPase at various KCl concentrations. The inset in Figure9 also shows that the enzyme is activated by low concentrationsof KCI. When these data are plotted for KCl concentrationsranging from 1 to 100 mm according to Lineweaver-Burk (30),a concave downward curve results (Fig. 10). It is apparentfrom these results that KCI activation of the enzyme doesnot obey typical Michaelis-Menten (32) kinetics. This isfurther illustrated when the data are plotted by the conventionsuggested by Eadie (6) and Hofstee (21) (Fig. 11) which permitsa greater concentration range to be evaluated (0.05-100 mM).The lack of linearity in both types of plots for KCI activationof the ATPase is not unique; many enzymes exhibit similarkinetics (24), and the significance of this will be consideredin the "Discussion."

Inasmuch as the KC1 kinetics of the ATPase are similar

Mg(mM) KCI (mM)

FIG. 8. Lineweaver-Burk plot of data in Figure 7. Kinetic con- FIG. 10. Lineweaver-Burk plot of data in Figure 9 (1-100 mMstants were calculated by linear regression analysis. KCI).

-VI..m

.0

6-

20

0,

E

a._

0

0E)

KCI (mM)FIG. 9. KCl-stimulated ATPase activity of plasma membrane fraction from oat roots at various KCI concentrations (0.5-100 mM). Inset is for

KCI concentrations from 0.01 to 0.35 mm. Reaction mixture contained 3 mm ATP (pH 6.5), 33 mm tris-MES (pH 6.5), 3.0 mm MgSO4, and KCIas indicated.

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LEONARD AND HODGES10

-

00.

C,

ot

E 2a._

00E-

00._

Plant Physiol. Vol. 52, 1973

VelocityKCI (mM)

FIG. 11. Eadie, Hofstee plot of data in Figure 9 (0.05-100 m.i KCl).

.-C

LL0'

y000E:I,

00

0

KCI (mM)

FIG. 12. 41K+ absorption by excised oat roots at various KCl concentrations. Inset is for KCl conicentrations from 0.01 to 0.35 mni. The ex-periment was conducted by J. D. Fisher (10) and the procedure is described in reference 11.

to the kinetics of ion absorption by roots (7, 27, 34), andsince it is possible that this enzyme may play a key role in ionabsorption, the kinetics of K+ absorption by oat roots is shownfor comparison. Figure 12 is a typical v versus S plot for 'Kabsorption by oat roots, and Figure 13 is an Eadie, Hofsteeplot which, as pointed out above, allows for a greater concen-tration range to be evaluated. The curved line in the Eadie,Hofstee plot indicates that the kinetics for '4K absorption byoat roots is similar to that reported for other ions and with avariety of tissues (7, 27, 34), and the similarity to the kineticsof KCI activation of the plasma membrane ATPase is striking.

DISCUSSION

The similarity in kinetics of KCI activation of the plasmamembrane ATPase and K+ absorption by roots representsanother correlation which is consistent with, but not proof of,the concept that an ATPase is responsible for the energy cou-pling involved in ion absorption bv roots (9-11. 18-20, 28, 29).

Although the ATPase described here has some similarities to"transport" ATPases of other organisms, the plant ATPaseis somewhat different. For example, mammalian transportATPase requires both K- and Na+ for activity, and it is in-hibited by ouabain (3, 35). In contrast, various combinationsof KCI and NaCl (Table I and Fig. 1) and the addition ofouabain (Table I) had little effect on the plasma membraneATPase of oat roots. This is not too surprising, however, sincethe existence and magnitude of a tightly coupled Na+- K+ trans-port in roots that is inhibited by ouabain is uncertain (4, 23).There is one report for barley roots that seems to demonstratea coupled Na+- K+ transport at the plasma membrane; how-ever, the possibility of an equally active Na+- Na+ exchangewas not ruled out, and there was little effect of ouabain on theK+-induced Nae efflux (23). Synergistic effects of Na+ andK+ in activating membrane-bound ATPase have been reportedfor halophytic plants (14, 25) and only after treating the mem-brane fraction with deoxycholate. The sensitivity of thisATPase to ouabain was not reported. The KCI- + NaCl-stim-

