C. O. Bewaji et al.

Biokemistri Vol. 1, No. 2, December, 1991 0795-8080/91 $3.00 + 0.00

Printed in Nigeria Klobex Academic Publishers

STUDIES ON ADENOSINE TRIPHOSPHATASE ACTIVITIES IN

HOMOGENATES OF RAT BRAIN AND LIVER

C. O. Bewaji*, K. S. Oyedotun and S. O. Malomo

Department of Biochemistry, University of Ilorin, Ilorin, Nigeria

(Received November 6, 1991)

ABSTRACT: Studies on ATP hydrolysis in homogenates of rat brain and liver indicated the presence
of three distinct ATPases: the Mg2+-ATPase, (Ca2+ + Mg2+)-ATPase and (Na+ + K+)-ATPase. The
maximal velocities of the three enzymes were significantly higher in the brain than in the liver (P <
0.01). However, the kinetics of putative ligand interactions of the ATPases followed identical pattern
in both tissues, except that the (Ca2+ + Mg2+)-ATPase of the liver had a higher affinity for ATP (P <
0.05) and a lower affinity for Ca2+ (P < 0.001) that the analogous enzyme in the brain. In addition, the
(Na+ + K+)-ATPase in both tissues and the (Ca2+ + Mg2+)-ATPase in the brain were potently inhibited
by orthovanadate with Ki ranging from 3.20 ± 0.32 M to 6.0 ± 0.25 M. However, the sensitivity of
the liver (Ca2+ + Mg2+)-ATPase was much reduced, while the Mg2+-ATPase was completely
unresponsive to orthovanadate inhibition. Although the apparent kinetic parameters of these
enzymes were affected by their state of purity, their responsiveness to classical inhibitors seemed to
follow the pattern usually reported for purified enzymes.

INTRODUCTION Furthermore, the molecular identity of

Kinetic studies on adenosine
triphosphatases have traditionally been
performed using membrane preparations
or purified enzymes reconstituted in
phospholipid liposomal systems (1-3).
Norgaard et al. (4) have observed that due
to difficulties in ensuring complete
recovery of ATPase activities, as well as
exposure of all available sites to ligands,
the use of isolated preparations might yield
false or lopsided information about the
total enzyme activity per gram of tissue.
Other workers (5,6) had earlier observed
that not less than 20 per cent of the
parameters of an enzyme show significant
differences between their in vitro and in
vivo values. This is a clear indication that
extrapolation of the behaviour of an
enzyme from in vitro to in vivo condition
can only be tentative and could sometimes
be quite misleading. The use of crude
homogenates, therefore, is more akin to
the in vivo condition than isolated
reconstituted systems.
the Mg2+-ATPase is, at present, not
clearly understood. Various attempts to
isolate and characterize this enzyme have
always proved abortive (7,8). Therefore,
the question has always been posed as to
whether the Mg2+-ATPase is a distinct
enzyme or just a background activity of the
various ATPases whose operations
absolutely require Mg2+ as a cofactor.
Some aberrant kinetic behaviour of the
liver Ca2+-ATPase have also been
reported (9,10). It is not clear whether such
behaviour is an artefact of purification or
an inherent property of the enzyme. In
order to clarify some of these issues, we
have characterized the various ATPases in
crude homogenates of rat brain and liver
with respect to all the putative ligands
necessary for enzymatic activities as well
as their inhibitor sensitivities, pH- and
temperature-dependence. It is anticipated
that this will provide information on novel
features of the ATPases which might have
been overlooked in studies involving
isolated systems. It will also provide data
on optimal assay conditions for ATPases

