Oxidative
phosphorylation
in
mitochondria
from germinating peas
A.J.
Zeevaart
Biochemisch
M.
GruberAND
M.H. van
Raalte
and
Laboratorium, Bloemsingel 10, Groningen
Botanisch
Laboratorium, Gro-
ningen.
SUMMARY
I.
The
fifth
2.
The
the
3.
1.
The
with
P/O ratio,
during the
first 2
succinate,
days
of
of mitochondria
germination; from
from
the
the
third
on
it
of peas
1.0-1.5
was
decreased
till
which
formed
at the
zero
day.
uncoupling was
caused
homogenization of
by
a
mixture of
long-chain fatty acids,
are
during
the tissue.
uncoupling, therefore,
is not due to
in
changes
the
mitochondria
during germination.
INTRODUCTION
The activities
of mitochondria from
with the
alter
age
of the
mitochondria from the
decreased after six
increased till the
endosperm
of
(1957)
days
tenth
fourth
found that these
day, reaching
Cherry
germinating
Stanley
germination,
The
capacity
beans
day (Servers
particles
at
zero
germination,
changes
in mitochondrial
materials
2.1.
followed
and
& Walker
the seventh
castor
day
of
decline. In the
a
oxidize
1956).
succinate,
oxidize citrate
to
fraction from the
succinate
shown
was
Akazawa &
bean showed
whereas the
day,
to
capacity
P/O
a
to
Beevers
maximum of
ratio declined from
germination.
activities of
phosphorylative
a
maximum after
the
present study
activity during germination
was
eight days
cause
of the
investigated.
methods
Germination
Pea seeds
placed
in
(var.
moist,
Alaska)
Mitochondria
All
operations
lings
sand,
were
sterilized
2.2
pH
by
seeds
particulate
a
isolated mitochondria from peanut seeds showed
of
of
oxidize
that the oxidative and
(1963) reported
to
capacity
pine
of
to
from the
the fifth
on
reported
found that the
whereas the
castor
day.
seeds have been
(1957)
of sugar
endosperm
of
oxidation
a-oxoglutarate
the fifth
seedling.
germinating
decline after the
2.
cotyledons
day
were
carried
and cooled in ice for
in
a
solution
7.4 with
7.2 After
out at
ca.
Oct.
1968
for
water
kept
at
They
were
ca.
The
pH
8
20-25
°
hrs.
of
They
were
cut
in
then
from the seedmortar
a
0,03
M-KH P0
the
homogenate
double cheese-cloth, the
were
in the dark.
homogenized
M-sucrose and
NaOH, (4 ml/g tissue).
17)5(,
in
and
0-4°. The cotyledons
2 hr.
containing 0,5
filtration through
Acta Bot. Neerl.
soaked
vermiculite,
2
homogenate
4
with
adjusted
was
was
6.9
to
to
centrifu-
349
A.
ged
a
at
1500
for 7
#
hand-driven
mitochondrial
2.3.
sucrose
taken up in buffered
uptake
was
made for the,
contained
in the
main
0.02 M-NaF, 0.012
The side
tissue),
was
H.
pellet
By
means
The
recentrifuged.
in the
RAALTE
of
suspended
was
The
VAN
intensely
pellet
green
experiments.
mined
was
30°.
No
The flasks
0.3
M-glucose,
mg/ml
hexokinase.
M-sucrose,
M-sucrose and 0.06
containing
for
M-sodium succinate.
5-9 mg. of
10 min. the
1
In
according
Slater
to
determined
the
(1953).
2
g
tipped
was
trichloroacetic
20%
was
experiments
some
protein (from
substrate
ml
inorganic phosphate
(1963).
measured
of 0.2
stopped by adding
solution
& Becking
was
ml
at
respiration.
M-sodium-ADP and 1
equilibration
the method of Lowry
by
1
in air
manometer
7.4 with NaOH. To the main compartment
pH
to
reaction
deproteinized
phate produced
Warburg
suspension,
added. After
method of Hurst
the
ml of 0.5
adjusted
were
in. After 30 min. the
by
the
glucose-6-phos-
Protein
deter-
was
(1951).
c.s.
Extraction of supernatant
Protein
removed
was
the
centrifugation,
with ether. The
of
stream
by precipitation
acetone
diminished pressure.
or
g
relatively low, endogenous
compartment
ml of the mitochondrial
acid. In the
a
M.
for 20 min.
g
12000
and
AND
(0.5 ml/g tissue).
immediately
M-MgCl 2,0.0015
contained 1
arm
Both solutions
2.4.
used
was
measured in
was
correction
of
the
(2 ml/g tissue)
solution
GRUBER
Respiration
Oxygen
1
solution
M.
