BIOCHEMICAL
Vol. 70, No. 4, 1976
EFFECTS
AND BIOPHYSICAL
PRODUCED
ON
MITOCHONDRIA
BY
Virgl/nia
B. C.
RESEARCH COMMUNICATIONS
ISOLATED
RAT
BERYLLIUM
Junqueira
HEART 192
SULFATE
and
M.
Bacila
D epartam ento de Bioq&mica, Instituto de Q&mica, Universidade de Szo Paulo, Caixa Postal 20.780 05508 - SZo Paulo, Brasil Received
April
rate of oxygen is proportional limited number the mitochondrial to an uncoupling
28,1976 Summary. Beryllium sulfate increases the uptake by isolated rat heart mitochondria. This effect to the Be2+ concentration. Beryllium binds to a of sites on the isolated mitochondria and stimulates ATPase. These effects, which appear to be related of respiration, cause swelling of the mitochondria.
The has been
the
et al. (Z),
subject
and Gorlin
Be’+
was
mainly
heart,
striated
toxicity
of beryllium
for
of previous
reports
by Aldridge
(3). found
muscle
In 1968, in liver
and brain
In the present action
of Be2+
on the properties
in biological
Witschi
systems
of isolated
new was
methods hearts
described removed
by Medina and immediately
(4) showed
with
evidence
obtained
that in kidney,
BeSO,. beariug
on the
by examining
AND
its
effects
fraction et al. (5). after
was
METHCQS prepared
Animals placed
were in ice-cold
according
1270
to the
decapitated,
the
extraction
1 This paper contains part of a Ph.D. Thesis presented at Universidade de S?io Paulo (V. B. C. Junqueira). 2 With a grant in aid from Funda$Ho de Amparo i Pesquisa Estado de ,550 Paulo (Projeto BICQ/FAPESP). Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any. form reserved.
Tepper
mitochondria.
MATERIALS Mitochondrial
et al. (l),
man,
andtoalesserextent
poisoned
paper,
including
and Aldridge
and spleen of rats
animals,
the do
BIOCHEMICAL
Vol. 70, No. 4,1976
medium
(10 mM
Rat hearts
were
homogenizer
was
0.25MmannitolandO
finely
minced
this
and the
centrifuged
15 min.
together
obtained
centrifuged
residue
collected
pH 7.4,
resuspended
was
the mitochondrial
Tris,
fraction
0.25M was
by measuring
suspension
twice.
of the preparations
ATPase
activity
Beryllium
was
was
was
assayed
measured
RESULTS When suitable rate
respiratory
was
found
of Be 2+ binding
sites
a maximum
rat heart
substrate
to increase
. Beryllium were of 6.0
prevented
by citrate
stimulated
(Fig.2),
AND
pmoles
per
(unpublished reaching
described
0.25M
obtained
was
Oxygen
uptake
Warburg
of
experiments,
of the mitochondrial Protein of Bticher
(6).
phosphate
by Vogel
(7).
(8).
DISCUSSION were
incubated
of BeSO4,
1)proportionally
(Fig.
the concentration
mg of protein. obs ervation). a maximum
1271
with
concentration 1) whose
of the ion came
Binding ATPas
a
the respiration
to the
by the mitochondria
when
and
Tris,
of inorganic
in the presence
is retained saturated
pH 7.4.
were
sediment
10 mM pellet
and
steps
medium
by the method
mitochondria
(Table
supernatant
Spectrophotometer.
by the release
by the method
The
in absorbance
DU-2
assayed
final
both
by conventional
the decrease
at 600 nm in a Beckman
xg.
with
The
The
medium
from
the extraction
mannitol,
assayed
xg.
extraction
at 10,000
resuspended
and centrifuged
in 10 mM
and swelling
with
then
the homogenization,
at 1,900
in the
pH 7.4).
a Potter-Elvehjem
supernatants
15 min.
and washed
in
15 min.
The
ElDTA,
Following
resuspended
at 1, 900 xg.
; the pellet
manitol,
medium.
and centrifuged
was
centrifuged
content
was
2 mM
and homogenized
extraction
suspension
collected
added
Tris,
with
the whole
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of
beryllium
e activity
at a concentration
to is
was
of BBS04
also
Vol. 70, No. 4, 1976
BIOCHEMICAL
Table The
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
stimulus in the respiration of the isolated mitochondria in the presence of beryllium 02 Uptake/mg
BeSO (M) None 1.8 3.6 5.4 7.2
mitochondria substrate, containing Temperature 15 minutes.
loo4 l(Y4 10’4 10’4
Protein
in 60 min.
