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Trace metal composition of fractions obtained by digitonin fragmentation of spinach chloroplasts
Vol. 17, No. 6, 1964
BIOCHEMICAL
TRACE METAL COMPOSITION
OF FRACTIONS
FRAGMENTATION
OF SPINACH
Jan M. Anderson,
RESEARCH COMMUNICATIONS
OBTAINED
BY DIGITONIN
CHLOROPLASTS
N.K.
Boardman
of Plant
Industry,
to current
concepts
the
involves
two distinct
Division
C.S.I.R.O.,
AND BIOPHYSICAL
and D.J.
David
Canberra,
A.C.T.
Australia
Receivedseptember
24, 1964
According photosynthesis
in
photochemical These
green
reactions
(cf. -
two reactions
absorbed
system
Absorption
by system of water
light
result
from
system
oxidation
of reduction
resultant phase the
digitonin chlorophyll different One type
after
of the
oxygen
of spinach
of particle
of particle
structural dg
a&
ratio
proteins (2.0).
of 5.0
remains
more
of the intact
the
ATP and oxygen
with that
the
associated
with
and Anderson, in
to 6.0; strongly
digitonin whereas attached
and has a lower
chloroplasts
685
complexes
chloroplasts
is
grana
products
two systems,
soluble
ratio
For
Thus
(Boardman
is more
of
and oxidation
evidence
chloroplasts
denoted
absorbed
chlorophyll-protein
obtained
of particles
light
cytochrome
of spinach
we have recently
a chlorophyll type
the
been
the reduction
NADPH2,
of the
light
1963).
while
of photosynthesis,
incubation
have
evolution.
co-operation
types
in
primary
1963).
2 (Duysens,
1 results
of
by the
which
a cytochrome,
By fractionating obtained
systems,
by system
with
of the
pigment
light
2 causes
by Clayton,
to be mediated
1 and pigment
of
NADP and the
review
are thought
by different
pigment
plants
process
this
two 1964).
and has the
other
to the chlorophyll ratio
was
Vol. 17, No. 6, 1964
3.0.
Since
ratio
are
the it
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
particles
with
the
able
electron
to photoreduce donor
couple
was speculated
system
that
1, while
the
communication
provides
fractions based
presence
contain soluble
evidence
dye),
digitonin
2.
This
that
these
1 and 2.
of
pigment
in
of system
to systems
the
d&
and indophenol
less part
distribution
chlorophyll in
particles
further
correspond on the
(ascorbate
particles
an integral
high
NADP only
these
may represent
the
This
of Cu, Mn and Fe in
present two
evidence the
is
two
fractions. Spinach leaves
in
sucrose
phosphate
purified
digitonin
suspended
O.OlM
was then
separated
following
manner;
(Gentry
phosphate
by differential
all
analysis,
homogenate was rigorously in
chloroform 1950).
chloroplasts (O.O5M, (twice
30 min
at O'C.
were pH 7.2) recrystalised The preparation
centrifugation
at 1000 x g,
and 60 min samples
0.3M
and Sherrington,
buffer
for
10 min
filtered
buffer
KC1 and 0.5% digitonin
at 50 000 x g,
chemical
the
spinach
and incubated
containing
8-hydroxyquinoline
of metals
purified
ethanol)
30 min
with
by blending
pH 7.2)
The phosphate
fragmentation, in
containing in
10 min.
traces
(O.O5M,
KC1 and sedimenting
by extraction
to remove
were prepared
buffer
and O.OlM
at 1000 x g for
For
chloroplasts
in
30 min
at 10 000 x g,
at 144 000 x g. (whole
Prior
chloroplasts, were washed
resuspension
and recentrifugation
at the
glass-distilled
appropriate
speeds,
For lyophilized of H2S04 digestion,
water and then
determination
fractions - HC104
(1+7)
of
(20-50
the
mg) were
was pipetted 686
components,
digested
and 5 ml of cont.
