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Vol. 245, No. 5, Issue of March 10, pp. 1115-1121, 1970

Printed in U.S.A.

Membrane Adenosine Triphosphatase from

Streptococcus faecalis

(Received for publication, July 2, 1969)


From the Department of Biochemistry, University of Colorado School of Medicine, Denver, Colorado 8O,%?O

SUMMARY was observed in polyacrylamide gels (7). From sucrose density

The membrane adenosine triphosphatase from Sfrepfo- sedimentation analysis it was estimated that the enzyme has a
COCCUS has been purified by heat treatment, gel
faecalis sedimentation coefficient of about 13 S and a molecular weight

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filtration through Agarose, and repeated chromatography on of about 350,000 (6). From gel electrophoretic analysis in urea
diethylaminoethyl cellulose. The purified ATPase appears we concluded that the enzyme is composed of nonidentical sub-
to be homogeneousas judged from gel electrophoresis and units (7). It was shown that the solubilized ATPase can be
sedimentation velocity studies. Sedimentation equilibrium reattached to membranes that were previously depleted of the
studies, which are presented in the succeeding paper, also enzyme and that Mg++ ions are an important, but not the sole
indicate that the purified preparation is homogeneous. factor in this enzyme-membrane interaction (8). It was found
Both products of the ATPase reaction, ADP and inorganic also that the membrane-bound form of the ATPase is strongly
phosphate, are competitive inhibitors of the enzyme with inhibited by dicyclohexylcarbodiimide but that the released en-
Ki values of 0.7 and 10 m&r, respectively, and the two have zyme is insensitive to this reagent (1). The entirely different
synergistic inhibitory effects. The purified enzyme does not response of the free and membrane-bound ATPase to DCCDl
catalyze an ADP-ATP exchange, and no evidence for a phos- seems completely analogous to the effects of DCCD and oligo-
phorylated enzyme intermediate could be found. No mycin on mitochondrial ATPase. The structural basis for this
cooperative homotropic effect of ATP on the purified enzyme phenomenon, termed “allotypy” by Racker (9), is not clear.
could be observed over the range of substrate concentrations Here we present a new and improved method for purifying the
tested. streptococcal membrane ATPase. This method has permitted
us to prepare the enzyme in amounts larger than were previously
obtained (7) and in a homogeneous state as judged by a variety
of criteria.
We have also examined the reaction mechanism of the purified
ATPase. It is well known that the plasma membranes of most
There is considerable evidence that the membrane adenosine animal cells contain a Na+- and K+-dependent ATPase which is
triphosphatase of Streptococcus faecalis is involved in the ener- believed to be involved in the linked active transport of these
gized transport of monovalent cations and other solutes across cations (10, 11). There is substantial evidence that the mech-
the plasma membrane (l-4). It may be expected, therefore, anism of these animal ATPases involves a Na+-dependent phos-
that the elucidation of the molecular properties of this enzyme, phorylation and a K+-dependent dephosphorylation of the en-
such as its physical structure, its reaction mechanism, and the zyme (11, 12). The membrane ATPase in S. fuecalis is also
way it interacts with other membrane components, might pro- thought to be involved in the active transport of monovalent
vide important insights into the mechanism of active transport. cations across the plasma membrane. This implication stems
Since the time it was first reported that an ATPase was associated mainly from the observation that DCCD inhibits the membrane-
with the bacterial plasma membrane (5), a considerable amount bound ATPase in vitro and also inhibits the net uptake of K+
of information about certain of these molecular aspects of the in vivo (1). However, in contrast to the animal ATPases, the
enzyme has been obtained, particularly after it was found that streptococcal enzyme does not seem to be specifically stimulated
the enzyme can be selectively released from the membranes in a by mixtures of Na+ and K+ ions (6, 13). In the present studies,
truly soluble form by a relatively mild extraction procedure (6-S). using the pure enzyme, we attempted but failed to demonstrate
We were able to purify the released enzyme, but only in very a phosphorylated intermediate in the reaction catalyzed by the
small amounts (1 mg or less), so that the degree of purity could streptococcal enzyme. On the contrary, from a study of inhibi-
not be established with certainty, although only one protein band tion by the products, ADP and inorganic phosphate, we conclude
that the reaction proceeds through a “Rapid Equilibrium Ran-
* This work was supported by United States Public Health Serv-
ice Grant GM 05810. 1 The abbreviation used is: DCCD, N,N’-dicyclohexylcarbodi-
$ To whom inquiries should be directed. imide.
1116 Purification and Mechanism of ATPase from S. faecalis Vol. 245, No. 5