Substrates oC-Ketoglutarate
Succinate x x x x
rat heart
16. 15 20.35 24. 57 27. 63 29.75
Pyruvate
4.20 6. 19 7.56 8.27 11.29
0.48 0.75 1. 72 1.92 3.00
System : In 3 ml reaction medium there was 0.2 ml of suspension containing from 2-5 mg protein, 3. 3 x 1003M BeSO in the indicated final concentrations and a buffer 1OmM Tris-0. 25M manitol, pH 7.4 to complete the volume. : 370C ; loo-120 shakings per minute. Time of equilibrium:
*
O.?O-
0 Fig.
Fig.
0
25 eo*+ ad&d (JImolrr 1
0
50 0
1. The
5
IO
[BOSOJ d M
beryllium retaining capacity of the mitochondrial fraction. System contained in 5.0 ml, 3 mg of mitochondrial protein, BeSO in the indicated concentrations, and 1OmM Tris0.25d manitol buffer, pH 7.4, to complete the volume. The systems were incubated 15 min. , at room temperature, centrifuged 15 min. at 10, 000 xg in a refrigerated centrifuge and the unbound beryllium assayed in the supernatant. 2. Stimulation of mitochondrial ATPas e activity by BeS04. The reaction was carried at 30°, in a system containing in 1.0 ml 10’2M ATP, BeSO in the indicated concentrations, 1.0 mg protein, and 0. 1M Tris-sulfate buffer, pH 7.4. Samples were preincubated 40 min. at 370 and the reaction started by the addition of ATP. After 30 min. at 30°, the reaction was stopped with 1.0 ml of 10 per cent trichloroacetic acid. Liberated Pi was measured in the apernatants of a 10 min. centrifugation at 10,000 xg. 1272
BIOCHEMICAL
Vol. 70, No. 4, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0 Time
Fig.
3. Effect
of 0. 5 mM suggest
concentrations
BeSO,
were
is an efficient
with
mitochondrial 2.5
swelling
but the final
; with
was
lower
decrease
must
transport
consider
highly
across
this
of Be2
has the smallest
it is highly
charged
and possesses
be related
tendency
to form
specific atomic covalent radius orbitals
complexes.
1273
of of
the rate
was
slower,
to the phenomenon
membranes.
atomic empty
rate
concentration
x 10m4M)
may
generating
Be2+
was
the same.
ion has the lowest
electronegative,
the highest
+
of a more
In addition,
shows
(2.5 was
respiration.
3). The
a Be 2+
the mitochondrial
the possibility
of Be2 + , because is also
effects
observation
of respiration (Fig.
with
concentration
in absorbance
The of Ca 2+
the uncoupling
maximum
These
of mitochondrial
of the mitochondria
swelling
x 10’3M
inhibitory.
uncoupler
As expected, associated
60
of BeSO, on the swelling of isolated rat heart mitochondria. System contained, in 1.0 ml, 0. 63 mg mitochondrial protein, appropriate volumes of 5. 1003M BeSO and Tris-manitol 10 mM-0.25 M buffer, pH 7.4. Changes in absorbance were followed at 600 nm in a quartz cuvette of 1 cm wide.
; higher
that
30 (minutes)
However,
mechanism
of the
number
in its
bonds in its with
with group
high
we effects
group,
end
other
atoms.
and because
energy,
it
Vol. 70, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCE8 1. Aldridge, W. N. ; Barnes, J.M. and Dena, F.A. (1949) Br. J. exp. Path. 30. 375.389. 2. Tepper, L. B. ; Hardy, H. L. and Chamberlin, R. I. (1961) Toxicity of beryllium compounds, Els evier, New York. 3. Gorlin, R. J. (1951) Oral Surg. 4 177-197. 4. Witschi, H. P. and Aldridge, W.N. (1968) Biochem. J. 106, 811-820. A. and Bacila, M. (1964) Biochem. Pharmacol. 5. Medina, H. ; Dmitraczenko, 13. 461-467. 6. Biicher, T. (1947) Biochim. biophys. Acta & 292-314. 7. Fiske, G.H. and Subbarow, Y. (1925) J.biol. Chem.66, 375-400. 3rd ed. 8. Vogel, A.I. (1961) A textbook of quantitative inorganic analysis. Longm an, London.