12000
at
homogenizer,
sucrose
suspension
ZEEVAART,
the supernatant
Potter-Elvehjem
in fresh buffered
was
min., and
J.
with
removed
was
4 volumes of cold
by
distillation under
The residual aqueous solution
extract was
the
nitrogen,
dried
on
remaining
CaCl
2
was
After
acetone.
nitrogen
gas
at
extracted three times
and after removal of the ether with
green oil
was
taken
up
in absolute ethanol
light petroleum (b.p. 60-80°)
2.5.
For
Chromatography
thin-layer chromatography,
Germany)
(b.p. 40-80°)
by spraying
For
as
was
the
2.6.
Free
with
a
1
-
acetic acid
and
fatty acids
of
HE
Kieselgel
were
(E. Merck, Darmstadt,
developed
(90:10:1, by vol.)
with
The spots
light
were
petroleum
made visible
iodine solution in methanol.
%
described
fatty
plates
chromatography,
by
stationary phase
Cotyledons
350
ether
gas-liquid
prepared
The
-
plates
The
prepared.
were
de
the
methyl
Boer & Backer
and helium
gas the
esters
of
the
fatty
acids
were
(1954). Diethyleneglycolsuccinate
mobile
phase.
acids
homogenates
could
not
be
were
extracted
titrated because
described
as
the
extract
Dole
by
had
Acta 801. Neerl.
a
(1956).
yellow-green
17(5),
Oct. 1968
IN
OXIDATIVE PHOSPHORYLATION
colour.
They
tography,
determined
were
acid
3.
used
was
from the other components
separated
were
After elution from the
above,
as
MITOCHONDRIA
by
as
of the
means
by thin-layer
chloroform the
gel with
copper method of Duncombe
chroma-
fatty
(1963);
acids
oleic
standard.
a
RESULTS
3.1.
Uncoupling
Table I
shows
that
diminishes with the
Table
1.
Oxidative
the
oxidative
esterified
(days)
((xmoles/30 min.)
1.34
6.3
7.6
0.83
1.6
8.8
0.18
5
0.4
6.4
0.06
6
-0.3
5.7
0
7
-0.1
3.7
0
8
-0.3
4.2
0
0.0
2.2
0
germinated
5-days
peas
the
the
first
presence
of
days
by adding
albumin in
Warburg
to
the other
the
by adding
of
hand,
solution
day
even
of
of
on,
(tables
of
a
3 and
super-
for
readily
restores
supernatant
capacity
4). Washing
albumin restored
serum
and
mitochondria from
amount
most
which
phosphorylating
germination,
requirement
are
factor
later
and,
albumin
serum
the
the mitochon-
supernatant from 2-days
supernatant
a
mitochondria
by adding
ever
uncoupling
by including
8-days
larger
peas
the
extra
were
amounts
factor present
on
of
the
during germination.
Identification of the
uncoupling
From the supernatant fraction of
17(5),
On
days.
From the
clear that the
mitochondria increases
Need.
uncoupled
germination
%
1
flasks,
phosphorylation.
albumin it is
a
the seventh
up
the
in table 2, the
shown
Restoration of the
bovine
with
pellet
phosphorylation
of
for five
uncoupling activity.
mitochondrial
As
become
days
recoupled
are
assuming
during
days.
two
two
with this supernatant. These observations
by washing
by
for
germinated
the
Acta Bot.
1.22
4
also obtained
3.2.
P/O
3
was
serum
uptake
2
5.2
with
capable
0
6.9
explained
peas.
((xatoms/30 min.)
8.4
dria from
factor
germinating
6.9
from peas
coupling
of
1
ty falls rapidly after about
or
cotyledons
2
from peas
peas,
cotyledons
phosphorylating capaci-
Phosphate
time
10
natant
mitochondria from
from
mitochondria
phosphorylation by
Germination
of
capacity
of germination, and that the
period
Oct.
1968
5-days
factor
peas, which
has
a
strong uncoupling
351
A. J.
Table 2.
Exp.
Effect of
oxidative
supernatant on
M. GRUBER
ZEEVAART,
AND
M.
H.
VAN
RAALTE
phosphorylation.
glucose-6-phosphate
no.