dom Bi Bi” mechanism, according to the terminology of Cleland Protein Concentrations--These were determined by the method
(14). of Lowry et al. (20).
EXPERIMENTAL PROCEDURES Gel Electrophoresis-This was carried out in vertical slabs of
7% polyacrylamide gel (Cyanogum 41) in the model 474 appa-
Preparation of Solubilized B TPase-The procedures for solu- ratus manufactured by the E-C Apparatus Corporation, Phila-
bilizing ATPase from membrane ghosts of X. faecalis were
delphia, Pennsylvania. Both the gels and electrode compart-
adapted and scaled up from those previously decsribed (6). In
ments contained a Tris-glycine buffer of pH 8.5 (0.015 M Tris and
essence, solubilization of the enzyme is achieved by repeatedly
0.07 M glycine) (7).
washing the membranes with low ionic strength buffers in the
Physical Studies-Sedimentation velocity experiments were
absence of Mg++ ions. This eventually leads to a relatively carried out in a Spinco model E ultracentrifuge equipped with
selective and abrupt “all or none” release of the enzyme (6).
schlieren optics and an RTIC temperature control unit. Spec-
For a typical preparation used in the present experiments, cells tral analyses were performed on a Beckman model DB spectro-
(8. faecalis, ATCC No. 9790) were grown at 38” in two 15-liter
batches of a glucose-tryptone-yeast extract-potassium phosphate
Exchange Reactions-ADP-ATP and Pi-ATP exchanges were
medium (15), which was agitated gently with a magnetic stirrer. measured by the incorporation of radioactivity into ATP from
The culture was chilled shortly after reaching stationary phase
3H-ADP and 32Pi, respectively. The incubation mixture con-
and harvested by centrifugation in a Lourdes CFR-1 continuous
tained 5 IrIM Mg-ATP, 0.1 M Tris-HCI, pH 7.5, 0.1 to 0.5 unit
flow rotor (Lourdes Instrument Corporation, Old Bethpage,
of enzyme, and 5 to 10 mM 3H-ADP or 32Pil or a mixture of both.
New York). After they were washed once with cold distilled
The reaction was stopped by the addition of an equal volume of
water, the cells were converted to protoplasts by the action of
2 M formic acid. Separation of ADP, ATP, and Pi was accom-
egg white lysozgme. The cells were suspended in 10 volumes per
plished by chromatography either on Whatman No. 1 paper,

