The Journal of Biochemistry, Vol. 65, No. 1, 1969
Accumulation of Arsenate-76 by Mitochondria*
By YASUO KAGAWA and
AKIKO KAGAWA
(From the Department of Biochemistry, Faculty of Medicine,
SKnshu University, Asahi, Matsumoto, Japan 390)
(Received for publication, June 10, 1968)
1. Accumulation of arsenate-76 by rat liver mitochondria was measured to study
phenomena which are not explained by the arsenolysis of a high energy intermediate.
2. Stimulation of ATPase [EC 3. 6. 1. 3] or respiration of mitochondria by arsenate
was accompanied by its accumulation. This reaction required energy and a divalent
cation.
3. Respiration-dependent arsenate accumulation required adenine nucleotide and
was inhibited by atractyloside but was stimulated by oligomycin. These were the marked
differences from phosphate accumulation.
4. The relationship between arsenate accumulation and phosphorylating mechanism
is discussed, since effects of arsenate have been considered to support the presence of a
high energy phosphorylated intermediate.
Inorganic arsenate (Asi)** is an interesttool to analyse oxidative phosphorylation,
since Asi resembles Pi, but Asi does not form
any stable bond like phosphate esters. If Asi
us substituted for Pi in substrate level phos-phorylation, a reaction called arsenolysis takes
place owing to spontaneous hydrolysis of the
;arsenate compounds.
Interaction of Asi with mitochondria
rresults in lowered P: O ratios (1), activation
• of latent ATPase [ATP phosphohydrolase EG
:3.6.1.3] (2), and stimulation of state 4 respiration (3). These effects have been interpret• ed by arsenolysis of an unknown high energy
* This work was presented at the 39th annual
-.meeting of Japanese Biochemical Society, at Kyoto in
INovember 1966.
** The following abbreviations were used;
EDTA: Ethylene diaminetetraacetate,
Asi: Inorganic arsenate,
Pi: Inorganic phosphate,
X ~ Y : A nonphosphorylated high energy compound produced in the process of oxidative phosphorylation,
X ~ P i : A stable high energy phosphate bond
derived from X~Y,
_X--*Asi: An unstable arsenate bond derived
from X~Y,
JJNP: 2,4-Dinitrophenol.
105
H,0
X~Asr
• X + As,
intermediate (X~Y) (2). However, the following phenomena are not explained by this
scheme of Asi effect: 1. a time lag is required to depress the P: O ratio (1, 4), but not
to induce ATPase (4). 2. The addition of
ADP is needed to release respiration (5).
Stimulation of ATPase by Asi is abolished
by the addition of an ATP-regenerating system (5). 3. The time-dependent decrease of
P: O ratio is prevented by EDTA ( 4), and
the Asi-induced ATPase activity is stimulated by Mg++ (2). 4. Asi liberates endogenous
"Pi (4).
It should be pointed out that both
arsenate-stimulated ATPase and respiratory
control were lost when the structure of intact
mitochondria was destroyed. Asi accumulation might be overlapped on the above uncoupling phenomena, since a possible phosphorylating site, which is coupling factor 1
(mitochondrial ATPase), is localized on the
matrix side of the inner membrane (6, 7).
Both uncoupling effect of Asi (3, 8) and
activity of coupling factor 1 are equally block-
106
Y. KAGAWA and A. KAGAWA
ed by oligomycin (9, 10). Stimulation of
anion accumulation by parathyroid hormone
has been reported (11), but Asi accumulation in the above mentioned conditions has
not been described.
Accumulation of Pi has been studied
extensively, but because of the presence of
large amounts of phosphate in mitochondria,
and rapid formation of phosphate esters, use
of Asi instead of Pi is advantageous. Accumulation of Asi or Pi might play an important role in oxidativep hosphorylation. Chemiosmotic theory, for instance, assumes that
Pi is present in the H+-accumulating compartment of anisotropic ATPase (12).
The purpose of this work is to determine
permeation and accumulation of Asi in mitochondria. Owing to the difficulties to determine the amount of Asi and Pi separately,
"Asi, "Pi and AT SJP were used.
