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Isolation of phosphorylating mitochondria from Astasia longa
Life Sciences Vol. 8, Part II, pp. 1099-1102, 1969. Printed in Great Britain
Pergamon Press
ISOLATION OF PHOSPHORYLATING MITOCHONDRIA FROM ASTASIA LONGA D. E. Buetow and P. J. Buchanan Department of Physiology and Biophysics, University of Illinois, Urbana, lllinois Biochemistry Research Laboratory, Veterans Administration Hospital, Baltimore, Maryland
(Received 30 June 1969; in final form 31 July 1969) Mitochondria which show oxidative phosphorylation have been isolated from higher plants, from animal tissues, and from certain microorganisms number of years.
for a
In contrast, biochemically active mitochondria have been
prepared from protozoa and algae only comparatively recently (i).
In the case
of Euslena gracilis, phosphorylating mitochondria were isolated by a technique wherein the cells were mixed with glass beads in a mortar and ground with a pestle, and the ground mixture was subjected to relatively low speed centrifugation (2, 3).
Recently, mitochondrial preparations were obtained from the
colorless euglenoid flagellate, Astasia lonsa, through a similar glass bead grinding technique showed oxidation,
(4, 5).
However, although these preparations
they did not phosphorylate
(4, 5).
from Astasia
In the present communi-
cation, we present an isolation method which does yield particles from Astasia that show oxidative phosphorylation.
Methods Astasla longa (strain J) were grown to the late logarithmic phase of growth in the dark at room temperature on a defined medium (6) with 20 mM sodium acetate as carbon source.
Cells from 4-6 liters of medium were collected
by centrifugation at 2 ° at 900g.
All subsequent steps were done at 2-4 °.
Packed cells were washed once with 0.25M sucrose (pH 7.4) and centrifuged as above, and the supernatant was discarded.
The pellet was transferred to a
cold mortar with a few ml. of 0,25M sucrose.
1099
A thick slurry was made with
II00
ISOLATION O F M I T O C H O N D R I A
acid--washed diameter),
glass beads
("Superbrjte",
Minnesota
Vol. 8, No~ 20
Mini~ig and ~dfg, ~:o., 0 28 m~.
and the mixture was grou~d with a pest]e
for (~5 ~ : ~ I ~ .
Th~ !~ ol
the ground mixture did not fall below 6.s. Cellular centrifuged
material was washed
for i0 minutes
natant was centrifuged pellet was washed 5 minutes
at 1000g,
and the pellet was discarded.
at 10,000g for I0 minutes.
each time and finally resuspended
The yield ranged Respiration
The resultant
twice with 0.25M sucrose with centrifugation
Total mitochondrial
gracilis
out of the beads wit!! 0.25M sucrose
protein was determined
(3) with respiration
In three experiments
in hexokinase
Thus,
the P:O ratio shown by Astasia lower than usually
flagellate,
Euglena
Homogenizing gentle
concentration
to approach
technique.
pellicle.
esterific~tion.
Composi-
as substrate,
('Fable I).
did not change
$racilis
the P:O ratio
(Table I).
with succinate
previousiy
as substrate,
with the closely related
(2, 3).
Astasia with glass ber~d~ as described The cells were not pulverized
When isolated
above
by the beads
were seen to leak out through
mitochondria
were then isolated
at a higher centrifugal
did not phosphorylate
is a relatively
by the glass beads, which
but rather were ruptured
cell particles
Astasia mitochondria
a P:O ratio
The use of a three-fold
mitochondria
1.0 as was observed
Phosphorylating
noted above.
disappear
observed with animal and higher plant mitochondria,
are larger than the cells, microscopically,
and phosphate
and Discussion
from 0.43 to 0.67 was observed
appeared
(7).
as in the case of Euglena
manometrically
with 6~7 mM succinate
range
though
for
medium is given in Table I.