02
uptake
produced
((j.moles/30 min.)
1
2-day
mitochondria
2-day
mitochondria
1
ml.
2
ml.
mitochondria
mitochondria
1 ml.
1,6
20,7
0,4
3,6
3,2
15,3
25,9
30,0
4,9
1,1
6,2
23,7
34,1
27,2
+
2-day supernatant
mitochondria;
2-day supernatant
1 ml.
mitochondria
5-day
5-day mitochondria,
washed
2-day supernatant
mitochondria
5-day
from
1
13,7
5-day supernatant
5-day
in
17,8
1,3
ml
Protein was
fered
sucrose
Table
3.
2-day
+
protein
22,9
supernatant
trichloroacetic
precipitated by
30,3
acid;
the sediment
5-day
mitochondria.
serum
serum
albumin
albumin
on
0
glucose-6-phosphate
added to the flasks
14,4
28,0
30
37,4
29,1
90
42,4
31,7
180
39,8
30,2
the active
supernatant
principle
from
in ethanolic
solution,
were
mixture of about 70
quantities
of C
conclude that
The
18
the
%
and
and
and
about
uncoupling capacity
10%
fatty
acids
extract
of
of
added,
component
one
long-chain
fatty
with
co-chromatographed
analysis
C
and
16
is
only
mixture
a
showed
C
each.
18:i
were
of mitochondria is
of
peas,
oleic acids
unknown substance
uncoupling
The
with methanol. When
gas-chromatographic
18:2
an
gel
1-day
identified as
stearic and
C
of
was
(table 5).
separated by thin-layer chromatography
eluted from the
It
way:
component,
was
mitochondria from
to
following
be extracted with ether
peas
uncoupling activity.
in the
the active
352
can
7-day
into 5 components. These
acids.
(fxatoms/30 min.)
2,3
10
showed
buf-
uptake
2
11,2
none
acids
twice with
produced
(u.moles/30 min.)
effect,
washed
was
solution.
Effect of bovine
mg bovine
the
min.)
+
supernatant
5-day
no
3
30
mitochondria;
no
1
5-day
(|j.atoms/
present.
caused
by
Bot.
Very
We
be
to
a
small
thus
may
long-chain fatty
demonstrated in
Acta
it
Neerl.
table 6 with
17(5),
Oct.
1968
OXIDATIVE
Table 4.
PHOSPHORYLATION IN
Restoration
MITOCHONDRIA
of oxidative
phosphorylation by
bovine
albumin
serum
(BSA) during
germination
Mitochondria
were
given
extra
were
washed
second
a
BSA is
wash
shown
under
taken
oxygen
sucrose
solution
buffered
up
in
in
30
enriched
sucrose solution
B. The mitochondria
esterified
AP, (xmoles phosphate
AO, (/atoms
in buffered
in the normal
were
of the
with
(A).
same
Table
1.
B
AO
AP
added
P/O
AP
AO
P/O
1.29
to flasks
1
8.8
2
6.1
5.5
3
7.3
7.8
4
9.3
9.3
5
8.3
9.5
6
9.2
7
8
5.
in
30 min.
(days)
Table
used
mg BSA
time
1
incubation
of including
%
min;
germination
1
(w/v). They
1
batches
A
10
BSA
The effect
time
Effect
6.2
1.42
30
8.0
6.2
1.12
30
7.4
5.6
1.31
30
7.2
7.4
0.97
0.94
30
8.3
7.8
1.05
0.88
30
9.3
8.8
1.05
11.7
0.80
30
12.0
10.5
1.14
1.1
8.9
0.12
30
7.5
10.3
0.73
0.9
8.5
0.10
90
8.9
0.63
1.1
5.3
0.21
90
was
60
1.00
5.6
0.5
6.7
0.08
min.
of extracts
from supernatants
on
oxidative
phosphorylation by 2-day
mito-
chondria.
0.1
ml of extract in
absolute
ethanol
corresponds
1.3
to
ml of
supernatant.
0
glucose-6-phosphate
uptake
2
produced
addition
to the
flask
(p.moles/30 min.)
0.1 ml. ethanol
0.1
ml.
extract
0.1 ml. extract
oleic acid.
2-day supernatant
16.4
20.0
15.8
5.3
serum
albumin is
acid. From the data in table 6 it
binds 6
24.2
5-day supernatant
Bovine
moles of oleic
moles bound
per
mole
can
shown
to
13.9
reverse
be calculated that
acid. This is in
serum
((xatoms/30 min.)
the
effect
1 mole of
good agreement
with the
albumin which has been found
by
of the
serum
fatty
albumin
number of 7
Björntorp
c.s.