MATERIALS AND METHODS
Mitochondria were isolated according to the method
of HOOEBOOM (13) from livers of Donryu strain male
albino rats, about 3 months old, and the suspension
was kept in an ice bath at a protein concentration of
about 20 mg per ml in 0.25 M sucrose. Mitochondria
were freshly prepared every time and the respiratory
control ratio (4 to 5) was checked with an oxygen
electrode.
7S
Asi was the product of Japan Research Reactor 3
(Uran-DjO, 10 Mega W) by means of the "As (n, f)
"As reaction. Since the half life of "As is 26.4 hr,
experiments were performed within 10 days after each
lot was prepared, and the purity was checked by a
400 channel pulse height analyser (Mitsubishi Electronic
Co., Tokyo, Model ND 1666) for gamma-ray spectroscopy, and by decay rate or chemical properties. "Ca,
32
P and 14C-ADP were purchased from Japan Isotope
Society, Tokyo. AT"P was synthesized with red blood
cells in the presence of glucose (14).
Nucleotides and antimycin A were the products
of Kyowa Hakko Co., Tokyo and valinomycin, rutamycin (oligomycin family) and atractyloside were the
gifts from Dr. S. Minakami. Blue dcxtran 2000 was
purchased from Pharmacia Co., Sweden. Other auxiliary reagents and enzymes were the same as described
in a previous paper (70).
Aerobic Ion Accumulation—The standard reaction
mixture contained 1 //mole of Na2H"AsO4 (about
10,000cpm), l//mole of CaCl,, 5/imoles of MgCl,,
2/imoles of ADP, 25^moles of sucrose, 10/Jg of rutamycin, 400/ig of cytochrome c and fresh mitochondria
(5 mg of protein), in a total volume of 1.0 ml. In some
experiments 48Ca, "C-ADP or "Pi were used in place
of nonradioactive constituents in the medium where
"Asi was omitted or cold Asi was added instead of
"Asi.
The reaction was started by the addition of a
mitochondrial suspension which corresponded to about
0.2 g of wet liver. The mixture was incubated in a
15 ml flat-bottom flask with a shaking rate of 1 cycle
per second at 37°C for lOmin. Then the reaction was
stopped by the addition of 15pg of antimycin A, and
the reaction mixture was transferred into a 5 ml transparent plastic tube (1 g), and centrifuged at 8000X£
for 5min at 0°. The resulting pellet was suspended
in 1 ml of 0.25 u sucrose at 0° and again centrifuged.
The pellet was suspended in 1 ml of distilled water,
poured in an alminum planchet (0.7 g), dried to constant weight and counted in an automatic gas-flow
counter (Japan Radio Co., Tokyo, Model LBC-22).
Wet and dry weights of the pellet were measured to
determine intramitochondrial water. When oxygen
consumption was measured polarographically, incubation volume was increased to 3.5 ml, with the same
concentration of the ingredients.
Anaerobic Ion Accumulation—The incubation medium
contained 5//moles of ATP and 15/ig of antimycin A
instead of rutamycin, succinate, cytcchrome c, and
ADP in the medium described in aerobic ion accumulation. The same procedures for incubation and isolation
of mitochondria were applied, except the use of 10/Jg
of rutamycin to stop the reaction.
Ion Permeation—The aerobic condition described
above with antimycin and Blue Dextran, without cytochrome c was used. After first centrifugation, the
weight and radioactivity of the pellet were measured.
Minor correction was made for extramitochondrial
water with Blue Dextran, which was measured by light
absorption at 620 mft after the pellet was dissolved with
3% cholate. The corrected wet weight minus dry
weight of the pellet was considered as intramitochondrial water space.
Other Determinations—ATPase activity was measured
with hydrolysis of AT"P, and the Pi was extracted
by the reported methods (15). Oxygen uptake was
measured polarographically (10), and protein concentration was measured as described previously (10 )•
RESULTS
Energy Requirements for
76
Asi Accumulation—
Substrate oxidation and ATP hydrolysis were
necessary for 78Asi accumulation in aerobic
and anaerobic conditions, respectively (Table
I). Asi accumulation was much less in the
anaerobic condition than in the aerobic. Es-
Accumulation of "As by Mitochondria
TABLE
107
I
Energy requirement for ?BAsi accumulation in mitochondria.