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 Biokemistri Volume 1, No. 2 (1991)
in crude homogenates, thereby obviating
the need for laborious purification of the
enzyme and/or membranes particularly in
laboratories where facilities for such work
are not available.
MATERIALS AND METHODS
Preparation of tissue homogenates
Adult male albino rats (Wistar strain)
weighing between 150 – 220g were used.
The conditions of animal care and
preparation of tissue homogenatesare as
previously described (1,2). Essentially, the
rats were sacrificed by cervical dislocation
and the brain and liver were quickly
dissected out into separate beakers
containing ice-cold solutions of 250 mM
sucrose, 10 mM Tris, pH 7.4. The tissues
were washed in the buffer until free from
blood and then homogenised in the same
buffer with 10 strokes of a tight-fitting
Teflon pestle in a Potter-Elvehjem glass
homogenizer operating at 1,000 rpm.
Twenty per cent suspensions of the tissue
homogenates were stored in
o 2+
microcentrifuge tubes at -20 C and used
for ATPase assay within one week. No
loss of enzyme activity was observed
within this period.
Determination of protein
Protein concentrations in the tissue
homogenates were determined by the
biuret method (13) using egg albumin as
standard.
Determination of ATPase activity
ATPase activity was assayed
spectrophotometrically under various
conditions of pH, temperature and ligand
concentrations, by measuring the release
of inorganic phosphate from ATP as
previously described (14). The following
conditions were common for the assay of
the three ATPases: 30 mM Tris, pH 7.4, 1
mM MgCl2, 1 mM ATP (disodium salt), and
10 – 20 g of tissue protein. The reaction
volume was 0.8 ml and the temperature
o Fig. 6 shows the synergistic effects of
was 37 C.
+ +
For the assay of (Na + K )-ATPase, the
assay medium also contained 140 mM
NaCl, 20 mM KCl and 0.5 mM EGTA. For
2+ 2+
(Ca + Mg )-ATPase: 120 mM KCl and
72
2+
50 M CaCl2. For the say of Mg -ATPase,
the content of the medium was similar to
+ +
that described for (Na + K )-ATPase,
except that the activity was measured in
the presence of 1 mM ouabain. Other
conditions are stated in the legends to the
figures for the individual experiments.
The mixture was pre-incubated for 3 min
at the required temperature and the
reaction was started by adding ATP (to a
final concentration of 1 mM). After 30 min
of incubation, with constant shaking, the
reaction was terminated with 0.2 ml of a
5% solution of sodium dodecyl sulphate.
The inorganic phosphate released was
determined by the procedure of Fiske and
SubbaRow (15).
RESULTS
2+
The Mg -dependence of the ATPase
activities in homogenates of rat brain and
liver is shown in Fig. 1. Maximal ATP
2+
hydrolysis was observed at a Mg
concentration of 1 mM in both tissues.
Mg concentrations greater than 1.6 mM
were inhibitory
The ATP-dependence of the three
2+ + +
ATPases: Mg -ATPase, (Na + K )-
2+ 2+
ATPase and (Ca + Mg )-ATPase in the
2+
presence of 1 mM Mg is shown in Figs. 2
and 3. In both tissues, maximal ATP
hydrolysis were observed at 1 mM ATP,
with higher concentrations being inhibitory.
In the experiment illustrated in Fig. 4, the
2+ 2+
ATP-dependence of the (Ca + Mg )-
ATPase was observed in the presence of 2
mM MgCl2. It was observed that any ATP
2+
in excess of the Mg present in the
medium was inhibitory. Maximal ATP
hydrolysis was observed in the presence
of equimolar concentrations of ATP and
2+
Mg .
2+
The Ca -dependence of ATPase
activities in rat brain and liver is illustrated
2+
in Fig. 5. The concentration of Ca
needed for maximal ATP hydrolysis was
higher in the liver than in the brain.
2+ +
Na+ and Ca on the activity of the (Na +
+
K )-ATPase in rat brain and liver. In both
tissues, maximal ATP hydrolysis was
observed when the ionic concentrations in
+
the medium was between 120 mM Na /40
 C. O. Bewaji et al.

Fig. 1 Mg2+-dependence of the Mg2+-ATPase in rat brain (O) and rat liver ()

Fig. 2: ATP-dependence of the Mg2+-ATPase (), (Ca2+ + Mg2+-)-ATPase () and (Na+
+ K+)-ATPase (O) in rat brain.

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 Biokemistri Volume 1, No. 2 (1991)

Fig. 3: ATP-dependence of the Mg2+-ATPase (), (Ca2+ + Mg2+-)-ATPase () and (Na+
+ K+)-ATPase (O) in rat liver.