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packed cell volume of 0.5 M glycylglycine, pH 7.2 (IS), conta,in- using an isobutyric acid-concentrated ammonium hydroxide-
ing 2 mM MgCl*, and incubated at 37” with 0.2 mg of lysozyme
water solvent (66:1:33 by volume) (21), or on DEAE-cellulose
per ml. After 45 min another 0.1 mg of lysozyme per ml was
paper, using a 0.6 M ammonium formate buffer (pH 3.1) as sol-
added, and 45 min after the second addition the protoplasts
vent (22). The ATP was located under ultraviolet light, cut out,
were collected by centrifugation at 36,000 x g for 20 min. Lysis
and assayed for 3H or azP. Corrections were made to account
of the protoplasts was accomplished in a Waring Blendor by for the losses of labeled ATP due to the concomitant ATPase
quick suspension into 2 to 4 volumes per packed protoplast reaction.
volume of a cold solution of 2 InM MgC&, containing 2 pg of Chemicals-ATP-+2P was prepared enzymatically by the
DNase per ml. The disruption of the protoplasts due to osmotic method of Glynn and Chappell (23), with the following modifica-
shock was virtually complete within 1 min as judged by the ap-
tion. The reaction was stopped by acidification with HCl, and
pearance of the ghosts under the phase contrast microscope.
the ATP was adsorbed to acid-washed Norit A. After washing
The membrane ghosts were washed in the cold, once with 2 mM the Norit on a Millipore filter, the ATP was eluted with a mix-
MgC&, twice with 2 M LiCl in 0.25 M Tris-HCl, pH 7.5, and once
ture of ethanol-water-concentrated ammonium hydroxide (50 :
with 20 mM Tris-HCl, pH 7.5. The washing was continued with 50: 1 by volume) which was then taken to dryness, 3H-,4TP
1 mM Tris-HCl, pH 7.5, until the enzyme was released. The
and 3H-ADP were purchased from Schwarz BioResearch, and
procedure was carried out by repeated centrifugation at 100,000 “Pi was obtained from New England Nuclear Corporation.
x g for 30 min and resuspension for 1 min in a Waring Blendor DNAse 1, RNAase, and lysozyme were obtained from Worthing-
in about 2 volumes per packed membrane volume of 1 mM Tris-
ton; ATP was from Sigma, and glycylglycine was from Pierce
HCI, pH 7.5. About 75% of the enzyme was released into the Chemical Company (Rockford, Illinois). All other chemicals
supernatants of the seventh to the tenth wash.
were reagent grade.
Enzyme Assays-Generally the enzyme activity was measured BuffersTwo buffers were used throughout most of the study
by the release of Pi from magnesium ATP as previously described and will be abbreviated as follows: Buffer TM 7.5, 0.02 ar Tris
(6). The method of Fiske and SubbaRow (17) was used for the and 0.01 M MgC12 titrated to pH 7.5 with HCI; Buffer SM 6.2,
determination of Pi. One unit of enzyme activity is defined as 0.02 M succinic acid and 0.01 M MgC12 titrated to pH 6.2 with
that amount of enzyme which catalyzes the release of 1 pmole of
inorganic phosphate from ATP per min at a magnesium ATP
concentration of 5 mM and at 38”. RESULTS
In order to examine the effect of inorganic phosphate on the
reaction, the activity was measured by the release of 32Pi from Enzyme Purijkation
ATP-y-a2P, using a modification of the method of Crane and
Lipmann (18). The incubation mixture contained 5 pmoles of Generally 30 liters of stationary phase culture were worked up
ATP-yJ2P, 5 pmoles of Mg++, and 100 pmoles of Tris-HCl, and yielded 135 to 150 g of cells (wet weight). The washes of
pH 7.5, in 1 ml, and enzyme. The reaction was stopped by the the membranes (see ‘<Experimental Procedures”) that contained
the released ATPase were pooled and supplemented with Tris-
addition of 1 ml of 1 N HCl; 200 mg of acid-washed Norit A were
HCl and MgC12 to a final concentration of 20 mM Tris-HCl,
then added in 1 ml of water, and after 10 min the suspension was
centrifuged. One milliliter of the supernatant, which was free pH 7.5, and 10 mM Mg++. The addition of buffer and Mg++
of organic phosphates, was counted in a Triton-toluene-based aided in stabilizing the enzyme and usually resulted in the forma-
scintillation fluid (19). Radioactivity was measured in a Beck- tion of a small precipitate. In order to remove contaminating
man model LS-133 liquid scintillation counter. The results ob- RNA, 0.5 mg of RNAse was added to these washes, which were
tained by this method were in very good agreement with those then dialyzed overnight against Buffer TM 7.5.
obtained by the calorimetric assay. Heat Treatment-The dialyzed material was heated to 55” in
Issue of March 10, 1970 H. P. Schnebli and A. Abrams 1117

-- Activil ‘Y
0% - Purification of ATPase

n Units/n II
- -
[AGAROSEI Total enzyme Specific activity
I5 _- .-
:: unilr unds/mg proreilt
Extracts. ............... 3050 1720 0.56
Heated extract .......... 880 1700 1.9
DEAEl................ 314 12.50 4.0
IO. Agarose ................. 91 875 9.6
DEAE 2. ............... 18 840 47
- -


FIG. 3. Schlieren patterns of purified ATPase. Photographs

were taken at 8-min intervals after attaining a rotor speed of
300 400 500 600 ml 56,000 rpm. The protein concentration was 0.6% in 0.02 M sodium
FIG. 1. Elution of ATPase from Agarose A 0.5. The concen- succinate buffer, pH 6.2, containing 0.01 M MgClz (Buffer SM 6.2).
trated protein solution (see text) was applied to a column of Aga- A double sector cell was used, and the temperature was 20”; the

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rose A 0.5 (Bio-Rad, Richmond, California; 2.5 X 165 cm) that analyzer angle was 70”. The protein was sedimenting from left
had been equilibrated with Buffer SM 6.2. Elution waswith the to right.
same buffer, and the flow rate was 16 ml per hour.
Buffer TM 7.5, and the reservoir contained 500 ml of the same
O%o - ---Activity buffer made0.6 M with respectto KCl. The flow rate was40 to
60 ml per hour. The enzyme emergedfrom the columnat about
0.3 0.27 M KCl. The fractions containing ATPase activity were
pooledand retained for further purification by gel filtration.