1274
Vol. 70, No. 4, 1976
EFFECTS
AND BIOPHYSICAL
PRODUCED
ON
MITOCHONDRIA
BY
Virgl/nia
B. C.
RESEARCH COMMUNICATIONS
ISOLATED
RAT
BERYLLIUM
Junqueira
HEART 192
SULFATE
and
M.
Bacila
D epartam ento de Bioq&mica, Instituto de Q&mica, Universidade de Szo Paulo, Caixa Postal 20.780 05508 - SZo Paulo, Brasil Received
April
rate of oxygen is proportional limited number the mitochondrial to an uncoupling
28,1976 Summary. Beryllium sulfate increases the uptake by isolated rat heart mitochondria. This effect to the Be2+ concentration. Beryllium binds to a of sites on the isolated mitochondria and stimulates ATPase. These effects, which appear to be related of respiration, cause swelling of the mitochondria.
The has been
the
et al. (Z),
subject
and Gorlin
Be’+
was
mainly
heart,
striated
toxicity
of beryllium
for
of previous
reports
by Aldridge
(3). found
muscle
In 1968, in liver
and brain
In the present action
of Be2+
on the properties
in biological
Witschi
systems
of isolated
new was
methods hearts
described removed
by Medina and immediately
(4) showed
with
evidence
obtained
that in kidney,
BeSO,. beariug
on the
by examining
AND
its
effects
fraction et al. (5). after
was
METHCQS prepared
Animals placed
were in ice-cold
according
1270
to the
decapitated,
the
extraction
1 This paper contains part of a Ph.D. Thesis presented at Universidade de S?io Paulo (V. B. C. Junqueira). 2 With a grant in aid from Funda$Ho de Amparo i Pesquisa Estado de ,550 Paulo (Projeto BICQ/FAPESP). Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any. form reserved.
Tepper
mitochondria.
MATERIALS Mitochondrial
et al. (l),
man,
andtoalesserextent
poisoned
paper,
including
and Aldridge
and spleen of rats
animals,
the do
BIOCHEMICAL
Vol. 70, No. 4,1976
medium
(10 mM
Rat hearts
were
homogenizer
was
0.25MmannitolandO
finely
minced
this
and the
centrifuged
15 min.
together
obtained
centrifuged
residue
collected
pH 7.4,
resuspended
was
the mitochondrial
Tris,
fraction
0.25M was
by measuring
suspension
twice.
of the preparations
ATPase
activity
Beryllium
was
was
was
assayed
measured
RESULTS When suitable rate
respiratory
was
found
of Be 2+ binding
sites
a maximum
rat heart
substrate
to increase
. Beryllium were of 6.0
prevented
by citrate
stimulated
(Fig.2),
AND
pmoles
per
(unpublished reaching
described
0.25M
obtained
was
Oxygen
uptake
Warburg
of
experiments,
of the mitochondrial Protein of Bticher
(6).
phosphate
by Vogel
(7).
(8).
DISCUSSION were
incubated
of BeSO4,
1)proportionally
(Fig.
the concentration
mg of protein. obs ervation). a maximum
1271
with
concentration 1) whose
of the ion came
Binding ATPas
a
the respiration
to the
by the mitochondria
when
and
Tris,
of inorganic
in the presence
is retained saturated
pH 7.4.
were
sediment
10 mM pellet
and
steps
medium
by the method
mitochondria
(Table
supernatant
Spectrophotometer.
by the release
by the method
The
in absorbance
DU-2
assayed
final
both
by conventional
the decrease
at 600 nm in a Beckman
xg.
with
The
The
medium
from
the extraction
mannitol,
assayed
xg.
extraction
at 10,000
resuspended
and centrifuged
in 10 mM
and swelling
with
then
the homogenization,
at 1,900
in the
pH 7.4).
a Potter-Elvehjem
supernatants
15 min.
and washed
in
15 min.
The
ElDTA,
Following
resuspended
at 1, 900 xg.
; the pellet
manitol,
medium.
and centrifuged
was
centrifuged
content
was
2 mM
and homogenized
extraction
suspension
collected
added
Tris,
with
the whole
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of
beryllium
e activity
at a concentration
to is
was
of BBS04
also
Vol. 70, No. 4, 1976
BIOCHEMICAL
Table The
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
stimulus in the respiration of the isolated mitochondria in the presence of beryllium 02 Uptake/mg
BeSO (M) None 1.8 3.6 5.4 7.2
mitochondria substrate, containing Temperature 15 minutes.
loo4 l(Y4 10’4 10’4
Protein
in 60 min.