Fig. 4: ATP-dependence of the (Ca2+ + Mg2+-)-ATPase in rat brain (O) and rat liver ()
at a fixed concentration of Mg2+ (2 mM).

74
 C. O. Bewaji et al.

Fig. 5: Ca2+-dependence of the (Ca2+ + Mg2+)-ATPase in homogenates of rat brain (O)

and rat liver ().

Fig. 6: Rates of ATP hydrolysis as a function of Na+ and K+ concentrations in

homogenates of rat brain (O) and rat liver ().

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 Biokemistri Volume 1, No. 2 (1991)
+ + +
mM K and 140 mM Na /20 mM K . The
+
ATPase was sensitive to K concentrations
greater than 40 mM. Although both cations
were required for optimim ATPase activity,
some residual ATP hydrolysis (about 7.4%
and 9.5% in liver and brain respectively)
+
was observed in the absence of either Na
+ + +
or K . The kinetics of Na and K activation
of the enzyme is illustrated in Fig. 7 (A and
B).
+ +
The (Na + K )-ATPase in brain and
2+ 2+
liver, as well as the (Ca + Mg )-ATPase
in brain, were sensitive to
inhibition by vanadate (Figs. 8 – 10). The
pattern of inhibition was competitive as
shown by the Dixon plots, with apparent
inhibition constants of 3.20 ± 0.32 M and
+ +
3.50 ± 0.05 M for the (Na + K )-ATPase
in the brain and liver respectively. The Ki
2+ 2+
for the (Ca + Mg )-ATPase in the brain
was 6.0 ± 0.25 M. 50% inhibition was
achieved at a vanadate concentration of
+ +
7.5 M for the (Na + K )-ATPase in the
+ +
brain, 7.0 M for the (Na + K )-ATPase in
2+
the liver and 10 M for the (Ca + Mg )-
2+
ATPase in the brain. Vanadate, up to 100
M, had no effect on the Mg2+-ATPase in
the brain and liver (Fig. 11).
A summary of the kinetic constants of
the three ATPases is presented in Table
1. The specific activities of the ATPases
are significantly higher in the brain than in
the liver (P < 0.01). However, the apparent
Km values for the putative ligands in both
2+
tissues are identical, except for the (Ca +
2+
Mg )-ATPase which has a higher affinity
2+
for Ca (P < 0.001) and a lower affinity for
ATP (P < 0.05) in the brain than in the
liver.
DISCUSSION
Cleland (16) has provided a convenient
set of criteria for distinguishing between
various enzymic reactions, based on their
kinetic behaviour. The results of the
present study show that In crude
homogenates of rat brain and liver, the
2+ + +
activities of the Mg -ATPase, (Na + K )-
2+ 2+
ATPase and (Ca + Mg )-ATPase are
76
distinguishable from each other and can
be conveniently assayed. This conclusion
is derived from the kinetics of ligand
interactions of the ATPases and further
confirms the notion that the enzymatic
activities are expressed by distinct
proteins.
It has been observed in several
laboratories that the plasma membrane
2+ 2+ 2+
(Ca + Mg )-ATPase caontains two Ca -
2+
stimulable sites (17,18). The high Ca -
2+
affinity state is stimulated by Ca in the
2+
micromolar range, while the low Ca -
2+
affinity state is stimulated by Ca in the
millimolar range. In the present study, only
2+ 2+ 2+
the high Ca -affinity (Ca + Mg )-Atpase
activity was detected.
The low Ca2+-affinity component
usually observed in many preparations
probably represents a calmodulin
stimulable component which is removed
during membrane isolation or purification.
Calmodulin is a low molecular weight,
2+
acidic, Ca -binding protein which has
been shown to transform plasma
2+ 2+
membrane (Ca + Mg )-ATPase from a
2+ 2+
low Ca -affinity to a high Ca -affinity
state (17). Inadvertent removal of this
protein could be responsible for the low
affinity component usually detected in
isolated or purified systems.
2+
The kinetic properties of the liver (Ca +
2+
Mg )-ATPase distinguishes it from its
counterpart in the brain. The liver enzyme
2+
has a lower affinity for Ca (apparent Km =
14.28 ± 0.12 M) than the brain enzyme
(with a Km of 6.62 ± 0.05 M), and a higher
affinity for ATP (apparent Km = 0.25 ± 0.1
mM) than the brain enzyme (apparent Km
– 0.45 ± 0.01 mM). This is in agreement
with results from other laboratories on
purified enzyme systems (9,10,19). There
is a general tendency to ascribe the
different kinetic behaviour of the purifies
2+ 2+
liver (Ca + Mg )-ATPase to artefacts of
purification. The demonstration of this
phenomenon in crude homogenates
renders this suggestion invalid.
 C. O. Bewaji et al.

