The Journil of Blochemiitry, Vol. 63, No. 5, 1968
Magnesium-Adenosine Triphosphatase Activated
by a Low Concentration of Calcium
in Brain Microsomes
By YOSHIAKI NAKAMARU
(From the Department of Biology, College of General Education,
Tohoku University, Sendai)
1. The distribution of the strophanthin-sensitive ATPase (SS-ATPase)
and the Mg+Ca-ATPase, and the effect of deoxycholate (DOC) on these
enzymes activities, were investigated in several fractions of pig brain.
2. The presence of the Mg+Ca-ATPase in the brain microsomal
fraction was clearly shown. It was also shown that the Mg+Ca-ATPase
was very similar to the SS-ATPase in its distribution pattern in several
fractions of the brain homogenate and in its sensitivity to DOC.
3. The microsomal fraction extracted with 0.1% DOC solution
exhibited high Mg+Ca-ATPase activity and high SS-ATPase activity.
For maximum activity of the strophanthin-insensitive ATPase, 3X10~ J M
Ca was necessary in the presence of 2x10"'M Mg, and 4X10~*M Mg
was necessary in the presence of 3X10" ! M Ca.
The presence of two different ATPases
[EC 3.6.1.4, ATP phosphohydrolase] in the
microsomal fraction of the brain is well
known. Considerable information is available
on the Na + K-ATPase which is activated by
Mg, Na and K, inhibited by g-strophanthin
and is thought to participate in active ion
transport (1—4). However, the enzymatic
properties of the Mg-ATPase, which is activated by Mg alone and is insensitive to
g-strophanthin, are still obscure. In the
previous paper (5) it was demonstrated that
a low concentration of Ca slightly stimulated
the Mg-ATPase [EC 3.6.1.4] of the brain
microsomal fraction which was isolated with
0.3 M mannitol solution. However, the preparation was contaminated with a small
amount of mitochondria (5). Ca-activated
ATPase is also present in the mitochondria
of various tissues {6—10). Thus there would
be a possibility that the Mg+Ca-ATPase
activity in the microsomal fraction might be
derived from the contaminating mitochondrial
fragments.
626
This paper demonstrates the actual presence
of a Mg+Ca-ATPase [EC 3.6.1.4] in the
brain microsomal fraction. Thus, there was
a higher Mg+Ca-ATPase activity in the
microsomal fraction than in the mitochondrial fraction using deoxycholate (DOC) treatment. To confirm the presence of the Mg+
Ca-ATPase in the microsomal fraction, the
distribution pattern of the Mg+Ca-ATPase
was compared with that of the Na+K-ATPase
(strophanthin-sensitive ATPase) in several fractions from the brain homogenate, as the highest
Na+K-ATPase activity is known to be present in the microsomal fraction (4 ). If there
were a parallel between the distributions of
Mg+Ca-ATPase and the Na+K-ATPase,
then the Mg+Ca-ATPase might exist with
the Na+K-ATPase on the membrane structures.
MATERIALS AND METHODS
Fractionation of Brain Homogenate—All procedures
were carried out at 0—4°C. The gray matter of the
pig brain was isolated, as previously described (77),
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(Received for publication, August 7, 1967)
ATPase of Brain Microsomes
The microsomal fraction (D) was prepared by the
following procedures. The residual supernatant after
removal of P, was recentrifuged at 15,000x« for 1 hr.
Then the supernatant was carefully decanted and
•centrifuged at 64,000x« for 1 hr. The resulting pellet
was suspended in a suitable amount of 5 mM Trismaleate buffer (pH 7.2) with a Teflon-glass homogenizer, and centrifuged at 105,000X£ for 1 hr. The
pellet was then separated, resuspended in the same
ibuffer (2—3 mg protein/ml), and used as the microaomal fraction (D).
When desired, fractions A, B, C and D were subsequently treated with DOC as follows. A suspension
of the fraction (2—5 mg protein/ml) was mixed with
.an equal volume of 0.08% DOC solution at room
temperature. After ten minutes 0.5 ml of the mixture
was used as enzyme sample in the reaction mixture.
The microsomal fraction was also prepared in the
presence of 0.1% DOC as described previously ( 7 / ) .
Enzymi Assays—ATPase activity was estimated as
•described previously (5).
Strophanthin-insensitive
ATPase (SI-ATPase) was assayed in the presence of
130m«Na, 20msiK and 1.25X10~'M «-strophanthin.
-Strophanthin-sensitive ATPase (SS-ATPase) activity
was calculated as the difference between the total
ATPase activity, assayed in the presence of 130 mM
Na and 20 mM K and in the absence of ^-strophanthin,
and the SI-ATPase activity. Succinate dehydrogenase
[EC 1.3.99.1] was measured colorimetrically by
SLATER and BONNER'S method (/6"). Acetylcholine.sterase [EC 3.1.1.7] was measured by HESTRIN'S
method (17, 18). Measurements were performed at
_37°C, and pH 7.4 obtained with Tris-HCl buffer.