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11PLASMA MEMBRANE-BOUND ATPase OF OAT ROOTS

3- 20

0''oI)E

>W10 _

0

@~~~~ I

0 10 20

FIG. 13. Eadie, Hofstee plot of d

ulated ATPase activity of purified oat root plasma membranes,as isolated in the present study, is such a minor componentrelative to the single salt-stimulated activity (Fig. 2), we be-lieve it to be of little significance.The oat root ATPase described here appears to be a cation-

activated ATPase (Table III) which is similar to the transportATPase of bacterial plasma membranes (1, 3, 15). Both en-zymes are stimulated to varying degrees by monovalent cationsin the presence of Mg", show little (Na+ + K+)-activation, areinsensitive to oligomycin and are inhibited by DCCD. Thefact that the root plasma membrane ATPase is activated bya variety of monovalent cations does not diminish the potentialrole of the enzyme in transport, since roots do transport avariety of cations; however, the significance of the specificitymust await further investigations.The bacterial ATPase was implicated in transport because

K+ transport and the ATPase were inhibited by DCCD in wildtype Streptococcus faecalis, while neither K4 transport norplasma membrane ATPase were inhibited by DCCD in mutantsresistant to DCCD (1, 15). In oat roots, K+ transport was assensitive to DCCD as plasma membrane ATPase (unpublishedresults); however, mitochondrial ATPase was also inhibited bythe compound. Thus, DCCD may inhibit transport by directlyinhibiting plasma membrane ATPase or by impairing mito-chondrial ATP production.

Since oligomycin did not inhibit ATPase of oat plasma mem-branes (Table I), it probably inhibits ion transport (2, 17, 22)by inhibiting ATP formation in mitochondria (2).With respect to the kinetic studies reported here, two points

seem to be of most significance. The first is the similarity be-tween the kinetics of KCl activation of the plasma membrane-ATPase and K+ absorption by oat roots and the implicationsthis has regarding energy transduction in ion transport. Thesecond is the complex nature of the kinetics and the implica-tions of this with respect to the mechanism of KCl activationof the ATPase and the mechanism of ion absorption.

Regarding the possibility that the ATPase is the energy-transducing agent involved in ion transport, we have nowshown that the ATPase is associated with the plasma mem-brane (20), its ion-stimulated activity is highly correlated withion absorption by roots of four plant species (11), it appearsto be activated by cations (Table III), and there is considerableevidence that cation transport is driven by, ATP in plant sys-

Velocity<CI (mM)

lata in fig. 12 (0.05-50.0 mm KCl).

tems (18), and finally, as reported here, there is a similarity be-tween the kinetics of K+ activation of the plasma membrane-ATPase and K+ absorption by roots. Taken together, theseresults make a strong case for the view that cation transportacross the plasma membrane is driven by ATP and that anATPase mediates the energy coupling.The second point, the complexity of the kinetics of K+ ac-

tivation of the ATPase and K+ absorption, is not unique butit appears to be extremely important. Kinetics of this type havebeen reported for several enzymes (24) and even in thefirst application of the Michaelis-Menten analysis to ion ab-sorption data, Epstein and Hagen (8) observed this com-plexity. The kinetics of transport were partially resolved insubsequent studies and they have led to the concept that twoseparate "mechanisms" are involved in the transport of aparticular ion species; mechanism I was described as a highaffinity system and thus functioned at low external ion con-centrations (0-0.5 mM), and mechanism II was described asa low affinity system and functioned at high external ion con-centrations (0.5-50 mM). Epstein and associates (7) believethat mechanisms I and II occur in parallel at the plasma mem-brane, whereas Laties and associates (27) believe that thetwo mechanisms function in series with mechanism I residingat the plasma membrane and mechanism II residing at thetonoplast. The present results are relevant to this controversy.The absorption period for 4K by oat roots was 30 min whichis sufficiently long enough for transport to have occurredacross both the plasma membrane and tonoplast (5). Now, ifthe ATPase is involved in ion transport, and the results dis-cussed above support this interpretation, then K+ activationof this plasma membrane enzyme throughout the concentrationrange of mechanisms I and II (i.e. 0.01-100 mM) supports theconcept that energy-dependent ion transport occurs across theplasma membrane over both the low and high concentrationranges. The present studies do not, however, provide any in-formation about the properties of ion transport across thetonoplast.The concept of two distinct "mechanisms" being involved

in transport of a given ion species has recently been questionedby Nissen (34). He found that the rates of sulfate absorptionby barley roots and leaves increased at certain critical ex-ternal ion concentrations. Similar results had previously beenreported for other ions and tissue (see 7, 27), however, Nissen