Fig. 7: (Na+ + K+)-ATPase activity in rat brain (O) and liver () as a function
of (A) K+ and (B) Na+ concentrations.

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 Biokemistri Volume 1, No. 2 (1991)

Fig. 8: Dixon plot for the inhibition of (Na+ + K+)-ATPase in rat brain by vanadate.

Fig. 9: Dixon plot for the inhibition of (Na+ + K+)-ATPase in rat liver by vanadate.

78
 C. O. Bewaji et al.
The kinetic properties of the Mg -
ATPase in both tissues, with respect to its
2+
interaction with Mg and ATP, is
consistent with the notion that MgATP is
the true substrate for the enzyme, as well
as for other ATPases. For all the ATPases
2+ 2+
(except for the (Ca + Mg )-ATPase of
the liver), maximal ATP hydrolysis was
observed when the concentrations of ATP
2+
and Mg in the assay medium were
equimolar. Inmost enzymic reacions in
which ATP is utilised, the true substrate
2-
has been shown to be MgATP (20,21).
Furthermore, most ATP-dependent
enzymes have a higher affinity for
2-
MgATP than the uncomplexed ATP4-.
Vincenzi and Hinds (8) have reported
2+
that Mg alone, in the absence of the
enzyme, can induce some ATP hydrolysis.
The existence of a stable enzyme-MgATP
complex has been demonstrated for the
2+ 2+
erythrocyte (Ca + Mg )-ATPase (22) and
+ +
for the (Na + K )-ATPase (23).
Pedemonte and Balegno (22) further
observed that in the operation of
2+
erythrocyte membrane (Ca
ATPase three different effectors are
2-
required: MgATP (acting as substrate)
2+ 2+
and free Mg and Ca as essential
cofactors or activators. However, in the
present study, it does not appear that free
2+
Mg is involved in the operation of the
2+ 2+
(Ca + Mg )-ATPase. This can be
2+
deduced from the fact that Mg in excess
of ATP was inhibitory and vice versa.
+ +
Na and K are clearly antagonistic in
their interactions with the ATPase. The
binding of one reduces the affinity of the
enzyme for the other. This is in agreement
with what is usually reported for purified
systems.
The general trend in this study was that
the Vmax of the ATPases were significantly
higher in the brain than in the liver (P <
0.01). This observation is of physiologica;
significance since the cations translocated
by the enzyme are also involved in the
generation of membrane potential and in
neurotransmitter release. The brain, being
an excitable tissue, has to deal with more
2+ +
Ca and Na fluxes than the liver. It has
been observed that the maximal activity of
2+ 2+
the (Ca + Mg )-ATPase is generally in
excess of what is required for the extrusion
2+
of Ca from non-excitable cells (19).
2+
Sensitivity to vanadate provides another
criteria for the characterization of the
ATPases in crude homogenates. Under
2-
the optimal assay conditions defined in the
present study, the ATPases could be
classified into three categories: (i) those
that are potently inhibited by vanadate (the
+ +
(Na + K )-ATPase of the brain and liver
2+ 2+
and the (Ca + Mg )-ATPase of the
brain); (ii) those that are relatively
2+ 2+
insensitive to vanadate (the (Ca + Mg )-
ATPase of the liver; and (iii) those that are
completely insensitive to vanadate (the
2+
Mg -ATPase in both tissues).
It is now generally agreed that vanadate
competes with phosphate for the same
binding site on the enzyme. The
insensitivity of the Mg2+-ATPase to
vanadate (up to 100 M) confirms that it is
2-
a separate enzyme distinguishable from
the (Na+ + K+)-ATPase or the (Ca2+ +
Mg2+)-ATPase. This opens the question
about the mechanism of action (or
enzymatic cycle) of the Mg2+-ATPase.
Although it is not an ion pump, a detailed
2+
+ Mg )- study of this enzyme could throw more
light on how a pumping event is achieved
by the cation-pumping ATPase. This can
be achieved by a comparative study of the
enzymatic cycle of the Mg2+-ATPase
(which is not a pump) and any of the cation
pumps.
REFERENCES
1. Carafoli, E. and Scarpa, A. (Eds.) (1982)
Transport ATPases. Ann. N. Y. Acad. Sci.
402, 1 – 604.
2. Nakao, M.; Nakao, T.; Hara, Y.; Nagai, F.;
Yagasaki, S.; Koi, M.; Nakagawa, A. and
Kawai, K. (1974) Purification and
properties of (Na+ + K+)-ATPase from pig
brain. Ann. N. Y. Acad. Sci. 242, 24 – 30.
3. Niggli, V.; Adunyah, E. S.; Penniston, J.
T. and Carafoli, E. (1981) Purified (Ca2+ +
Mg2+)-ATPase of the erythrocyte
membrane: Reconstitution and effect of
calmodulin and phospholipids. J. Biol.
Chem. 256, 395 – 401.
4. Norgaard, A.; Kjeldsen, K. and Hansen,
O. (1984) (Na+ + K+)-ATPase activity of
crude homogenates of rat skeletal muscle
as estimated from their K+-dependent 3-
O-methylfluorescein phosphatase activity.
Biochim. Biophys. Acta 770, 203 – 209.
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 Biokemistri Volume 1, No. 2 (1991)