‘, L*
Gel Filtration-The enzyme was concentratedfor this step by

:‘Y :
: \
I5 precipitation with ammoniumsulfate. The solidsalt wasadded
to make the solution 85% saturated. After 30 min to 1 hour
the precipitate wascollectedby high speedcentrifugation, redis-
solvedin SM 6.2 buffer, and dialyzed againstthe samebuffer for
: ‘\. \ - 10
2 to 4 hours. The concentrated protein solution (about 4 ml

0.1 1


containing about 300 mg of protein)
columnof AgaroseA 0.5, as illustrated in Fig. 1.
was then fractionated

DEAE Chromatography %-The ATPase from the Agarosecol-

umn was finally chromatographed on a secondDEAE-cellulose
on a

‘\ \ column, but now at pH 6.2. A typical elution profile is shown

in Fig. 2.
The fractions containing the enzyme with a constant specific
100 200 ml activity were pooled, while the small shoulderwith a lower spe-
FIG. 2. Second chromatography of ATPase on DEAE-cellulose. cific activity was discarded. The enzyme was again concen-
The pooled fractions containing ATPase activity from the Agarose trated by precipitation with 85% saturated ammoniumsulfate,
column were applied directly to another DEAE-cellulose column
(Carl Schleicher and Schuell; 1 X 18 cm) which had previously dialyzed extensively againstSM 6.2 buffer, and storedat 4’. A
been washed with SM 6.2 buffer. The enzyme was eluted with 400 summary of the purification procedure is presented in Table I.
The enzyme obtained in the last step was purified over 50-fold
ml of a linear salt gradient of 0.1 to 0.4 M KC1 in the same buffer.
The flow rate ~86 6 to 10 ml per hour, and the enzymeemergedat from the extracts of the membraneghostsand representsa yield
about 0.2 M KCl. of 25 to 30%.
It shouldbe pointed out that our starting material, the pooled
a water bath and maintainedat this temperaturefor 10 in. The washesof isolatedmembranescontaining the solubilisedenzyme,
heating and coolingperiodswere lessthan 5 min each. A white, wasalready greatly enrichedin ATPase. This is why a purifica-
inactive precipitate, removed by centrifugation and filtration tion of only 85-fold resultedin a homogeneous preparation, aswe
through glasswool, was discarded. All subsequentsteps were showbelow.
carried out in the cold.
Purity of Enzyme
DEAE ChromatographyI-The heat-treated extracts, gener-
ally 400 to 600 ml, were placed directly on a DEAE-cellulose The specificactivity of the enzyme obtained from the second
column (Whatman DE 32, 2.5 x 40 cm) which was previously DEAE chromatography wasalways 45 to 50 units per mg of en-
washedand equilibrated with Buffer TM 7.5. All of the ATPase zyme and could not be increasedfurther by purification through
wasadsorbedto the column,which wasthen washedwith 200 ml chromatography on DEAE-cellulose at various pH values, with
of the samebuffer. The protein waseluted with a linear gradi- hydroxylapatite or A&08, or by treatment with alumina Cy.
ent of 0 to 0.6 M KCl. The mixing chambercontained 500ml of This preparation was thus judged to be maximally purified.
Puri$cation and Mechanism of ATPase from S. faecalis Vol. 245, No. 5

FIQ. 5. Specific release of the terminal phosphate from ATP.
The incubation mixtures contained 5 mM magnesium-ATP-@*P,
0.1 M Tris-HCl. DH 7.5. and 0.03 to 0.12 unit of enzyme. The reac-
tion wasstoppk;l by the addition of an equalvolume of 1 N HCl.
The total Pi released was determined by the calorimetric method,

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and the radioactive Pi released was determined by the procedure
of Crane and Lipmann (18) (seeExperimental Procedures).