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LEONARD AND HODGES

(34) interpreted these results to indicate that only one trans-port system, located at the plasma membrane, underwent dis-crete phase transformations at critical external concentrations.For each unique concentration range, the transport systempossessed a different Km and Vmax. The curved lines resultingfrom the kinetic plots ofK+ activation of the ATPase (Figs. 10and 11) andK+ absorption by oat roots (Fig. 13) would beconsistent with this interpretation. Thus, Km and V,,,x ofboth K+-ATPase and K+-transport continually increase as theexternal KCI concentration increases. For ion transport it isdifficult to know whether only one system is involved as sug-gested by Nissen (34). However, in the case of the ATPase, itwould appear that only one enzyme was being measured. Thisconclusion is based on the fact that if several ATPases wereresponsible for the various kinetic constants for KCl activa-tion, one would also expect various kinetic constants for bothATP and Mg2+. This was not the case, since standard Michaelis-Menten kinetics were observed for ATP and Mg2+ (Figs. 6 and8). Thus the view that there is only one ATPase and onetransport system which has several different affinities formonovalent cations appears to be a more feasible interpretationthan the one involving two distinct mechanisms for the trans-port of a single ion species.As mentioned previously, several purified enzymes exhibit

kinetics similar to those of ion transport by plants and for theK+ activation of the plasma membrane-ATPase. Koshland (24)refers to this type of kinetics as negative cooperativity andhe has described a model similar to that proposed by Monodet al. (33) for interpreting sigmoidal-type (positive coopera-tivity) kinetics. The negative cooperativity model assumes amultisubunit enzyme in which the binding of a ligand (cation inthis case) to the first subunit brings about conformationalchanges of the remaining subunits such that the binding sitesof these subunits have a lower affinity for the ligand. Thus,each successive binding of ligand to a subunit makes it moredifficult for the next ligand to bind. Enzymes that exhibitnegative cooperativity kinetics always have Lineweaver-Burkplots that are concave downward (as in Fig. 10) and haveHill coefficients (16) less than 1.0 (24). The Hill coefficients forK+ transport into oat roots and for the K+-stimulated ATPaseof plasma membranes were 0.56 and 0.52 (using the data ofFigs. 9 and 12), respectively. Thus, it appears that the Koshlandmodel provides a reasonable interpretation of ion transportkinetics and should be evaluated more thoroughly.

In conclusion, purified plasma membranes of oat rootscontain an ion-stimulated ATPase that has kinetic propertiessimilar to the kinetics of monovalent cation transport. This,along with other results discussed, provides strong evidencefor a role for this ATPase in ion absorption by plants. Thekinetic results presented here are also consistent with the con-cept that only one transport system, consisting of bindingsites possessing varying affinities for ions, is involved in theabsorption of ions from very low (0.01 mM) to very high (100mM) concentrations.

LITERATURE CITED

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bound aelenosine triphosphatase from plant roots. In: S. P. Colowsiek andN. 0. Kaplan, eds., Methods in Enzymology, Academic Press, N\ewv York.In press.

20. HODGEs, T. K..R. T. LEON-ARD, C. E. BRACKER, AN-D T.W. KEEN-AN'. 1972.Purification of an ion stimulated ATPase from plant roots: A-sociationwith plasma membranes. Proc. Nat. Acad. Sci. U.S.A. 69: 3307-3311.

21. HOFSTEE, B. H.J. 1952. On the evaluation of the constants V-sto andKIn i

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12 Plant Physiol. Vol. 52, 1973

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