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P 700 and cytochrome F in particles obtained by digitonin fragmentation of spinach chloroplasts
Vol.
23, No. 6, 1966
BIOCHEMICAL
P 700 AND CYTOCHROME
AND
Department
of Plant Biology,
RESEARCH
-F IN PARTICLES
OBTAINED
OF SPINACH
CHLOROPLASTS
FRAGMENTATION Jan M. Anderson
BIOPHYSICAL
*, D. C. Fork Carnegie
BY DIGITONIN
+
and J. Amesz
Institution
COMMUNICATIONS
of Washington,
Stanford,
California
May 27, 1966 Boardman
and Anderson
plasts
with
digitonin
which
could be separated
the photochemical
caused
resulted
separation
1 and 2 Lcf review
at 10,000 x g were
sedimentable
at 144, 000 x g were
light-induced
changes
P 700 as untreated particles.
systems
* On leave from + On leave from
that small
chloroplasts
As there
1 (Kok and Hoch, chemical
in optical
indicate
is evidence
1961),
into particles In view
density
and three
of these
systems 1965).
in system
sizes, composition,
particles
particles”
2, and the “small
of system
1.
approximately more
that P 700 acts as the reaction that a partial has been achieved
separation
particles”
Experiments
contain
times
(Board-
that digitonin
“Large
around
to four
chloro-
of photosynthesis
in the region
particles
this indicates
of photosynthesis
and Avron,
representative
of different
they postulated
of the two pigment
enriched
of spinach
of the pigment
content
-et -.al ’ 1964),
by Vernon
sedimentable
in this paper,
that incubation
and the manganese
1964; Anderson
in a physical
(systems
a fragmentation
by centrifugation.
activities
man and Anderson,
(1964) reported
on
700 rnp reported twice
as much
than the large center
of system
of the two photo-
by digitonin
fragmentation.
the Division of Plant Industry, C. S. I. R. O., Canberra, Australia. the Biophysical Laboratory, University of Leiden, the Netherlands
Q?A
Vol.
23, No.
6, 1966
Measurements agreement
of absorption with
After digitonin were for
BIOCHEMICAL
changes
incubation
separated
of spinach
and Anderson,
by differential
The sediments changes
previously
1964),
described
upon illumination.
of optical
density
Changes
beam were 144,000
of optical
to measure
were
assumed
selectively
to be neglible.
at 702 and 683 rnp indicating
observed
absorbing
at 683 rnp especially
amount
of P 700 bleached
obtained
1965).
30 cm
signal. of
Since the intensity
than that of the actinic excited
by the measuring
difference
spectra
of the
of reduced
DAD
acts as an effective
electron
The spectra
maxima
large
the 10, 000 x g fraction,
875
intensity
by subtraction
showed
of P 700 and possibly
Since a realtively
light and
was placed
in the presence which
the oxidation
with
of actinic
shut off.
lower
1 shows
observed
Since the high actinic
of fluorescence
and Pistorius,
at 683 mp.
700 rnp were
for fluorescence
Fig.
The
of an apparatus
the fluorescence
beam was
a change of yield
1 (Trebst
by means
the photomultiplier
lo4 times
x g for
-KC1 buffer.
around
beam low.
the measuring
1000 x g
1964).
density
corrected
x g and 10,000 x g fractions
donor for system
speeds;
to apply a high intensity
beam was at least
caused
recorded
the maximum
to minimize
density when
in a phosphate
(2, 3, 5, 6-tetramethyl-p-phenylenediamine),
a pigment
in
particles
x g for 30 min and 144,000
fluorescence,
in order
obtained
errors
changes
chlorophyll
of the measuring light,
were
for 30 min at 0” with 0.5%
and Fork,
of the measuring
of optical
the signal
of the spectrum
at the following
were
(de Kouchkovsky
it was necessary
to keep the intensity
the cuvette
50,000
resuspended
In order
upon illumination,
strong
COMMUNICATIONS
the chlorophyll-containing
centrifugation
were
In all fractions
caused
RESEARCH
in the blue region
chloroplasts
10 rnin, 10,000 x g for 30 min,
light-induced
BIOPHYSICAL
this conclusion.
(Boardman
60 min.
from
AND
the bleaching
fluorescence
signal
the measurements
of was in
Vol. 23, No. 6, 1966
BIOCHEMICAL
X+!Suap
Im!+do
/$SUap
j0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
&UD~~
Itqdo
j0
876
diUDc(3
BIOCHEMICAL
Vol. 23, N:o. 6, 1966
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
this area. may be less reliable. The amount of P 700 in the various fractions by means of a French press was calculated at 702 rn,p upon illumination extinction
coefficient
The chlorophyll
intensity.