25 mM Pi

FIG. 4. Electrophoretic analysis of purified ATPaee. Sample

A, 5 rg, and sampleB, 100pg of protein. The gel wasrun for 43
hours at 300 volts in a Tris-glycine buffer of pH 8.5 and stained
with Amido black. See “Experimental Procedures” for details
of the preparation of the gel.
10 mM Pi

The final enzyme preparation wasexaminedwith respectto its

homogeneityby a numberof criteria. Fig. 3 is a seriesof photo-
graphs representingthe boundary of the enzyme during a sedi-
mentation analysis. The purified protein sedimentedas a single
componentwith a symmetrical schlierenpeak at all concentra- No Pi
tions tested. The szo,u,extrapolated to zero concentration was
13.4 (seesucceedingpaper, Reference24).
Analysis of the purified enzyme by acrylamide gel electropho-
resisshowsoneprotein band (Fig. 4), again indicating homogene-
ity. In the succeedingpaper, someadditional criteria of purity
are presented(24). In sedimentationequilibrium experiments
with the purified enzyme, linear plots of In c versusx2 were ob- 0:5 1.0 (mhr’)
tained, and the molecularweight determinedby this method was A
independentof the initial protein concentration. Furthermore, [Mg _ATP]

the enzyme hasa very uniform appearancewhen examinedunder FIQ. 6. Competitive inhibition of the ATPase by inorganic
the electron microscope(24). phosphate. Purified ATPase was assayed with varying concen-
trations of magnesium ATP-y-**P in the presence of 10 and 25 lll~
Propertiesof Enzyme Pi and in the absence of Pi. The abscissarepresents reciprocal
substrate concentration and the ordinate the reciprocal of the
We found that a variety of salts, such as NaCl, KCl, and reaction rate.
NH&l, or buffers such as Tris-HCl, pH 7.5, and sodiumsucci-
nate, pH 6.2, at concentrationsof 0.01 to 0.1 M, aided in stabi- The ultraviolet spectrum of the enzyme in Buffer TM 7.5
lizing the enzyme, prolonging its half-life 5- to lo-fold over that exhibits a maximumat 275 a trough at 250 m, and rises
in 1 rnM Tris-Cl, pH 7.5. Concentrationsof 0.002 to 0.01 M sharply below 240 mp. The ratio of Az(o:A~o is 0.68, typical
Mg* alsohad a markedstabilizing effect; sulfhydryl compounds for proteins.
at 0.002M had no effect on the stability of the enzyme, but de- ReactionMechanism
stroyed the activity at a concentration of 0.1 M. The half-life of
the pure ATPase storedat (r-4” in Buffer SM 6.2 or Buffer TM 7.5 Specijicity of Reaction-Previously, it was observedthat the
is 15 to 25 days. Freezing of the enzyme for a short period of ATPase from S. jaecalis is specific toward purine nucleoside
time causesextensive lossof activity. The enzyme can, how- triphosphates,and that pyrimidine nucleosidetriphosphatesare
ever, be stored for severaldays at room temperature or at O-4’. not hydrolyzed (6,7).
Issue of March 10, 1970 H. P. Schnebli and A. Abrams 1119

Synergistic inhibitory effect of ADP and Pi on the ATPase
The incubation mixture contained 5 mM ATP-?-azP, 0.1 M Tris-
HCl, pH 7.5, and enzyme. ADP and Pi were added as indicated.

Enzyme activity Inhibition

pmolc =p/10 Twin %

Control. .. . 0.52
+0.35 maa ADP.. 0.40 23
+lO mM Pi.. _. . 0.30 42
+0.35 mM ADP and 10 mM Pi. . 0.09 83

Exchange reactions a I a I
0.4 0.8
The incubation mixture contained, in 1 ml, 5 rmoles of Mg-ATP,
log MS-ATP-cont. (mM1
a.1 mmole of Tris-HCl, pH 7.5, and enzyme. Reaction A con-
tained, in addition, 10 rmoles of 3H-ADP; reaction B contained FIG. 7. Hill plot to determine n, the “order with respect to
10 pmoles of “Pi; and reaction C contained 5 pmoles each of 3H- Mg-ATP,” for the reaction catalyzed by membrane ATPase from
ADP and “‘Pi. The reaction was stopped after 1 hour. 6’. jaecalis. The reaction mixture contained 0.1 M Tris-HCl, pH
7.5,0.04 unit of enzyme, and magnesium ATP at the concentrations

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Exchange indicated.