Fig. 10: Dixon plot for the inhibition of (Ca2+ + Mg2+)-ATPase in rat brain by vanadate.

Fig. 11: Vanadate sensitivities of the Mg2+-ATPase in rat brain (O), Mg2+-ATPase in rat liver (), and
the (Ca2+ + Mg2+)-ATPase in rat liver ().

80
 C. O. Bewaji et al.

Table 1: Apparent kinetic constants of ATPases in homogenates of rat brain and liver.

Constants Tissues Mg2+-ATPase (Ca2+ + Mg2+)- (Na+ + K+)-

ATPase ATPase

Km (Mg2+) (mM) Brain 0.40 ± 0.04

Liver 0.42 ± 0.02
2+
Km (Ca ) (mM) Brain 6.67 ± 0.05
Liver 14.28 ± 0.12
Km (ATP) (mM) Brain 0.38 ± 0.14 0.45 ± 0.01 0.29 ± 0.02
Liver 0.40 ± 0.06 0.25 ± 0.10 0.27 ± 0.04
+
Km (Na ) (mM) Brain 25.00 ± 0.025
Liver 26.31 ± 1.31
Km (K+) (mM) Brain 4.55 ± 0.03
Liver 4.35 ± 0.23
Vmax (mol/mg. Brain 2.98 ± 0.06 3.85 ± 0.21 4.81 ± 0.30
prot./hr)
Liver 2.14 ± 0.18 2.70 ± 0.10 2.95 ± 0.17

The Km and Vmax were calculated from double reciprocal plots. Each value represents the
mean ± S.D. of triplicate determinations.