RESULTS
Comparison of Enzymatic Properties of Microsomes with Those of Other Fractions—Fig. 1 shows
the succinate dehydrogenase, acetylcholinesterase and SS-ATPase activities in various
fractions of the brain homogenate. Succinate
dehydrogenase activity decreases in the order
fraction A>B>G and D, whereas acetylcholinesterase increases in the above order. SSATPase activity is the lowest in fraction D,
and the highest in fraction B.
In the presence of g-strophanthin the
brain microsomes split a considerable amount
of ATP. This ATP splitting could be increased by Ca contaminating the microsomes
(77). As shown in Fig. 2, the Mg-ATPase
activity in each fraction is considerably lower
in the presence of 0.125 mM glycoletherdiaminetetraacetate (EGTA) than in the presence of 3X10" 5 M Ca. A difference is seen
in the distributions of these two activities.
Thus Mg+Ca-ATPase activity is the highest
in fraction B, and the lowest in fraction A.
The Mg-ATPase activity which is insensitive
to EGTA is, however, high in fraction A.
\
L5
5
'
1
[l.Oi
i
;o.5-
B
C
FRACTION
Fio. 1. Distribution of SS-ATPase, succinate
dehydrogenase and acetylcholinesterase in fractions
A, B, C and D. Fractions were prepared as described in " MATERIALS AND METHODS."
—O— Repraents SS-ATPase activity in the
presence of 2 mu Mg, 130 nm Na, 20 mM
K and 1.25 mu ATP.
— • — Succinic dehydrogenase activity.
- - • - - Acetylcholinesterase activity.
Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
and homogenized in a waring Blendor with 10 volumes
of 0.32 M sucrose solution for 20 sec. After centrifugation at 1500X/ for lOmin, the resultant supernatant
was further centrifuged at 15,0OOX£ for 15 min. The
precipitate (P,) obtained, including most of the mitochondria, myelin fragments, nerve endings and some
of the heavy microsomes (12—15), was mixed with
2 M sucrose to give a 1.2 M sucrose solution. Then an
equal volume of 1.2 M sucrose was added to this suspension. Subsequent centrifugation at 105,000Xf for
2 hr gave three fractions; a precipitate (P t ), a middle
turbid layer (S,), and a white, creamy top layer (S,).
Each of these fractions was separated and suspended
in four to five volumes of 5 mM Tris-maleate-NaOH
buffer (pH7.2) with the aid of a Teflon homogenizer.
In the case of fraction S!, 3 volumes of Tris-maleate
buffer were added. After centrifugation at 105,000Xf
for 1 hr, each precipitate was thoroughly homogenized
with 5 mM Tris-maleate buffer (pH 7.2), yielding fraction A from P j , fraction B from S!, and fraction C
from Si.
627
628
Y. NAKAMARU
Effect of DOC Treatment on ATPase Activity
C
FRACTION
D
FIG. 2. Distribution of SI-ATPase in fractions
A, B, C and D.
The mixture contained 5 mM Mg, 130 mM Na,
20 mM K, 1.25 mM ATP and 1.25x10-'M gstrophanthin, pH 7.4, at 37°C.
—O— In the pretence of 3 X 1 0 " 5 M Ca.
— • — 1.25X1O-*M EGTA.
- - • - - Difference regarded as Mg+Ca-ATPase
activity.
A
B
C
FRACTION
Fio. 3. Effect of DOG treatment on SSATPase in fractions A, B, C and D.
Activity was measured in the presence of 2 mil
Mg, 130 mM Na, 20 mM K and 1.25 mM ATP at
pH 7.4, at 37°C.
—O— Without DOC treatment.
— • — After 0.08% DOC treatment.
- - • - - Difference.
B
C
FRACTION
Fio. 4. Effect of DOC treatment on MgATPase in fractions A, B, C and D in the
presence of g-strophanthin and EGTA.
Conditions for determination of activity were:
as in Fig. 2.
—O— Without DOC treatment.
— • — After 0.08% DOC treatment.
--%-- Difference.
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B
—It is known that DOC stimulates the Na +
K-ATPase of brain microsomes, both during
isolation of the enzyme (4, 11) and on
treatment of the final preparation (19). Fig.
3 illustrates the effect of DOG treatment on
the SS-ATPase activities of the four fractions.
Before treatment fraction B shows the highest
activity and fraction D shows the lowest,
whereas the activity in fraction D is markedly stimulated by treatment with 0.08% DOG.