Results
ranging
at 10,000g
prote.in per 109 ce~!s,
ance from the medium used as the measure of phosphate tion of the respiration
miLochondrial
by the method of Lowry et al.
were measured
determined
The super
in about 2 ml. of 0.25M sucrose.
from 18-28 mg mitochondrial and phosphorylation
.3nd
(4, 5).
force,
so that,
the surface
at 10,000g as e.g.,
A similar
21,000-27,000g
loss of
Vol. 8, No. 20
ISOLATION OF MITOCHONDRIA
1101
TABLE I
Astasia Lonsa Mitochondria, ~xidative Phosphory!ation
Mitochondrial protein (mg. per vessel)
Yeast hexokinase (mg. per vessel)
Respiration (~atoms 0 per vessel)
Phosphorylation (~moles Pi per vessel)
4.05
0.5
2.15
0.93
0.43
2.38
0.5
1.50
1.01
0.67
4.05
1.5
2.46
1.36
0.55
P:0
Each vessel contained 6.7 mM phosphate (pH 7.2), 6.7 mM MgCI2 { 14.0 mM NaF, 6.7 mM succinate as substrate, 2 mM ATP and, as phosphate acceptor, 0.5 or 1.5 mg yeast hexokinase (Sigma, Type III) dissolved in 0.I ml of 0.3 mM glucose. Mitochondrial protein in 0.4-0.9 ml of 0.25M sucrose was added and vessels were made to a final volume of 3 mi with 0.25M sucrose. Incubation, 30 min., 30 °.
phosphorylating ability was previously noted for Euglena mitochondria at centrifugal forces above lO,O00g (I).
isolated
Thus, both the present and previous
results (I, 4, 5) indicate that the isolation of phosphorylating mitochondria from euglenoid flagellates requires both relatively gentle cell homogenization and relatively low centrifugal forces.
Acknowledgement These experiments were performed at the Gerontology Research Center, National Institutes of Health, Baltimore City Hospitals, Baltimore, Maryland.
References I.
D. E. Buetow in D. M. Prescott, (ed.), Methods in Cell Physiology, vol. IV, Academic Press, 1969 (in press).
2.
D. E. Buetow and P. J. Buchanan, Exptl. Cell Research 36, 204 (1964).
3.
D. E. Buetow and P. J. Buchanan, Biochim. Biophys. Aeta 96, 9 (1965).
4.
N. Begin-Heick and J. J. Blum, Biochem. J. 105, 813 (1967).
5.
V. Kahn and J. J. Blum, Biochemistry ~, 817 (i967).
6.
D. E. Buetow, J. Cell. Comp. Physiol. 66, 235 (1965).
1102
7.
ISOLATION OF MITOCHONDRIA
Vol. 8, No, 20
O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J° R~ndall, J~ Biol. Chem~ 19_.3, 265 (1953).
Pergamon Press
ISOLATION OF PHOSPHORYLATING MITOCHONDRIA FROM ASTASIA LONGA D. E. Buetow and P. J. Buchanan Department of Physiology and Biophysics, University of Illinois, Urbana, lllinois Biochemistry Research Laboratory, Veterans Administration Hospital, Baltimore, Maryland
(Received 30 June 1969; in final form 31 July 1969) Mitochondria which show oxidative phosphorylation have been isolated from higher plants, from animal tissues, and from certain microorganisms number of years.
for a
In contrast, biochemically active mitochondria have been
prepared from protozoa and algae only comparatively recently (i).
In the case
of Euslena gracilis, phosphorylating mitochondria were isolated by a technique wherein the cells were mixed with glass beads in a mortar and ground with a pestle, and the ground mixture was subjected to relatively low speed centrifugation (2, 3).
Recently, mitochondrial preparations were obtained from the
colorless euglenoid flagellate, Astasia lonsa, through a similar glass bead grinding technique showed oxidation,
(4, 5).
However, although these preparations
they did not phosphorylate
(4, 5).
from Astasia
In the present communi-
cation, we present an isolation method which does yield particles from Astasia that show oxidative phosphorylation.
Methods Astasla longa (strain J) were grown to the late logarithmic phase of growth in the dark at room temperature on a defined medium (6) with 20 mM sodium acetate as carbon source.
Cells from 4-6 liters of medium were collected
by centrifugation at 2 ° at 900g.
All subsequent steps were done at 2-4 °.
Packed cells were washed once with 0.25M sucrose (pH 7.4) and centrifuged as above, and the supernatant was discarded.
The pellet was transferred to a
cold mortar with a few ml. of 0,25M sucrose.
1099
A thick slurry was made with
II00
ISOLATION O F M I T O C H O N D R I A
acid--washed diameter),
glass beads
("Superbrjte",
Minnesota
Vol. 8, No~ 20
Mini~ig and ~dfg, ~:o., 0 28 m~.
and the mixture was grou~d with a pest]e
for (~5 ~ : ~ I ~ .
