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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
photometer.
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,
TABLE I
-- Activil ‘Y
0% - Purification of ATPase
n Units/n II
- -
[AGAROSEI Total enzyme Specific activity
I5 _- .-
:: unilr unds/mg proreilt
?a
!
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
- -
5’
‘, L*
Gel Filtration-The enzyme was concentratedfor this step by
0.2
:‘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
:
:
:
“11,
\
\
‘1.
‘\
u
-IDEAE
\
21
5
containing about 300 mg of protein)
columnof AgaroseA 0.5, as illustrated in Fig. 1.
was then fractionated
TOTAL PHOSPHATE
RELEASED
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,
(min./pMole)
25 mM Pi
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 ml.land 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
TABLE II
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.
TABLE III
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
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
DISCUSSION
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-
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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