EH-ADP-ATP ‘2P i-ATP preparation are probably due to other enzymes, such as ad-
enylate kinase, nucleoside diphosphate kinase, and polynu-
m/mwles/unit of enzyme/hr
cleotide phosphorylase, and that the ATPase as such does not
Washed membranes.. . . 2960 (A) 190 (B)
catalyze exchange reactions. It is also possible, although less
Purified enzyme. .. 6 ((3 <1 0
I likely, that the membrane-bound ATPase catalyzes exchange re-
actions but that the soluble enzyme is unable to do so.
Here we wish to demonstrate that the ATPase specifically Reaction Intermediates-The purified ATPase failed to catalyze
cleaves the terminal pyrophosphate bond of adenosine triphos- an ADP-ATP exchange, as indicated in the previous section.
phate. As can be seen in Fig. 5, the amount of radioactive phos- This practically excludes the existence of a phosphorylated inter-
phate cleaved from ATP-T-~~P is identical with the total amount mediate in the reaction. Nevertheless, several attempts were
of phosphate released. Furthermore, no phosphate is released made to demonstrate an acid-stable phosphorylated enzyme.
from ADP when incubated with enzyme (6). All experiments which employed the methods that have been
Product Inhibition-Cleland has shown (14,25) that the study used to label animal Na+- and K+-dependent ATPases by reac-
of product inhibition kinetics is a useful tool in understanding the tion with ATP-T-~~P (11, 27) failed to give any indication of the
mechanism of an enzymatic reaction. Inorganic phosphate is a occurrence of such an intermediate in the reaction catalyzed by
competitive inhibitor, as can be seen in Fig. 6. The double re- the purified membrane ATPase from S. fuecalis.
ciprocal plots of initial velocity against substrate concentration Cooperativity-It is shown in the succeeding paper (24) that
(26) give straight lines with a common intercept on the ordinate. the ATPase is made up of 12 subunits of two kinds. It was,
The Ki for orthophosphate determined from the data of Fig. 6 therefore, of interest to look for possible allosteric properties of
was 10 mM. ADP has previously been shown to be a competitive the enzyme. However, the kinetics of the reaction catalyzed
inhibitor of the ATPase (6). The Ki for ADP determined with by the ATPase is of the classical type. In the plot of rate against
the highly purified enzyme was 0.7 mM, similar to the value ob- magnesium ATP concentration we did not detect any sigmoidic-
tained previously with crude preparations of the enzyme. The ity over the range tested. From the Hill equation (28) the
K, for the substrate, magnesium ATP, was found to be 2.5 InM, apparent “order of the reaction with respect to substrate” (29),
in agreement with the previously reported value (6). Roth also called the “interaction coefficient” (30), can be determined.
products of the reaction, ADP and Pi, inhibit the enzyme com- Fig. 7 shows a Hill plot of the data obtained by measuring initial
petitively with respect to ATP when tested individually. Their velocities at various concentrations of the substrate, magnesium
inhibitory effects, however, are more than additive when both ATP. It gives a straight line with a slope of n = 0.94. There-
products are tested simultaneously, as seen in Table II. These fore, we conclude that either there is no homotropic cooperativity
results suggest a “Rapid Equilibrium Random” mechanism (14, between multiple active sites, or else there is only 1 active site
25). per molecule. This conclusion is based on experiments with the
&change Reactio?as-Table III presents typical data obtained soluble enzyme, and it should be pointed out that the membrane-
from ADP-ATP and Pi-ATP exchange experiments. Almost bound ATPase might behave differently. Different behavior
no detectable exchange is observed with the highly purified sol- toward DCCD of the two forms of the enzyme has been observed
uble ATPase. However, washed membranes have a high capac- previously (1).
ity to exchange ADP into ATP and a lesser capacity to exchange
Pi into ATP. The ADP-ATP exchange rate in the membrane
preparation was 5% of the ATPase reaction rate. From this We have devised a new and improved method for the purifica-
it is concluded that the exchange reactions in the membrane tion of the membrane ATPase from S. faecalis that allows the
1120 PuriJication and Mechanism of ATPase from X. faecalis Vol. 245, T\‘o. 5