Th~ !~ ol
the ground mixture did not fall below 6.s. Cellular centrifuged
material was washed
for i0 minutes
natant was centrifuged pellet was washed 5 minutes
at 1000g,
and the pellet was discarded.
at 10,000g for I0 minutes.
each time and finally resuspended
The yield ranged Respiration
The resultant
twice with 0.25M sucrose with centrifugation
Total mitochondrial
gracilis
out of the beads wit!! 0.25M sucrose
protein was determined
(3) with respiration
In three experiments
in hexokinase
Thus,
the P:O ratio shown by Astasia lower than usually
flagellate,
Euglena
Homogenizing gentle
concentration
to approach
technique.
pellicle.
esterific~tion.
Composi-
as substrate,
('Fable I).
did not change
$racilis
the P:O ratio
(Table I).
with succinate
previousiy
as substrate,
with the closely related
(2, 3).
Astasia with glass ber~d~ as described The cells were not pulverized
When isolated
above
by the beads
were seen to leak out through
mitochondria
were then isolated
at a higher centrifugal
did not phosphorylate
is a relatively
by the glass beads, which
but rather were ruptured
cell particles
Astasia mitochondria
a P:O ratio
The use of a three-fold
mitochondria
1.0 as was observed
Phosphorylating
noted above.
disappear
observed with animal and higher plant mitochondria,
are larger than the cells, microscopically,
and phosphate
and Discussion
from 0.43 to 0.67 was observed
appeared
(7).
as in the case of Euglena
manometrically
with 6~7 mM succinate
range
though
for
medium is given in Table I.
Results
ranging
at 10,000g
prote.in per 109 ce~!s,
ance from the medium used as the measure of phosphate tion of the respiration
miLochondrial
by the method of Lowry et al.
were measured
determined
The super
in about 2 ml. of 0.25M sucrose.
from 18-28 mg mitochondrial and phosphorylation
.3nd
(4, 5).
force,
so that,
the surface
at 10,000g as e.g.,
A similar
21,000-27,000g
loss of
Vol. 8, No. 20
ISOLATION OF MITOCHONDRIA
1101
TABLE I
Astasia Lonsa Mitochondria, ~xidative Phosphory!ation
Mitochondrial protein (mg. per vessel)
Yeast hexokinase (mg. per vessel)
Respiration (~atoms 0 per vessel)
Phosphorylation (~moles Pi per vessel)
4.05
0.5
2.15
0.93
0.43
2.38
0.5
1.50
1.01
0.67
4.05
1.5
2.46
1.36
0.55
P:0
Each vessel contained 6.7 mM phosphate (pH 7.2), 6.7 mM MgCI2 { 14.0 mM NaF, 6.7 mM succinate as substrate, 2 mM ATP and, as phosphate acceptor, 0.5 or 1.5 mg yeast hexokinase (Sigma, Type III) dissolved in 0.I ml of 0.3 mM glucose. Mitochondrial protein in 0.4-0.9 ml of 0.25M sucrose was added and vessels were made to a final volume of 3 mi with 0.25M sucrose. Incubation, 30 min., 30 °.
phosphorylating ability was previously noted for Euglena mitochondria at centrifugal forces above lO,O00g (I).
isolated
Thus, both the present and previous
results (I, 4, 5) indicate that the isolation of phosphorylating mitochondria from euglenoid flagellates requires both relatively gentle cell homogenization and relatively low centrifugal forces.
Acknowledgement These experiments were performed at the Gerontology Research Center, National Institutes of Health, Baltimore City Hospitals, Baltimore, Maryland.
References I.
D. E. Buetow in D. M. Prescott, (ed.), Methods in Cell Physiology, vol. IV, Academic Press, 1969 (in press).
2.
D. E. Buetow and P. J. Buchanan, Exptl. Cell Research 36, 204 (1964).
3.
D. E. Buetow and P. J. Buchanan, Biochim. Biophys. Aeta 96, 9 (1965).
4.
N. Begin-Heick and J. J. Blum, Biochem. J. 105, 813 (1967).
5.
V. Kahn and J. J. Blum, Biochemistry ~, 817 (i967).
6.
D. E. Buetow, J. Cell. Comp. Physiol. 66, 235 (1965).
1102
7.
ISOLATION OF MITOCHONDRIA
Vol. 8, No, 20
O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J° R~ndall, J~ Biol. Chem~ 19_.3, 265 (1953).