Experimental conditions were the same as described in " METHODS " except that antimycin was replaced
by 1/imole of KCN in anaerobic medium, and additions or omissions indicated in the table.
Asi in mitochondria (protein 1,9 mg)
Condition
Experiment 1
Experiment 2
Aerobic
(m/imoles)
Complete
—succinate
-ATP
+ DNP
+ antimycin (15/ig)
+oligomycin1> (5/ig)
-Mg++
111
15
Complete - K + , Na+
+KC1 10/imoles
+KC1, +valinomycin 1/ig
129.5
123.5
124.2
Anaerobic
(m/imoles)
24
7.4
2.4
9.5
13.1
10.0
13.2
6.2
1) Rutamycin.
sentially the same results were obtained when
succinate was replaced by 10/*m.oles of glutamate. Since the reaction was abolished by
0.5/anoles of DNP in both aerobic and
anaerobic systems, this inhibition of Asi accumulation may be caused by the uncoupling of oxidative phosphorylation. Valinomycin, an uncoupler which induces K+ accumulation (16), did not inhibit Asi uptake.
Respiratory inhibitors such as KCN or antimycin A stopped the aerobic Asi accumulation. A requirement for Mg++ was noticed
in both conditions, although addition of Mg++
was not essential for oxidative phosphorylation in intact mitochondria. Most of the
radioactivity in the mitochondria was identified as Asi by extraction and formation of
arsenomolybdate. Since only a small part
of added radioactivity was accumulated, and
"As decayed rapidly, it was essential to confirm the purity of radioactivity in mitochondria by the pulse height analyser (Fig. 1).
,0.56MeV
Back Scatter
2.06 MeV
t / \ .
100
200
300
CHANNEL NUMBER (Energy of r-R«y)
400
F i o . 1. r ' R a Y S p e c t r u m of " A s accumulated
in m i t o c h o n d r i a . A pulse hight analyzer equipped
with 400 channels of energy level was ujed. T h e
detector was N a l containing T l .
T w o standards were u s e d ; 1>7 Cs (0.6616 MeV)
corresponded to 130th channel, a n d M C o (1.1728
and 1.3325 M e V ) to 234th a n d 265th channel respectively.
Time Course of the Reaction—The reaction
rate was constant except for the first few
minutes under the aerobic condition (Fig. 2).
The amount of Asi accumulated was proportional to the amount of mitochondria
added. Owing to both the incompleteness
of rutamycin inhibition and liberation of Pi
from ATP, the accumulation of Asi was not
proportional to time under the anerobic condition.
Metal Requirement—Divalent cations were'
108
Y. KAGAWA and
A. KAGAWA
1,200 -
10
TIME (min)
Fio. 2. Time course of Asi accumulation.
Experimental conditions were as described in
" aerobic ion accumulation ", with different incubation time.
—O— "Asi accumulation without CaCl 2 .
—-•— "Asi accumulation with CaClj.
TABLE
II
6
Mttal rtquiremtnts for ? Asi accumulation
in mitochondria.
Experimental conditions were aj described in
"aerobic ion accumulation", except for addition
of 1/imole of divalent cation (Cl salt) and mitochondrial protein 2.0 mg.
Asi in mitochondria (m/imoles)
+ Mg ++
None
Ca ++
Co ++
Mn ++
Ba ++
28.0
81.5
37.6
251.2
226.0
-Mg++
- M g + + , +EDTA
8.0
7.5
21.5
36.4
7.0
9.1
104.0
193.0
8.7
70.7
effective in supporting Asi accumulation. Effect of Mg + + was weak, and 20—30% of the
accumulated Asi was lost during washing.
Omission of Mg ++ and addition of EDTA
caused marked decrease in Asi accumulation
(Table II). Asi accumulation was promoted
when concentration of Ca ++ was increased
Fio. 3. Influence of CaClj concentration on
"Asi accumulation. Experimental conditions were
as in the legend for Fig. 2, with different amount
of CaCl2 and ADP as indicated in the figure.