Substrates oC-Ketoglutarate
Succinate x x x x
rat heart
16. 15 20.35 24. 57 27. 63 29.75
Pyruvate
4.20 6. 19 7.56 8.27 11.29
0.48 0.75 1. 72 1.92 3.00
System : In 3 ml reaction medium there was 0.2 ml of suspension containing from 2-5 mg protein, 3. 3 x 1003M BeSO in the indicated final concentrations and a buffer 1OmM Tris-0. 25M manitol, pH 7.4 to complete the volume. : 370C ; loo-120 shakings per minute. Time of equilibrium:
*
O.?O-
0 Fig.
Fig.
0
25 eo*+ ad&d (JImolrr 1
0
50 0
1. The
5
IO
[BOSOJ d M
beryllium retaining capacity of the mitochondrial fraction. System contained in 5.0 ml, 3 mg of mitochondrial protein, BeSO in the indicated concentrations, and 1OmM Tris0.25d manitol buffer, pH 7.4, to complete the volume. The systems were incubated 15 min. , at room temperature, centrifuged 15 min. at 10, 000 xg in a refrigerated centrifuge and the unbound beryllium assayed in the supernatant. 2. Stimulation of mitochondrial ATPas e activity by BeS04. The reaction was carried at 30°, in a system containing in 1.0 ml 10’2M ATP, BeSO in the indicated concentrations, 1.0 mg protein, and 0. 1M Tris-sulfate buffer, pH 7.4. Samples were preincubated 40 min. at 370 and the reaction started by the addition of ATP. After 30 min. at 30°, the reaction was stopped with 1.0 ml of 10 per cent trichloroacetic acid. Liberated Pi was measured in the apernatants of a 10 min. centrifugation at 10,000 xg. 1272
BIOCHEMICAL
Vol. 70, No. 4, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0 Time
Fig.
3. Effect
of 0. 5 mM suggest
concentrations
BeSO,
were
is an efficient
with
mitochondrial 2.5
swelling
but the final
; with
was
lower
decrease
must
transport
consider
highly
across
this
of Be2
has the smallest
it is highly
charged
and possesses
be related
tendency
to form
specific atomic covalent radius orbitals
complexes.
1273
of of
the rate
was
slower,
to the phenomenon
membranes.
atomic empty
rate
concentration
x 10m4M)
may
generating
Be2+
was
the same.
ion has the lowest
electronegative,
the highest
+
of a more
In addition,
shows
(2.5 was
respiration.
3). The
a Be 2+
the mitochondrial
the possibility
of Be2 + , because is also
effects
observation
of respiration (Fig.
with
concentration
in absorbance
The of Ca 2+
the uncoupling
maximum
These
of mitochondrial
of the mitochondria
swelling
x 10’3M
inhibitory.
uncoupler
As expected, associated
60
of BeSO, on the swelling of isolated rat heart mitochondria. System contained, in 1.0 ml, 0. 63 mg mitochondrial protein, appropriate volumes of 5. 1003M BeSO and Tris-manitol 10 mM-0.25 M buffer, pH 7.4. Changes in absorbance were followed at 600 nm in a quartz cuvette of 1 cm wide.
; higher
that
30 (minutes)
However,
mechanism
of the
number
in its
bonds in its with
with group
high
we effects
group,
end
other
atoms.
and because
energy,
it
Vol. 70, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCE8 1. Aldridge, W. N. ; Barnes, J.M. and Dena, F.A. (1949) Br. J. exp. Path. 30. 375.389. 2. Tepper, L. B. ; Hardy, H. L. and Chamberlin, R. I. (1961) Toxicity of beryllium compounds, Els evier, New York. 3. Gorlin, R. J. (1951) Oral Surg. 4 177-197. 4. Witschi, H. P. and Aldridge, W.N. (1968) Biochem. J. 106, 811-820. A. and Bacila, M. (1964) Biochem. Pharmacol. 5. Medina, H. ; Dmitraczenko, 13. 461-467. 6. Biicher, T. (1947) Biochim. biophys. Acta & 292-314. 7. Fiske, G.H. and Subbarow, Y. (1925) J.biol. Chem.66, 375-400. 3rd ed. 8. Vogel, A.I. (1961) A textbook of quantitative inorganic analysis. Longm an, London.
1274