A specific
for P 700 at 702 m,u was applied, the
5 in 80% acetone at 663 rnp (MacKinney,
content of the fractions
80 x acet:one (Arnon,
fragmented
from the decrease of optical density
with blue light of saturating
of 82 1 g-t cm-r
same as that of chlorophyll
and in chloroplasts
was calculated after extraction
1941). with
1949).
Table 1 shows that the highest ratio of P 700 was found in the 144,000 x g fraction
(one P 700 for about 200 chlorophyll
Table 1. Comparison of the relative
Fraction
In the 10,000 x g
amounts of chlorophyll
plasts fragments and for the centrifugal tion, in the presence of 8 x lo-’
molecules).
fractions
to P 700 for chloro-
obtained by digitonin fragmenta-
M DAD and 1.2 x 10m3M sodium ascorbate. Chlorophylla
and -b
Chl -a
P 700
Chl -b
Chloroplast fragments:
460 420
2. 82 2. 82
10,000 x 1;
650* 730
2.31 2. 40
50,000 x g
330
4. 10
144,000 x g
235 175
5. 42 5.88
144,000 x g supernatant
980
3. 86
* With 1.2 x lo-’ fraction
M N-methylphenazonium
methosulphate
there was only one P 700 for every 700 chlorophylls.
to P 700 ratio for untreated chloroplasts
instead of DAD The chlorophyll
is in agreement with the value reported
877
Vol. 23, No. 6, 1966
by Kok (1961). individually
BIOCHEMICAL
The calculated
the chlorophyll
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
chlorophyll
to P 700 ratio obtained by summing
and P 700 contents of the fractions,
was 420.
These results are in agreement with the hypothesis that digitonin fragmentation of chloroplasts
brings about a partial
also indicated by measurements difference
separation of systems 1 and 2. This was The
of absorption changes in the blue region.
spectrum had a maximum at 425 rnp which indicated overlap of P 700 Kok, 1961; Witt ----.T et al
(430 mp maximum: (420 rnp maximum: cytochromef
Duysens,
1965) and cytochromef
oxidation
1955); a positive change at 405 rnp confirmed
was being oxidized.
that
Fig. 2, traces (a) and (d) show the kinetics
of the absorbance change at 437 mp, which indicate in agreement with the results at 702 mu, that little P 700 is present in the 10,000 x g fraction. (b), (c), (e) and (f) indicate that there is relatively chrome -f in the 10,000 x g fraction at both saturating
It should however,
change of optical density at 420 rnp in both fractions
Our experiments
oxidation of cyto-
as compared to the 144,000 x g fraction,
and low light intensities.
ed if P 700 and cytochrome
very little
The lower traces
be noted that the
is lower than would be expect-
-f were present in equal amounts.
are evidence for the actual separation of system 1 from
system 2, rather than an inactivation
of system 2 in the small particles.
assumed that P 700 is associated with system 1, and that the chlorophylls approximately
actually found for the 144,000 x g fraction.
particles
would indicate that these
contain about 70 s of system 2 and 30 s of system 1, in good agreebased on the photochemical
1966).
878
activities
to
This value was
The figure of one P 700 for about
molecules in the 10,000 x g fraction
ment with the estimation Boardman,
are
evenly divided between system 1 and 2, the ratio of chlorophyll
P 700 would be expected to be about 200 for system 1 particles.
700 chlorophyll
If it is
(Anderson and
Vol. 23, No. 6, 1966
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References Anderson, J. M. and Boardman, N. K., Biochim. Biophys. Acta, 112, 403 (1966). Anderson, J. M., Boardman, N. K. and David, D. J., Biochem. Biophys. Res. Commun., 17, 685 (1964). Ax-non, D. I., Plant Physiol., 24, 1 (1949). Boardman, N. K. and Anderson, J. M., Nature, 203, 166 (1964). Duysens, L. N. M. , Science, 121, 210 (1955). Kok, B., Biochim. Biophys. Acta, 48, 527 (1961). Kok, B. and Hoch, G., in “Light and Life”, McElroy, W. D. and Glass, B., Eds. (Johns Hopkins Press, Baltimore, 1961) p. 397. de Kouchkovsky, Y., and Fork, D. C., Proc. Natl. Acad. Sci., 52, 232 (1964). MacKinney, G., J. Biol. Chem., 140 315 (1941). Trebst, A. and Pistorius, E., Z. Naturforschg., z, 143 (1965). Vernon, L. P. and Avron, M., Ann. Rev. Biochem., 34, 269 (1965). Witt, H. T., Rumberg, B., and Schmidt-Mende, P., Angew. Chem., 77, 821 (1965)
879
23, No. 6, 1966
BIOCHEMICAL
P 700 AND CYTOCHROME
AND
Department
of Plant Biology,
RESEARCH
-F IN PARTICLES
OBTAINED
OF SPINACH
CHLOROPLASTS
FRAGMENTATION Jan M. Anderson
BIOPHYSICAL
*, D. C. Fork Carnegie
BY DIGITONIN
+
and J. Amesz
Institution
COMMUNICATIONS
of Washington,
Stanford,
California
May 27, 1966 Boardman
and Anderson
plasts
with
digitonin
which
could be separated
the photochemical
caused
resulted
separation
1 and 2 Lcf review
at 10,000 x g were
sedimentable
at 144, 000 x g were
light-induced
changes
P 700 as untreated particles.