(1964).
3.3.
Source of the
The
question
ce
of the
now
amount
strongly
Acta Bot.
arose
on
fatty
in the
acid
acids
whether the
homogenization,
shows that the
The
fatty
or
were
content
homogenate
the fourth
Need. 17(5),
day,
Oct.
in
1968
fatty
acids
already
were
present
liberated
in the
of the seeds remains low
is also low
parallel
during
with the
as
consequen-
Fig.
1
during germination.
the first three
uncoupling
a
intact seed.
days,
but rises
of the mitochondria.
353
A.
Table
6.
Effect of oleic
A solution
stream of
Oleic
acid
of oleic
acid
acid
and bovine
in hexane
was
serum
J.
ZEEVAART, M.
albumin
pipetted
into
GRUBER
(BSA)
the
on
AND
2-day
M.
H.
and the solvent removed
flasks,
by
a
nitrogen.
to the
flask
Phoshate
Oa
uptake
P/O
(nmoles/30 min.)
(natoms/30 min.)
16.1
none
10.8
0,3 nmole
12.6
9.9
0,9 nmole
6.6
10.0
0.0
6.2
11.0
10.8
3,6 fxmole
It is
RAALTE
mitochondria.
esterifled
3,6 nmole
VAN
+
30 mg BSA
concluded that the
loss of
during germination normally
1.49
1.28
0.65
0.0
1.02
phosphorylating capacity
does
not
occur
in
vivo,
of the
but is
1.
Fig.
mitochondria
artifact of pre-
an
their
acid
Fatty
of
tent
con-
and
cotyledons
du-
homogenates
ring germination.
Cotyledons
genized
in
Blendor
for
homo-
were
Waring
a
30
The
sec.
homogenate was kept
for 30 min.
ice
sequently,
of
the
selves
extracted
was
(Dole 1966).
Extraction
cotyledons
was
carried
homogenizing in
of
sence
tion
of
the
was
4.
no
accumulation of
at
fatty
the tenth
day
acids in the
of
after
30
min.
P/O
ratio
may have another
homogenate
by
the preextrac-
o,
content
•,
homogenate
at
0°; A,
(taken
from
1).
cause,
since there
10-day peas.)
DISCUSSION
Our
results show
also in
respiration
chondria
that the
-
themselves,
decrease in
phosphorylation
during germination, is
but
to an
not
due
that the decrease
354
to
-
and
probably is
not a
to
(1957),
fatty
we
extent
acids
on
the
therefore belie-
physiological phenomenon,
Acta
some
alterations in the mito-
increased concentration of free
isolated mitochondria. Unlike Akazawa & Beevers
ve
of
themout
cotyledons;
content
table
paration. (The uncoupling
Dole’s
medium,
in
and sub-
Bot. Need.
but
an
17(5),
artifact
Oct.
1968
OXIDATIVE PHOSPHORYLATION
IN
of isolation which is due
days
of
the
to
metabolic
changed
state
of the tissue after several
Similar effects have been observed in
germination.
tissues (Wojtczak
MITOCHONDRIA
&
Lehninger
1961; Wojtczak &
number of animal
a
Wojtczak
An effect of
acids
endogenous fatty
mitochondria from
on
plant
genates has been demonstrated by Dalgarno & Birt (1962).
fatty
Che-
1960;
1964, 1966).
furka
acids in
homogenates
peanuts after
of
of
days
some
from
cotyledons
germination
beans,
castor
has been observed
tissue homo-
Accumulation of
cotton
St.
by
seeds and
Angelo &
Altschul (1964).
Our results are,
With the
(1960).
found
In
a
however,
same
ratio of 0.4
P/O
conclusion,
our
“Lipase action is
in
the tenth
on
results
methods,
same
c.s.
these authors still
day.
illustrate the
made
point
present problem
an ever
with those of Young
complete disagreement
of peas and the
variety
in
by
of
analysis
Benson
(1964)
cell
plant
that,
fractions”.
ACKNOWLEDGEMENT
The
investigation
Advancement
carried out with financial
was
of Pure
Research
(Z.
W.
aid from the Netherlands
Organization for
the
O.).
REFERENCES
T. & H.
Akazawa,
bean:
tor
Beevers,
A
H.
Beevers
& D.