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 Biokemistri Volume 1, No. 2 (1991)
5. Kornberg, H. L. and Reeves, R. E. (1972)
Correlation between hexose transport and
phosphodiesterase activity in Escherichia
coli. Biochem. J. 126, 1241 – 1243.
6. Sols, A.; Felicy, J. A. and Aragon, J. J.
(1975) Study of enzyme activity in animal
cells in situ. In: Metabolic Interconversion
of Enzymes. (Shaltiel, S. ed.). Springer-
Verlag, Berlin, Heidelberg, New York. Pp.
191 – 197.
7. Rosenthal, A. S.; Kregenow, F. M. and
Moses, H. L. (1970) Some characteristics
of a Ca2+-dependent ATPase activity
associated with a group of erythrocyte
membranes which form fibrils. Biochim.
Biophys. Acta 196, 254 – 262.
8. Vincenzi, F. F. and Hinds, T. R. (1976)
Plasma membrane calcium transport and
membrane-bound enzymes. In: The
Enzymes of Biological Membranes,
(Martonosi, A. ed.). Vol. 3, pp. 261 – 281.
Plenum Press, New York.
9. Lotersztajn, S.; Itanoue, J. and Pecker, F.
A. (1981) High affinity calcium-stimulated
magnesium-dependent ATPase in rat liver
plasma membranes: dependence on an
endogenous protein activator distinct from
calmodulin. J. Biol. Chem. 256, 11209 –
11215.
10. Iwasa, Y.; Iwasa, T.; Higashi, K.; Matsui,
K. and Miyamoto, E. (1982)
Demonstration of a high affinity Ca2+-
ATPase in rat liver plasma membrane.
Biochem. Biophys. Res. Commun. 105,
488 – 494.
11. Bewaji, C. O. and Odutuga, A. A. (1987)
Interactions of some local anaesthetic and
antisickling drugs with calcium-carrier
proteins in biomembranes. Nig. J. Pur.
Appl. Sci. 2, 6 – 13.
12. Bewaji, C. O.; Ojo, C. I.; Malomo, S. O.
and Odutuga, A. A. (1991) Effects of
some antimalarial drugs on adenosine
triphosphatase activities of rat liver and
brain. Biosci. Res. Commun. 3, 243 –
251.
13. Gornal, A.; Barbwill, C. J. and David, M.
M. (1949) Determination of serum proteins
by means of the biuret reaction. J. Biol.
Chem. 177, 751 – 766.
82
14. Bewaji, C. O.; Olorunsogo, O. O. and
Bababunmi, E. A. (1987) Further
characterization of the membrane-bound
(Ca2+ + Mg2+)-ATPase from porcine
erythrocytes. Int. J. Biochem. 19, 721 –
724.
15. Fiske, C. H. and SubbaRow, Y. (1925)
The colorimetric determination of
phosphorus. J. Biol. Chem. 66, 375 – 400.
16. Cleland, W. W. (1963) The kinetics of
enzyme-catalyzed reactions with two or
more substrates and products. Biochim.
Biophys. Acta 67, 104 – 137.
17. Carafoli, E. (1984) Calmodulin-sensitive
calcium-pumping ATPase of plasma
membranes: Isolation, reconstitution and
regulation. Fed. Proc. 43, 3005 -3010.
18. Condrescu, M.; Osses, L. and DiPolo, R.
(1984) Partial purification and
characterization of the (Ca2+ + Mg2+)-
ATPase from squid optic nerve plasma
membrane. Biochim. Biophys. Acta 769,
261 – 269.
19. Carafoli, E. (1984) Plasma membrane
Ca2+ transport and Ca2+ handling by
intracellular stores: an integrated picture
with emphasis on regulation. In:
Mechanisms of Intestinal Electrolyte
Transport and Regulation by Calcium,
(Opie, L. H. ed), pp. 121 – 134.
20. Lipman, F. (1941) Metabolic generation
and utilization of phosphate bond energy.
Adv. Enzymol. 18, 99 – 162.
21. Pedersen, P. L. and Carafoli, E. (1987)
Ion motive ATPases. I. Ubiquity,
properties and significance to cell
function. Trends Biochem. Sci. 12, 146 –
150.
22. Pedemonte, C. H. and Balegno, H. F.
(1981) Ca2+-stimulated ATPase from red
cell membranes: Studies on the kinetic
mechanism. Comp. Biochem. Physiol. 70,
559 – 564.
23. Jorgensen, P. L. (1982) Mechanism of the
Na+/K+ pump protein structure and
conformation of the pure (Na+ + K+)-
ATPase. Biochim. Biophys. Acta 694, 27
– 68.