10 ml of water
twice
lyophilized. metal
with
HN03. into
to
the
10 000 x g and 144 000 x g fractions) in
the
flasks,
the 2 ml
After the
by
Vol. 17, No. 6, 1964
contents No.
BIOCMEMICAL
thoroughly
42 papers were
of them,
against
standards
to sample
atomic
absorption
instrument
O-3;
filtrates
and
or dilutions
in parts
and Mg,
and H2S04
solutions.
dry Whatman
manganese
containing
Mn,
O-6;
RESEARCH COMMUNICATIONS
through
iron,
on these
intervals
alent
:
each
at suitable
at a concentration
equiv-
A Perkin
spectrophotometer
O-l
per million
Elmer
Model
303
was used with
the
following
settings: cathode
20 ma for Slit
Copper,
determined
Fe,
concentration
Hollow
and filtered
(9 cm).
magnesium
Cu, O-0.5;
mixed
AND BlOPHYSlCAt
tube
Mn,
width:
Flame:
current:
15 ma for
18 ma for
Cu, 40 ma for
Fe,
Mg.
1.0 mm for
Cu, Mn and Mg;
Stoichiometric
air-acetylene
0.3 mm for
mixture,
Fe.
10 cm
path-length. Resonance,lines: Scale
Cu 3247 i,
expansion:
unexpanded
2-fold
for
for
of
striking
a system 4.5
magnesium
content)
evolving Spencer
times
sequence
higher
than
in
Mg 2852 g. Cu,
synthetic
electron
we believe
to be more representthan
system
(relative
1 particle).
From
to be required
of photosynthesis
the
for
of the
cytochromes
the
(representevidence,
the
oxygen1957;
other
hand
the
144 000 x g fraction Iron
are involved
(Duysens, 687
1 particle
to
(Kessler,
10 000 x g fraction.
transport
other
On the
1961).
I.
case of manganese.
144 000 x g fraction
the
since
the
02-evolving)
contents
implicated
was in
as the
and Possingham, and iron
Table
as much manganese
is believed
copper
are shown in
which
2 (i.e.
of a system
manganese
for
partitioning
contained
ative
Fe and Mn, 5-fold
obtained
The 10 000 x 8 fraction ative
Mn 2795 i,
Mg.
The results The most
Fe 2483 i,
1963).
is in
were of course photo-
Copper
is
Vol. 17, No. 6, 1964
BlOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE: I Trace
Metal
Content
of Chloroplast
Fractions
The Mn, Fe and Cu contents of the fractions are given relative to Mg content. Magnesium was shown to be nearly lOO$ equivalent to the chlorophyll content of these fractions. The magnesium content in umoles/g dry wt. was 90.4 for chloroplasts, 102.4 for 10 000 x g fraction and 62.5 for 144 000 x g fraction respectively. --
Fraction
Mg
Mn
Fe
CU
Chloroplasts
100
1.37
4.16
1.88
3.0
10 000 x g
100
1.93
2.11
1.00
2.0
144
100
0.41
3.41
1.62
5.5
115
1
6
3
2.3+
000
x g
Quantosomez? w
Park
also
& Pon (1963)
--et al.
system
is
located
Recent
(unpublished)
on the
since
has been
(1962).
Smillie
It
# Lichtenthaler
known to be present
plastocyanin
& Calvin
by Katoh that
of the
of interest
transport
pathway.
that
iron
the
fraction.
Comparison
metal
are closer
values
they
are somewhat It
adsorption plast
of the of Park
to those
of heavy
of buffers-used
of intact
and copper
than content
and Pon
located
(1963)
in
the
contents 10 000 x g
of these
fractions
show that
their
chloroplasts,
although
higher.
is worthwhile
fragments
and
electron
are higher
quantosomes
(unpublished) is
144 000 x g fraction
the
by Katoh
plastocyanin
of the
with
(1964)
protein,
in chloroplasts
works
4;
a copper-containing
indicate
1 side
therefore
Chlorophyll
is for
to note
metal very
ions
the
to chloroplasts
great,
isolation
that
and therefore is
688
essential.