preparation of 10 to 20 mg of pure material in about 1 week. be a competitive inhibitor, and ADP should be a noncompetitive
The ATPase we have isolated appears to be homogeneous by a inhibitor, which was not the case.
qumber of different criteria. These include a single symmetrical It is interesting to compare our results with the findings made
schlieren peak upon sedimentation in the ultracentrifuge (Fig. 3), on other systems. The ATPase from the inner membrane of
a single band of protein by gel electrophoresis (Fig. 4), and a bovine heart mitochrondria has also be solubilized and purified.
constant specific activity throughout a peak upon chromatog- This mitochrondrial enzyme, like the streptococcal ATPase,
raphy on DEAE-cellulose (Fig. 2). Other criteria of homoge- does not catalyze exchange reactions (31), and no evidence for a
neity are discussed in the succeeding paper (24) and include sedi- phosphorylated intermediate has been found.2 The ATPase
mentation equilibrium studies and electron micrographs. It is from the aerobe Micrococcus lysodeikticus has been solubilized
of interest to note that this is the only ATPase derived from and partially purified (32-34). Like the streptococcal and mito-
plasma membrane preparations which has thus far been obtained chondrial enzymes, it does not catalyze an ADP-ATP exchange
in a form satisfying the usual criteria of purity. after partial purification (33).
In previous studies, small quantities of this enzyme were The mechanism of the Na+- and K+-dependent ATPases from
purified (7) using sucrose gradient centrifugation. It appeared animal cells, first described by Skou (35), appears to involve the
to be homogeneous on gel electrophoresis but was resolved into formation of a phosphorylated enzyme intermediate (11, 12).
three bands when it was electrophoresed in 8 M urea. The two The streptococcal membrane ATPase, which like the animal
major bands were designated ar and /3, and a minor band was ATPase is involved in monovalent cation transport, seems to
called y. As we show in the accompanying paper (24), the have a different reaction mechanism.
ATPase prepared by the method described here is made up of
Acknowbdgment-We thank Dr. John P. Perkins, Department
only two different kinds of subunits, namely cy and 6. The y
of Pharmacology, University of Colorado School of Medicine,
protein component, seen in our earlier preparations (7), is re-