(Fig. 3). Release of state 4 respiration by
Asi plus ADP was only slightly depressed by
the omission of Mg ++ or by the addition of
EDTA.
Specificity of Asi Among Aninons—Pi sup-
ported "Ga + + accumulation in the absence
of ADP, while Asi did not (Table III). Addition of adenine nucleotide was essential
for Asi accumulation. Accumulation of Ga ++
stimulated by sulfate or borate was less than
that by Asi in the presence of 1 mM ADP.
Uncoupling action of Asi was specific among
aninons {cf. ref. 2). In the aerobic condition, Asi accumulation was greatly stimulated by the addition of an energy-transfer
inhibitor, while that of Pi was little (Table
IV), if ADP was replaced by ATP to prevent
competition between ion accumulation and
ATP formation {cf. ref 16).
Adenine Nucleotide Requirement—Adenine
nu-
cleotide was specifically required for Asi accumulation (Table V). Addition of 100 /anoles
glucose and 20 mg of hexokinase [ATP: r>
hexose 6-phosphotransferase EG 2.7.1.1] inhibited Asi transport with or whitout rutamycin. Since all the adenine nucleotides were
Accumulation of "As by Mitochondria
TABLE
109
III
Differtnu bttwetn Asi and Pi in aerobic 45Ca accumulation.
Experimental conditions were the same as described in "aerobic ion accumulation" in which anions
were as indicated in the table with 5.2 mg of mitochondrial protein. In Experiment 1, ADP was omitted
and 2/imoles (6800 cpm) of "CaCl, was used. In Experiment 2, ADP was replaced by ATP and l/<mole
(3400cpm) of "CaClj was used.
"Ca ++ in mitochondria (cpm)
Anion (nw)
Experiment 1
Experiment 2
+Mg++
-Mg++
-ADP
Asi
Pi
0
0
5
5
0
5
0
5
Asi
Pi
-ATP
+ATP
0
5
5
0
1569
63
3030
187
5746
167
5101
TABLE
-ADP
338
383
321
320
-Mg++
+ATP
690
878
2177
IV
Effect of oligomydn on ?6Asi and MPi accumulation without Ca+*.
Experimental conditions were as described in "aerobic ion accumulation", except that CaCl2 was
omitted and ADP was replaced by 2/imoles ATP, with anions indicated in the table and 4.0 mg of mitochondrial protein.
Anion in mitochondria (m/imoles)
Concentrations of Pi
or Asi
'•Asi
"Pi
H
—oligomycin '
+oligomycin
— oligomycin
+oligomycia
l
11.5
14.5
70.2
71.9
2
20.5
29.8
105.0
110.9
5
33.0
65.0
171.0
195.3
1
° Rutamycin.
•converted into AMP by ATP: AMP phosphotransferase [EG 2.7.4.3] and hexokinase,
A M P remaining in the assay system may not
rsupport accumulation of Asi.
Effect of Atractyloside—Atractyloside inhibited accumulation of Asi, Ga ++ and ADP (Table
"VI). Release of state 4 respiration caused by
Asi and ADP was markedly depressed by
.atractyloside.
Effect of ATP
Regenerating
System—Addi-
tion of phosphoenolpyruvate (10/anoles, pH
7.4) and ATP: pyruvate phosphotransferase
[EG 2.7.1.40, 20 pg] markedly inhibited accumulation of Asi, in the aerobic as well as
anaerobic conditions (Table VII). The same
inhibition was shown when Ga ++ was omitted
from the medium. These effects were not
caused by the addition of sodium pyruvate.
In a comparable experiment (Experiment 3
in the Table VII) accumulation of Ga ++ was
not impaired by the addition of ATP regenerating systems. ATP regenerating system inhibited Asi-released respiration and activated
ATP hydrolysis which was not stimulated by
110
Y. KAGAWA and A. KAGAWA
tion of Asi with a counter cation, or exchange diffusion of Asi with release of an
intramitochondrial anion.