likelihood
of
and chloroprior
purification
A similar
Vol. 17, No. 6, 1964
BIOCHEMICAL
experiment
to
fractions
that
were
resulted
in
data
for
the
content
also
with
the
distribution
30 min.
not
only
fractions
is
obtained
consistent
a physical
particles
which 1.
with
of the
determine
in
different photochemical
the
two chloroplast that
of system
1 particles
we have from
of system
of these work
structure,
composition
particles
2 than
content
the
chlorophyll-containing
Moreover
our hypothesis
cytochrome
of
activities,
metals.
between
of
amounts
Further
of these
all
chlorophyll-
interest.
to
be published
in
great
progress
buffer,
high,
results
metals
three
fragmentation
are more representative
be of
metals
of trace
separation
Analysis will
trace
with
ratios
of these
the
phosphate
digitonin
for
different
in which
was particularly
2 and & and different
but
will
I,
unpurified
shows that
particles
chlorophyll
fractions
iron
Table
with
values
chloroplasts
containing
system
isolated
the This
spinach
shown in
higher
fractions;
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is
in
and function
and full
details
elsewhere.
References
Boardman, N.K., and Anderson, J.M., Nature, Ann. Rev. Plant Physiol., 14, Clayton, R.K., Duysens, L.M.N., Proc. Roy. Sot. B., 157,301 and Sherrington, L.G.,nalyst, ~a;;;~,SC.H.Rt, Shiratori, I., and Takamiya, A., , "51, 32 (1962). Kessler, G Planta, 49, 435 (1957). Lichtenthaler, H.K., and Calvin, M., Biochim. 79, 30 (1964). Park, R.B., and Pon, N.G., J. Mol. Biol, 6, J.V., Biochiz. Spencer, D., and Possingham, 52, 379 (1961).
689
203, 166 (1964). 159 (1963). (1963). 75, 17 (1950). 5. BEchem., Biophys.
Acta,
105, (1963). Biophys. Acta,
BIOCHEMICAL
TRACE METAL COMPOSITION
OF FRACTIONS
FRAGMENTATION
OF SPINACH
Jan M. Anderson,
RESEARCH COMMUNICATIONS
OBTAINED
BY DIGITONIN
CHLOROPLASTS
N.K.
Boardman
of Plant
Industry,
to current
concepts
the
involves
two distinct
Division
C.S.I.R.O.,
AND BIOPHYSICAL
and D.J.
David
Canberra,
A.C.T.
Australia
Receivedseptember
24, 1964
According photosynthesis
in
photochemical These
green
reactions
(cf. -
two reactions
absorbed
system
Absorption
by system of water
light
result
from
system
oxidation
of reduction
resultant phase the
digitonin chlorophyll different One type
after
of the
oxygen
of spinach
of particle
of particle
structural dg
a&
ratio
proteins (2.0).
of 5.0
remains
more
of the intact
the
ATP and oxygen
with that
the
associated
with
and Anderson, in
to 6.0; strongly
digitonin whereas attached
and has a lower
chloroplasts
685
complexes
chloroplasts
is
grana
products
two systems,
soluble
ratio
For
Thus
(Boardman
is more
of
and oxidation
evidence
chloroplasts
denoted
absorbed
chlorophyll-protein
obtained
of particles
light
cytochrome
of spinach
we have recently
a chlorophyll type
the
been
the reduction
NADPH2,
of the
light
1963).
while
of photosynthesis,
incubation
have
evolution.
co-operation
types
in
primary
1963).
2 (Duysens,
1 results
of
by the
which
a cytochrome,
By fractionating obtained
systems,
by system
with
of the
pigment
light
2 causes
by Clayton,
to be mediated
1 and pigment
of
NADP and the
review
are thought
by different
pigment
plants
process
this
two 1964).
and has the
other
to the chlorophyll ratio
was
Vol. 17, No. 6, 1964
3.0.