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for the preparation of ATP-+*P.
moved from the ATPase during the second DEAE chromatog-
raphy (Fig. 2). The sedimentation rates of ATPase prepara- REFERENCES
tions containing the protein y and of preparations devoid of y 1. HAROLD, F. M., BuRD.~, J. R., BARON, C., AND ABRBMS, A.,
were identical. From this we conclude that y is either a con- J. Bid. Chem., 244, 2261 (1969).
taminant or possibly some altered form of the cy or /? subunit. 2. HSROLD, F. M., AND BURDA, J. R.,J. &zcleriol.,%, 816 (19G8).
3. ARR.ZMS, A., J. Biol. Chem., 236, 1281 (1960).
The finding that both products, Pi and ADP, are competitive 4. ZBRLENGO, M., AND SCHULTZ, S. G., Biochim. Biophys. Acta,
. I
inhibitors of the ATPase reaction, together with the failure to 126, 308 ‘(1966).
observe exchange reactions or a phosphorylated intermediate, 5. ABRAMS. A.. McN.~MAR,\. I'.. AND JOHNSON. F. B.. , J. Biol.
suggest that the reaction mechanism is “Rapid Equilibrium Chem.; 236, 3659 (1960): ’
Random Bi Bi,” according to the nomenclature of Cleland (14). 6. ARRSMS, A., J. Biol. Chem., 240, 3675 (1965).
7. ABR.\MS, A., END B.IRON, C., Biochemistry, 6, 225 (1967).
In this mechanism all possible enzyme-substrate and enzyme- 8. AHRNVIS, A., AND BARON, C., Biochemistry, 7, 501 (1968).
product complexes dissociate rapidly and reversibyly, and the 9. RECITER, E., Fed. Proc., 26, 1335 (1967).
only slow step is the interconversion of the enzyme-substrate to 10. ALDERS, It. W., Annu. Rev. Biochem., 36, 727 (1967).
the enzyme-products complex. There is no obligatory sequence 11. POST, R. L., SEN, A. K., AND ROSENTHAL, A. S., J. Biol. Chem.,
240, 1437 (1965).
in which the products leave the enzyme. Both products can 12. SKOU, J. C., Physiol. Rev., 46, 596 (1965).
bind to the free enzyme, which is consistent with the observation 13. NEUUHR, H. Y., H~NSSON, E., AND FERM, It., Acta Chem.
that both products are competitive inhibitors of the enzyme hand., 21, 182 (1967).
when tested individually. There is no unique interpretation for 14. CLEL~ND, W. W., Biochim. Biophys. Acta, 67, 104 (1963).
the observed synergistic inhibitory effect of ADP and inorganic 15. ARR~MS, A., 1. Biol. Chem., 230, 949 (1958).
16. A~R:YMS, A., .\ND McN,YM,\R,~, P., J. Biol. Chem., 237, 170
phosphate. We feel that the more than additive inhibition may (1962).
be due to the fact that, in the presence of one another, the prod- 17. FISKE, C. H., AND Sur~r~.~Itow, Y., J. Biol. Chem., 66, 375
ucts can each bind to two forms of the enzyme; e.g. in the pres- (1925).
ence of Pi, ADP can still bind to the free enzyme, but also to the 18. CR.~NE, R. K., .\ND LIPM‘INN, F., J. Biol. Chem., 201, 235
enzyme-Pi complex. It is interesting to speculate that the syner- 19. PATTERSON, M. S., IND GREEN, R. C., Anal, Chem., 37, 854
gistic inhibitory effect of ADP and Pi plays a role in the control (1965).
of the ATP utilization in Go. The proposed “Rapid Equilib- 20. Lo~RY,O.H.,ROSEUROUGH,N.J.,F.~RR, A.L., ANDR~ND.~LL,
rium Random” mechanism is also in agreement with the failure It. J., J. Biol. Chem., 193, 265 (1951).
of the enzyme to catalyze exchange reactions. Exchange reac- 21. WADKINS, C. L., .\ND LEHNINGER, A. L., in S. P. COLOWICK
AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol.
tions require that the reaction be reversible, that the products VI, Academic Press, New York, 1963, p. 265.
leave sequentially, and that the second product leave relatively 22. MORRISON, J. F., Anal. Biochem., 24, 106 (1968).
slowly, compared with the first. 23. GLYNN, I. M., AND CHAPPELL, J. B., Biochem. J., 90,147 (1964).
Finally, the failure to observe a phosphorylated enzyme inter- 24. SCHNEDLI, H. P., V‘ZTTER, A. E., AND ARRAMS, A., J. Biol.
Chem., 246, 1122 (1970).
mediate is consistent with both the incapability of the enzyme to
25. CLELAND, W. W., Biochim. Biophys. Acla, 67, 173 (1963).
catalyze an ADP-ATP exchange (Table III) and the kinetics of 26. LINEWEAVER, H., AND BURK, I>., J. Amer. Chem. sot., 66,658
product inhibition. It will be realized that a phosphorylated (1934).
enzyme intermediate would also be an intermediate in the ADP- 27. SCHONER, W., BEUSCH, R., I\NDKRAMER, R.,Eur. J. Biochem.,
ATP exchange. The occurrence of a covalent intermediate 7, 102 (1968).
28. HILL, A. V., Biochem. J., ‘7, 471 (1913).
would further imply that the products leave the enzyme sequen-
tially. This leads to the prediction that, in this case, Pi should 2 E. Racker, personal communication.
Issue of March 10, 1970 H. P. Schnebli and A. Abrams 1121

29. ATKINSON, D. E., HATHAWAY, J. A., AND SMITH, E. C., J. Biol. 32. ISHIKAWA, S., AND LEHNINGER, A. L., J. Biol. Chem., 237,
Chem., 240, 2682 (1965). 2401 (1962).
30. CHANCEUX, J. I’., Cold Spring Harbor Symp. Quant. Biol., 28, 33. ISHIKAWA, S., J. Biochem. (Tokyo), 60, 598 (1966).
497 (1963). 34. MUNOZ, E., SALTON, M. R. J., Mo, M. H., AND SCHOB, M. T.,
31. PULLMANN, M. E., PENEFSKY, H. S., DATT.4, A., AND RACKER, Eur. J. Biochem., 7, 490 (1969).
E., J. Biol. Chem., 236, 3322 (1960). 35. SKOU, J. C., Biochim. Biophys. Acta, 23, 394 (1957).

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Membrane Adenosine Triphosphatase from Streptococcus faecalis :
Hans P. Schnebli and Adolph Abrams
J. Biol. Chem. 1970, 245:1115-1121.

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