Asi (5).
Competition between Asi and Pi—The
amount
of 76Asi in mitochondria was lowered by the
addition of Pi (Table VIII). "Pi accumulation was inhibited by addition of Asi to a
lesser extent.
Permeation
of
Asi
into
Stoichiometric Relationships in Asi Accumula-
tion—The amount of Asi accumulated was
proportional to the Ca++ accumulated (Asi r
Ca= 1:1.5) in aerobic condition, but it wasnot in anaerobic condition owing to the Pi
liberated from ATP. In this assay condition
hydrolysis of 1 mole of ATP caused the accumulation of about 0.3 mole of Asi and a
part of energy released by arsenolysis might
be utilized for ion accumulation, since higher
arsenate concentration caused larger Asi accumulation and AT^P hydrolysis. Stoichiometric relationships to substrate were not
obtained owing to the uncoupling effects of
Asi, release of Asi, and formation of ATP
from ADP by adenylate kinase.
Mitochondria—An
energy-independent permeation of Asi was
noticed when mitochondrial pellet was not
washed (Fig 4). Intramitochondrial water
space was slightly increased by higher concentration of Asi. Intramitochondrial concentration of Asi was close to that of the
medium. The result suggested free permeaTABLE V
Nucleotidt specificity of aerobic KAsi accumulation.
Experimental conditions were as described in
"aerobic ion accumulation", except that ADP
was omitted and 2/imoles of nucleotides were added.
In the last 2 experiments Ca++ was omitted.
Addition
Effect of Asi and Pi on Exchange Reaction—
ATP-ADP exchange reaction of intact mitochondria was stimulated (100%) by 10mM Pi,,
and inhibited (55%) by 20 mM Asi. Removal
of Pi by sucrose phosphorylase [disaccharideglucosyltransferase EG 2.4.1.7] purified from
Asi in mitochondria
(protein 4.0 mg)
(mpmoles)
Leuconostoc dextranicum caused decrease in this,
81
490
113
83
143
96
224
106
ATP
GTP
ITP
CTP
UTP
ATP, - C a + +
GTP, - C a + +
exchange reaction without Mg++, and inhibition of Pi controlled state 4 respiration. "PiATP exchange reaction was also inhibited
by Asi, but the inhibition was only 32% at
Asi/Pi ratio of 5 in submitochondrial particlesAsi showed no stimulatory effects on ATPaseactivity of purified coupling factor 1 or mitochondrial preparations which had lost res-
TABLE
VI
Effect of atractyloside on ion accumulation.
Experimental conditions were as described under "METHODS", using aerobic or anaerobic media with
components described in the table.
Accumulation in mitochondria (m^moles)
Aerobic
Atractyloside
(MS)
"Asi
Anaerobic
"C-ADP
"Ca
"Asi
+ADP
-ADP
-Asi
+Asi
+Asi
0
257
22.6
8.5
16.9
382
119.2
10
24
18.5
6.5
12.1
55
4. 1
Accumulation of "As by Mitochondria
TABLE
111
VII
Effect of ATP regenerating system on Ion accumulation.
Experimental conditions were as described under " M E T H O D S " , except oligomycin wa3 omitted, and
additions indicated in the table. PK and PEP stand for pyruvate kinase (20/ig) and phosphoenolpyruvate
(10/imoles., pH7.4), respectively. Mitochondrial protein was 1.9mg for Experiments 1 and 2, 5.0 mg for 3.
In Experiment 3, Asi was substituted by 1/imole of P i .
Ions in mitochondria
(m/imoles)
Conditions
Experiment 1 Anaerobic
Ions
++
Ca
61
27
338
625
9
8
10
6
79
653
14
14
Pi 5mu
Pi 20 mw
91.1
48.9
17.6
152
Permeation of Arsenate in Mitochondria
1 5
Ca
, Mg
Pi + +
DISCUSSION
Pi OmM
213
Mg
piratory control.