systems
* On leave from + On leave from
that small
chloroplasts
As there
1 (Kok and Hoch, chemical
in optical
indicate
is evidence
1961),
into particles In view
density
and three
of these
systems 1965).
in system
sizes, composition,
particles
particles”
2, and the “small
of system
1.
approximately more
that P 700 acts as the reaction that a partial has been achieved
separation
particles”
Experiments
contain
times
(Board-
that digitonin
“Large
around
to four
chloro-
of photosynthesis
in the region
particles
this indicates
of photosynthesis
and Avron,
representative
of different
they postulated
of the two pigment
enriched
of spinach
of the pigment
content
-et -.al ’ 1964),
by Vernon
sedimentable
in this paper,
that incubation
and the manganese
1964; Anderson
in a physical
(systems
a fragmentation
by centrifugation.
activities
man and Anderson,
(1964) reported
on
700 rnp reported twice
as much
than the large center
of system
of the two photo-
by digitonin
fragmentation.
the Division of Plant Industry, C. S. I. R. O., Canberra, Australia. the Biophysical Laboratory, University of Leiden, the Netherlands
Q?A
Vol.
23, No.
6, 1966
Measurements agreement
of absorption with
After digitonin were for
BIOCHEMICAL
changes
incubation
separated
of spinach
and Anderson,
by differential
The sediments changes
previously
1964),
described
upon illumination.
of optical
density
Changes
beam were 144,000
of optical
to measure
were
assumed
selectively
to be neglible.
at 702 and 683 rnp indicating
observed
absorbing
at 683 rnp especially
amount
of P 700 bleached
obtained
1965).
30 cm
signal. of
Since the intensity
than that of the actinic excited
by the measuring
difference
spectra
of the
of reduced
DAD
acts as an effective
electron
The spectra
maxima
large
the 10, 000 x g fraction,
875
intensity
by subtraction
showed
of P 700 and possibly
Since a realtively
light and
was placed
in the presence which
the oxidation
with
of actinic
shut off.
lower
1 shows
observed
Since the high actinic
of fluorescence
and Pistorius,
at 683 mp.
700 rnp were
for fluorescence
Fig.
The
of an apparatus
the fluorescence
beam was
a change of yield
1 (Trebst
by means
the photomultiplier
lo4 times
x g for
-KC1 buffer.
around
beam low.
the measuring
1000 x g
1964).
density
corrected
x g and 10,000 x g fractions
donor for system
speeds;
to apply a high intensity
beam was at least
caused
recorded
the maximum
to minimize
density when
in a phosphate
(2, 3, 5, 6-tetramethyl-p-phenylenediamine),
a pigment
in
particles
x g for 30 min and 144,000
fluorescence,
in order
obtained
errors
changes
chlorophyll
of the measuring light,
were
for 30 min at 0” with 0.5%
and Fork,
of the measuring
of optical
the signal
of the spectrum
at the following
were
(de Kouchkovsky
it was necessary
to keep the intensity
the cuvette
50,000
resuspended
In order
upon illumination,
strong
COMMUNICATIONS
the chlorophyll-containing
centrifugation
were
In all fractions
caused
RESEARCH
in the blue region
chloroplasts
10 rnin, 10,000 x g for 30 min,
light-induced
BIOPHYSICAL
this conclusion.
(Boardman
60 min.
from
AND
the bleaching
fluorescence
signal
the measurements
of was in
Vol. 23, No. 6, 1966
BIOCHEMICAL
X+!Suap
Im!+do
/$SUap
j0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
&UD~~
Itqdo
j0
876
diUDc(3
BIOCHEMICAL
Vol. 23, N:o. 6, 1966
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
this area. may be less reliable. The amount of P 700 in the various fractions by means of a French press was calculated at 702 rn,p upon illumination extinction
coefficient
The chlorophyll
intensity.