A.
Walker
germinating castor
A. A.
Benson,
Chem.
Boer,
&
and
The
(1956):
R. H.
in
the
endosperm
of the
germinatingcas-
J. 67:115-118.
oxidative
activity
of
fractions
particulate
from
J. 62:114-120.
lipids. Ann.
Bradford
phosphorylation
Rev. Plant
Physiol.
Albumin
(1964):
reactions
in
liver
rat
15:1-13.
antagonism
of
fatty acid;
mitochondria.
J.
Biol.
239:339-344.
Th. J.
Rec.
Biochem.
Plant membrane
H. A. Ells
oxidation
on
Mitochondria
beans. Biochem.
(1964):
Björntorp, P.,
effects
(1957):
developmental study.
de &
H.
Trav. Chim.
W.
Chefurka,
to in vitro
J. Backer
(1954):
A
new
method
for the
preparation
of diazomethane.
73:229-234.
Pays-Bas
(1964): Dinitrophenol-induced adenosinetriphosphatase activity
aging
of mitochondria.
Abstracts 6th Int.
Congress
Biochem.
in
relation
(New York)
VIII:
646.
W. & T. Dumas
Chefurka,
J.
Cherry,
peanut
H.
Dole,
V.
on
P.
isolated
& H.
and tissues.
Duncombe,
Hanson,
J.
J.
W.
Biochem. J.
J.
Exp.
acid,
mitochondria,
(1962):
(1960):
Chem.
(1963):
Free
mitochondria.
Meinertz
Biol.
G.
B., C.
Bot.
J.
Birt
carrot
fatty
in
vitro
aged
mitochondria.
and
changes
enzyme
in
cotyledons
of
38:440-446.
acids in
Biochem.
carrot-tissue
preparations
and their
J. 87:586-596.
Microdetermination
of
longchain fatty
acids
in
plasma
235:2595-2599.
The
colorimetric
microdetermination
M.
Wilson,
of
long-chain fatty
acids.
Chrispeels,
W. A.
Krueger
respiratory
& H.
senescence
R. Swanson
of
maize
(1965);
scutellum.
16:282-293.
Biochem.
Acta Bot. Neerl.
M. J.
and other factors involved in the
R. O. & G. C.
Canad.
phosphorylation in
88:7-10.
Ribonuclease
Hurst,
Oxidative
during germination. Plant Physiol.
L. & L. M.
Dalgarno,
effect
Nucleic
(1963);
seeds
(1966):
3904-3911.
Biochemistry 5:
17(5),
Becking
Physiol.
(1963): Hydrolysis
of calf
thymus DNA by pancreatic
DNAse
41:469-480.
Oct. 1968
355
A. J. ZEEVAART, M.
Lowry,
with
Slater,
O.
N. J.
H.,
the Folin
E.
C.
Rosebrough, A.
phenol
reagent.
Angelo,
A.
J.
& A.
R.
G.
seed. Plant
Wojtczak,
(1957):
M. Altschul
&
L.
AND
(1951):
M.
H.
VAN
RAALTE
Protein measurement
193:265-276.
determination
of
fructose-1,6-diphosphate,
hexose-
J. 53; 157-167.
(1964):
and the
Lipolysis
free
fatty
acid
pool
in
39:880-883.
Krebs
of mitochondria
cycle activity
from
endosperm
of sugar
pine
32:409-412.
Physiol.
inhibition of
Chem.
and ADP. Biochem.
seedlings. Plant Physiol.
Stanley,
L. Farr & R. J. Randall
Biol.
(1953): Spectrophotometric
monophosphates, ATP
St.
J.
GRUBER
A. B.
Wojtczak
ATP-Pi exchange by
(1959):
a
Uncoupling
of
oxidative
phosphorylation
substance from insect mitochondria.
Biochim.
and
Biophys.
Acta 39:277-286.
Wojtczak,
L.
&
uncoupling
A. L.
factor
Lehninger
Formation
(1961):
during swelling
and
contraction
and
disappearance
of mitochondria.
of
an
endogenous
Biochim.
Biophys.
Acta 51:442-456.
Young,
J.
356
C.
Huang,
S.
enzyme
synthesis
in
L.,
affecting
R.
Vanecko,
J.
cotyledons
D.
of
Marks &
J.
E.
germinating seeds.
Varner
Plant
(1960):
Physiol.
Acta Bot. Neerl.
Conditions
35:288-292.
17(5),
Oct.
1968