Asi in mitochondria (m/imoles)
1.0
++
16
Experimental conditions were as described in
"aerobic ion accumulation", except that CaClj
•was omitted and washing of mitochondrial pellet
was omitted, with 5.0 mg mitochondria.
0.5
++
24
TABLE VIII
Inhibition of 76Asi accumulation by Pi
in Ca free media.
0
++
Ca , Mg
+ P K , P E P oligomycin
272
++
Mg++
+ P K , PEP
Asi 10 mu
<6
Asi
Ca + + , M g + +
Complete
1 mu
Experiment 3 Aerobic
78
"Asi
Counter Ion
Asi
Experiment 2 Aerobic
2.0
" A s i IN MEDIUM (HIM)
FIG. 4. Permeation of Asi in mitochondria.
Experimental conditions were as described in
" METHODS " , with 25 mg of mitochondrial protein.
"Asi could penetrate into mitochondrial
membrane, which were impermeable to smaller ions such as H+, K+ or Cl" (12). The
conditions for Asi accumulation (Table I)
were similar to that of Pi (16), except for the
effect of oligomycin (Table IV) and requirement for adenine nucleotide (Tables III and
V). Asi accumulation seems to be an active
transport process, because of its energy requirement, its uphill nature of ion concentration, and its anion specificity. However, a
divalent cation was required for this phenomenon (Table II), and accumulation of the
cation is considered as a primary event (77).
Accumulation of Asi might explain several phenomena accompanied by uncoupling
effect of Asi. Liberation of endogenous 81Pi
by Asi (4) was caused by entrance of Asi.
Protective effect of EDTA against progressive
uncoupling (4) paralleled with metal requirements for Asi accumulation. Rate of
ATPase activity induced by Asi is slower than
the respiratory rate stimulated by Asi, and
the concentration of Asi to induce ATPase is
much higher than that to release respiration.
When energy is supplied from ATP hydrolysis,
not only X ~ P | but also Pi are produced in
mitochondria, and Pi impedes both Asi accumulation (Tables I and VIII) and interac-
112
Y. KAGAWA and A. KAGAWA
tion of Asi with the phosphorylating site. An
ATP regenerating system containing Mg++
increases ATPase, because ADP, a strong inhibitor of coupling factor 1 is removed, and
the resulting ATPase is not stimulated by
Asi since intramitochondrial Asi is lowered
(Table VII).
ADP requirement for release of tightly
coupled respiration by Asi has been explained
by removal of Pi (3), but formation of
ADP ~ Asi and Asi accumulation might be
the contributing factors (Tables III, V and
VI). Inhibition of Asi accumulation by
atractyloside (Table VI) might be closely
related to ADP requirement.
Considering the Asi concentration around
the phosphorylating site, evidence for the
presence of stable X~Pi bond (2, 5, 8) is
not conclusive. On the other hand, the effect
of Asi is explained by a hypothetical intermediate Asi~X—ADP or Pi-X—ADP (19)
since the presence of ADP is necessary to
uncouple phosphorylation by Asi (3). But
an unstable intermediate of ADP~Asi is
equally possible, as origin of 18O in Asi in
oxidative phosphorylation is entirely different
from that in arsenolysis of substrate level
phosphorylation (20).
Uncoupling by Asi was observed in intact mitochondria or digitonin particles (2),
where ion accumulation might take place.
"Pi-ATP exchange was depressed by Asi in
sonicated particles, in which only transient
ion accumulation was reported (12). However, Asi accumulation and uncoupling by
Asi are different process, since oligomycin
increased accumulation (Table IV) but lowered P : O ratio (3), and addition of EDTA
did not prevent uncoupling by Asi (19).
Data in Fig. 4 suggest that "Asi is accumulated. Release of Asi from mitochondria should
be rapid as shown by rapid exchange of 18O
in Asi by mitochondria (20). Specific penetration of large ions such as Pi and Asi, which
are competing, may be explained by the presence of a specific carrier.
The authors wish to thank Dr. S. Ishikawa and
Dr. Y. Kaziro in University of Tokyo for their valuable
discussions. Thanks are also due to Prof. T. Yamakawa of University of Tokyo for his supporting thi»
work.
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