The SS-ATPase activity of fraction A is not
affected by this treatment.
When the ATPase activity is determined
in the presence of g-strophanthin and EGTA,
stimulation of all fractions except fraction D
by DOC is observed, as shown in Fig 4.
Fig. 5 shows the effect of DOC treatment
on the Mg+Ca-ATPase activity which was.
estimated by the difference in activities as in.
Fig. 2. It can be seen that the low Mg+CaATPase activity of fraction D is increased.
6-fold by 0.08% DOC treatment. Moreover,,
distribution pattern of Mg+Ga-ATPase activity before and after DOC treatment issimilar to that of SS-ATPase activity shown
in Fig. 3, and entirely different from that of
a Mg-ATPase shown in Fig. 4. These results.
ATPase of Brain Microsomes
D
Fio. 5. Effect of DOC treatment on Mg+
Ca-ATPase in fractions A, B, G and D.
Conditions for determination of activity were
the same as for Mg+Ca-ATPase, in Fig. 2.
—O— Without DOC treatment.
— • — After 0.08% DOC treatment.
- - • - - Difference.
10
TIME (mln)
20
suggest a closer relationship of the Mg+CaATPase to the SS-ATPase than to the MgATPase.
The Mg+Ca-ATPase of DOC-extractcd Microsomes—Extraction of the brain microsomal
fraction with 0.1% DOG solution yielded a
fraction with high SS-ATPase and SI-ATPase
activities, as previously described {11). Fig.
6 shows the time course of orthophosphate
liberation from ATP by DOC-extracted microsomes. In the presence of 10~5 M Ca, the
activity is extremely low but it increased
greatly on addition of 2 mil Mg. However,
there was a considerable decrease in activity
in the presence of 6 X 1 0 ~ S M EGTA in addition to Mg. Similar results were obtained
in the absence of Na and K. The optimum
Mg and Ca concentrations for the SI-ATPase
of DOC-extracted microsomes are 4 X 1 0 ~ 3 M
Mg in the presence of 3X10~ 5 M Ca, and 3 x
10"5 M Ca in the presence of 2 X 1 0 " 8 M Mg,
as shown in Fig. 7. The high activity in the
30
Fio. 6. Time course of orthophosphate liberation by the microsomes extracted with 0.1%
DOC.
Conditions were ; 130 mu Na, 20 n u K,2.5mM
ATP, 1.25x10-* M g-strophanthin.
—O— In the presence of 2 mM Mg and 6 x 10"* M
EGTA.
—•— 2mu Mg and 10~s u Ca.
- - • - - IO-'M Ca.
K T 7 icr 6 icr 9 io~4 io~3 icr2 icr1
CONCENTRATION OF DIVALENT ION (M)
Fio. 7. The effect of Mg or Ca on the
ATPase activity of brain microsomes extracted
with 0.1% DOC.
The conditions were; 50 mu Tris-HCl (pH
7.4), 130 nm Na, 20 HIM K, 1.25 mu ATP and
1.25xlO-*u g-strophanthin, at 37°C.
— • — Ca in the presence of 2 mu Mg.
—O— Mg in the presence of 3xl0~ 5 u Ca.
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B
C
FRACTION
629
630
Y. NAKAMARU
absence of Ca shown in Fig. 7, is probably
due to Ca contaminating the microsomes,
and is decreased to some extent by addition
of EGTA, as shown in Fig. 6.
It has been demonstrated that Ca alone
can stimulate the ATPase of rat liver microsomes (21), pig brain microsomes ( 5 ) and
the relaxing granules of skeletal muscle (22).
In these cases the Ca concentration required
for maximum stimulation is above 5 HIM. A
low concentration of Ca can, however, increase
the activity in the presence of Mg, as reported in the case of the relaxing granules of
muscle (22). HASSELBACH and MAKINOSE
especially suggested the participation of this
extra-splitting of ATP by Ca, in active Ca
incorporation into relaxing granules (23),
though EBASHI and YAMANOUCHI could not
show any stoichiometric relation between Ca
uptake and ATP splitting at low ATP concentration (24). It has recently been reported
that the microsomal fraction of the brain
Konishiof the Biological Institute of this University, for
his continued encouragement and valuable suggestions
in the preparation of the manuscript. He also wiihes
to thank Prof. K. Aoki of the same institute, Prof.
K. Atoda and Prof. T. Nagano of this college for
their encouregement.
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DISCUSSION
The SI-ATPase of brain microsomes can
It is generally accepted that the N a + K be
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ATPase
is
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!
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M
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Several investigators have, however, suggested
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(Fig. 3). These facts suggest that some of the
SS-ATPase in fraction B was derived from
The author is much indepted to Asst. Prof. K.
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ATPase of Brain Microsomes
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631