A specific
for P 700 at 702 m,u was applied, the
5 in 80% acetone at 663 rnp (MacKinney,
content of the fractions
80 x acet:one (Arnon,
fragmented
from the decrease of optical density
with blue light of saturating
of 82 1 g-t cm-r
same as that of chlorophyll
and in chloroplasts
was calculated after extraction
1941). with
1949).
Table 1 shows that the highest ratio of P 700 was found in the 144,000 x g fraction
(one P 700 for about 200 chlorophyll
Table 1. Comparison of the relative
Fraction
In the 10,000 x g
amounts of chlorophyll
plasts fragments and for the centrifugal tion, in the presence of 8 x lo-’
molecules).
fractions
to P 700 for chloro-
obtained by digitonin fragmenta-
M DAD and 1.2 x 10m3M sodium ascorbate. Chlorophylla
and -b
Chl -a
P 700
Chl -b
Chloroplast fragments:
460 420
2. 82 2. 82
10,000 x 1;
650* 730
2.31 2. 40
50,000 x g
330
4. 10
144,000 x g
235 175
5. 42 5.88
144,000 x g supernatant
980
3. 86
* With 1.2 x lo-’ fraction
M N-methylphenazonium
methosulphate
there was only one P 700 for every 700 chlorophylls.
to P 700 ratio for untreated chloroplasts
instead of DAD The chlorophyll
is in agreement with the value reported
877
Vol. 23, No. 6, 1966
by Kok (1961). individually
BIOCHEMICAL
The calculated
the chlorophyll
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
chlorophyll
to P 700 ratio obtained by summing
and P 700 contents of the fractions,
was 420.
These results are in agreement with the hypothesis that digitonin fragmentation of chloroplasts
brings about a partial
also indicated by measurements difference
separation of systems 1 and 2. This was The
of absorption changes in the blue region.
spectrum had a maximum at 425 rnp which indicated overlap of P 700 Kok, 1961; Witt ----.T et al
(430 mp maximum: (420 rnp maximum: cytochromef
Duysens,
1965) and cytochromef
oxidation
1955); a positive change at 405 rnp confirmed
was being oxidized.
that
Fig. 2, traces (a) and (d) show the kinetics
of the absorbance change at 437 mp, which indicate in agreement with the results at 702 mu, that little P 700 is present in the 10,000 x g fraction. (b), (c), (e) and (f) indicate that there is relatively chrome -f in the 10,000 x g fraction at both saturating
It should however,
change of optical density at 420 rnp in both fractions
Our experiments
oxidation of cyto-
as compared to the 144,000 x g fraction,
and low light intensities.
ed if P 700 and cytochrome
very little
The lower traces
be noted that the
is lower than would be expect-
-f were present in equal amounts.
are evidence for the actual separation of system 1 from
system 2, rather than an inactivation
of system 2 in the small particles.
assumed that P 700 is associated with system 1, and that the chlorophylls approximately
actually found for the 144,000 x g fraction.
particles
would indicate that these
contain about 70 s of system 2 and 30 s of system 1, in good agreebased on the photochemical
1966).
878
activities
to
This value was
The figure of one P 700 for about
molecules in the 10,000 x g fraction
ment with the estimation Boardman,
are
evenly divided between system 1 and 2, the ratio of chlorophyll
P 700 would be expected to be about 200 for system 1 particles.
700 chlorophyll
If it is
(Anderson and
Vol. 23, No. 6, 1966
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References Anderson, J. M. and Boardman, N. K., Biochim. Biophys. Acta, 112, 403 (1966). Anderson, J. M., Boardman, N. K. and David, D. J., Biochem. Biophys. Res. Commun., 17, 685 (1964). Ax-non, D. I., Plant Physiol., 24, 1 (1949). Boardman, N. K. and Anderson, J. M., Nature, 203, 166 (1964). Duysens, L. N. M. , Science, 121, 210 (1955). Kok, B., Biochim. Biophys. Acta, 48, 527 (1961). Kok, B. and Hoch, G., in “Light and Life”, McElroy, W. D. and Glass, B., Eds. (Johns Hopkins Press, Baltimore, 1961) p. 397. de Kouchkovsky, Y., and Fork, D. C., Proc. Natl. Acad. Sci., 52, 232 (1964). MacKinney, G., J. Biol. Chem., 140 315 (1941). Trebst, A. and Pistorius, E., Z. Naturforschg., z, 143 (1965). Vernon, L. P. and Avron, M., Ann. Rev. Biochem., 34, 269 (1965). Witt, H. T., Rumberg, B., and Schmidt-Mende, P., Angew. Chem., 77, 821 (1965)
879