Since
ratio
are
the it
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
particles
with
the
able
electron
to photoreduce donor
couple
was speculated
system
that
1, while
the
communication
provides
fractions based
presence
contain soluble
evidence
dye),
digitonin
2.
This
that
these
1 and 2.
of
pigment
in
of system
to systems
the
d&
and indophenol
less part
distribution
chlorophyll in
particles
further
correspond on the
(ascorbate
particles
an integral
high
NADP only
these
may represent
the
This
of Cu, Mn and Fe in
present two
evidence the
is
two
fractions. Spinach leaves
in
sucrose
phosphate
purified
digitonin
suspended
O.OlM
was then
separated
following
manner;
(Gentry
phosphate
by differential
all
analysis,
homogenate was rigorously in
chloroform 1950).
chloroplasts (O.O5M, (twice
30 min
at O'C.
were pH 7.2) recrystalised The preparation
centrifugation
at 1000 x g,
and 60 min samples
0.3M
and Sherrington,
buffer
for
10 min
filtered
buffer
KC1 and 0.5% digitonin
at 50 000 x g,
chemical
the
spinach
and incubated
containing
8-hydroxyquinoline
of metals
purified
ethanol)
30 min
with
by blending
pH 7.2)
The phosphate
fragmentation, in
containing in
10 min.
traces
(O.O5M,
KC1 and sedimenting
by extraction
to remove
were prepared
buffer
and O.OlM
at 1000 x g for
For
chloroplasts
in
30 min
at 10 000 x g,
at 144 000 x g. (whole
Prior
chloroplasts, were washed
resuspension
and recentrifugation
at the
glass-distilled
appropriate
speeds,
For lyophilized of H2S04 digestion,
water and then
determination
fractions - HC104
(1+7)
of
(20-50
the
mg) were
was pipetted 686
components,
digested
and 5 ml of cont.
10 ml of water
twice
lyophilized. metal
with
HN03. into
to
the
10 000 x g and 144 000 x g fractions) in
the
flasks,
the 2 ml
After the
by
Vol. 17, No. 6, 1964
contents No.
BIOCMEMICAL
thoroughly
42 papers were
of them,
against
standards
to sample
atomic
absorption
instrument
O-3;
filtrates
and
or dilutions
in parts
and Mg,
and H2S04
solutions.
dry Whatman
manganese
containing
Mn,
O-6;
RESEARCH COMMUNICATIONS
through
iron,
on these
intervals
alent
:
each
at suitable
at a concentration
equiv-
A Perkin
spectrophotometer
O-l
per million
Elmer
Model
303
was used with
the
following
settings: cathode
20 ma for Slit
Copper,
determined
Fe,
concentration
Hollow
and filtered
(9 cm).
magnesium
Cu, O-0.5;
mixed
AND BlOPHYSlCAt
tube
Mn,
width:
Flame:
current:
15 ma for
18 ma for
Cu, 40 ma for
Fe,
Mg.
1.0 mm for
Cu, Mn and Mg;
Stoichiometric
air-acetylene
0.3 mm for
mixture,
Fe.
10 cm
path-length. Resonance,lines: Scale
Cu 3247 i,
expansion:
unexpanded
2-fold
for
for
of
striking
a system 4.5
magnesium
content)
evolving Spencer
times
sequence
higher
than
in
Mg 2852 g. Cu,
synthetic
electron
we believe
to be more representthan
system
(relative
1 particle).
From
to be required
of photosynthesis
the
for
of the
cytochromes
the
(representevidence,
the
oxygen1957;
other
hand
the
144 000 x g fraction Iron
are involved
(Duysens, 687
1 particle
to
(Kessler,
10 000 x g fraction.
transport
other
On the
1961).
I.
case of manganese.
144 000 x g fraction
the
since
the
02-evolving)
contents
implicated
was in
as the
and Possingham, and iron
Table
as much manganese
is believed
copper
are shown in
which
2 (i.e.
of a system
manganese
for
partitioning
contained
ative
Fe and Mn, 5-fold
obtained
The 10 000 x 8 fraction ative
Mn 2795 i,
Mg.
The results The most
Fe 2483 i,
1963).
is in
were of course photo-
Copper
is
Vol. 17, No. 6, 1964
BlOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE: I Trace
Metal
Content
of Chloroplast
Fractions
The Mn, Fe and Cu contents of the fractions are given relative to Mg content. Magnesium was shown to be nearly lOO$ equivalent to the chlorophyll content of these fractions. The magnesium content in umoles/g dry wt. was 90.4 for chloroplasts, 102.4 for 10 000 x g fraction and 62.5 for 144 000 x g fraction respectively. --
Fraction
Mg
Mn
Fe
CU
Chloroplasts
100
1.37
4.16
1.88
3.0
10 000 x g
100
1.93
2.11
1.00
2.0
144
100
0.41
3.41
1.62
5.5
115
1
6
3
2.3+
000
x g
Quantosomez? w
Park
also
& Pon (1963)
--et al.
system
is
located
Recent
(unpublished)
on the
since
has been
(1962).
Smillie
It
# Lichtenthaler
known to be present
plastocyanin
& Calvin
by Katoh that
of the
of interest
transport
pathway.
that
iron
the
fraction.
Comparison
metal
are closer
values
they
are somewhat It
adsorption plast
of the of Park
to those
of heavy
of buffers-used
of intact
and copper
than content
and Pon
located
(1963)
in
the
contents 10 000 x g
of these
fractions
show that
their
chloroplasts,
although
higher.
is worthwhile
fragments
and
electron
are higher
quantosomes
(unpublished) is
144 000 x g fraction
the
by Katoh
plastocyanin
of the
with
(1964)
protein,
in chloroplasts
works
4;
a copper-containing
indicate
1 side
therefore
Chlorophyll
is for
to note
metal very
ions
the
to chloroplasts
great,
isolation
that
and therefore is
688
essential.
likelihood
of
and chloroprior
purification
A similar
Vol. 17, No. 6, 1964
BIOCHEMICAL
experiment
to
fractions
that
were
resulted
in
data
for
the
content
also
with
the
distribution
30 min.
not
only
fractions
is
obtained
consistent
a physical
particles
which 1.
with
of the
determine
in
different photochemical
the
two chloroplast that
of system
1 particles
we have from
of system
of these work
structure,
composition
particles
2 than
content
the
chlorophyll-containing
Moreover
our hypothesis
cytochrome
of
activities,
metals.
between
of
amounts
Further
of these
all
chlorophyll-
interest.
to
be published
in
great
progress
buffer,
high,
results
metals
three
fragmentation
are more representative
be of
metals
of trace
separation
Analysis will
trace
with
ratios
of these
the
phosphate
digitonin
for
different
in which
was particularly
2 and & and different
but
will
I,
unpurified
shows that
particles
chlorophyll
fractions
iron
Table
with
values
chloroplasts
containing
system
isolated
the This
spinach
shown in
higher
fractions;
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is
in
and function
and full
details
elsewhere.
References
Boardman, N.K., and Anderson, J.M., Nature, Ann. Rev. Plant Physiol., 14, Clayton, R.K., Duysens, L.M.N., Proc. Roy. Sot. B., 157,301 and Sherrington, L.G.,nalyst, ~a;;;~,SC.H.Rt, Shiratori, I., and Takamiya, A., , "51, 32 (1962). Kessler, G Planta, 49, 435 (1957). Lichtenthaler, H.K., and Calvin, M., Biochim. 79, 30 (1964). Park, R.B., and Pon, N.G., J. Mol. Biol, 6, J.V., Biochiz. Spencer, D., and Possingham, 52, 379 (1961).
689
203, 166 (1964). 159 (1963). (1963). 75, 17 (1950). 5. BEchem., Biophys.
Acta,
105, (1963). Biophys. Acta,