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Plant Cell Physiol. 38(2): 133-138 (1997)
JSPP © 1997
Isolation, Properties and a Possible Function of a Water-Soluble
Chlorophyll a/6-Protein from Brussels Sprouts
Yasumaro Kamimura, Takahiro Mori, Takenobu Yamasaki and Sakae Katoh
Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, 274 Japan
A water-soluble Chi a/6-protein (CP673) was isolated
and purified from Brussels sprouts (Brassica oleracea L.
var. gemmifera DC). The protein had a molecular mass of
78 kDa and an isoelectric point of 4.7, consisted of three or
four subunits of 22 kDa and was extremely heat-stable. Although CP673 contained about one Chi a per protein, the
blue and red absorption bands of Chi a that consisted of
three or four Chi a forms with different absorption maxima
suggested that there are several different modes or sites of
binding for Chi a. Chi a/b ratio of larger than 10 also indicated that Chi b is present only in a small fraction of
CP673. The heterogeneity of CP673 in terms of composition and binding of Chi suggests that Chi is not an intrinsic
component of the Chl-protein. Homology search showed
that the N-terminal amino acid sequence of CP673 is highly
homologous with that of a 22 kDa protein that accumulates in water-stressed leaves of two Brassicaceae plants,
rapeseed and radish, but not with those of the light-harvesting Chi a/6-proteins of photosynthesis. A possible function of the water-soluble Chl-protein was discussed.
ygen, a new absorption peak appeared at 743 nm concomitant with diminution of a Chi a band at 668 nm. Similar
photosensitive water-soluble Chl-proteins distribute among
several species of Chenopodiaceae and Amaranthaceae
(Takamiya 1972).
Water-soluble Chl-proteins that lack the photoconvertibility of Chi a have been isolated from inflorescence of
cauliflower (Murata et al. 1971) and leaves of wild mustard
and Lepidium virginicum (Murata and Murata 1971). The
Chl-proteins from these three Brassicaceae plants have molecular masses of 78 to 93 kDa (Murata et al. 1971, Murata
and Murata 1971, Murata and Ishikawa 1981). The Lepidium Chl-protein consists of several subunits of 17-20 kDa
(Murata 1976, Tabata et al. 1983) and carrys four Chi per
protein (Murata and Ishikawa 1981). Chi a/b ratio varies
with species (Murata et al. 1971, Murata and Murata 1971)
or even with organs of the same plant (Itoh et al. 1982).
An important question as to what the physiological
function of these water-soluble Chl-proteins is has remained unanswered. Krasnovsky (1965) suggested that CP668
might be produced by a catalytic action of chlorophyllase
present in Chenopodium leaves. Terpstra (1966) ruled out
this possibility by showing that the amount of CP668 does
not increase during a prolonged incubation of leaf extracts
at room temperature and suggested that CP668 is a denaturation product of a Chl-protein in leaves because no
photoconversion of Chi in the protein was detected in illuminated Chenopodium leaves. CP668 is present more abundantly in stems than in leaves (Takamiya 1972). Atriplex
hortensis CP668 is present in stems throughout the life cycle of plants, whereas the content of the protein in leaves
was very low in young plants and increased exponentially
at the onset of the reproductive stage (Hager and Nobs
1974). These observations, as well as the limited distribution of the water-soluble Chl-proteins among plant species,
cast a doubt on the notion that the Chl-protein is a component of photosynthesis.
Key word: Brassica oleracea L. var. gemmifera DC
(Brussels sprouts) — Chlorophyll a form — Drought-induced protein — Kiinitz type protease inhibitor — Watersoluble chlorophyll-protein.
There are a variety of Chl-protein complexes in green
leaves that function in the light-harvesting and the primary
photochemistry of the two photosystems. All the Chl-proteins related to the early events of photosynthesis are hydrophobic membrane proteins that can be solubilized only
with the aid of detergents. In 1963, Yakushiji et al. found a
water-soluble Chl-protein from leaves of Chenopodium
album (Yakushiji et al. 1963). The Chl-protein called
CP668 has a molecular mass of 78 kDa (Takamiya 1972) or
69 kDa (Oku and Tomita 1975) and is extremely heat-stable
(Oku et al. 1972). The molar ratio of Chi a to b was about
6.0 but the two different contents of Chi [7 Chi (Takamiya
1972) vs. 23 Chi (Oku and Tomita 1975)] have been
reported. On illumination of CP668 in the presence of ox-
In the present study, we report isolation and properties of a water-soluble Chl-protein from Brassels sprouts.
Evidence was obtained indicating that the Chl-protein is
heterogeneous in both composition and binding site of
Chi. Homology search of the N-terminal amino acid sequence suggested a possible function for the Chl-protein.
Abbreviation: CP673, a water-soluble chlorophyll-protein
from Brassica oleracea L. var. gemmifera DC; CP668, a watersoluble chlorophyll-protein from Chenopodium album.
133
134
A water-soluble Chl-protein from Brussels sprouts
Materials and Methods
The water-soluble Chl-protein was extracted from Brussels
sprouts (Brassica oleracea L. var. gemmifera DC) purchased from
local markets. About 1 kg of the sprouts were cut into small pieces
and homogenized with 2 liters of 50 mM phosphate buffer, 2 M
NaCl, 26 mM sodium ascorbate and 10 g polyvinylpyrrolidone
with a blender. Throughout extraction and purification procedures, phosphate buffer of pH 7.0 was used. The homogenate
was strained through four layers of gauze and the filtrate was centrifuged at 8,000 xg for 40min. To one liter of the supernatant
was added 176 g of ammonium sulfate and the resulting precipitate was removed by centrifugation. An additional 300 g of ammonium sulfate was added to the supernatant and the precipitate
appeared after standing for 2 h was collected by centrifugation at
8,000xg for 40 min and dissolved in 5 mM phosphate. Taking a
unique advantage that the water-soluble Chl-protein is extremely
heat-stable (see below), large amounts of extraneous proteins were
removed by heat-treatment at 90°C for 5 min and subsequent centrifugation. The supernatant was applied to a DEAE-cellulose column (3 x 20 cm) equilibrated with 5 mM phosphate. The charged
column was successively washed with 10 mM, 20 mM and 50 mM
phosphate and a green fraction containing the water-soluble Chlprotein was eluted by increasing concentration of phosphate to
100 mM. Then, the Chl-protein fraction was dialyzed against a
large volume of 5 mM phosphate and placed onto a column of hydroxyapatite (Bio-Rad) equilibrated with the same phosphate buffer. After intensive wash with 20 mM and 50 mM phosphate, the
Chl-protein was eluted with 100 mM phosphate. If necessary,
the Chl-protein was further purified by column chromatography
with Sephacryl s-200 (Pharmacia) or DEAE-cellulofine (Seikagaku
Kogyo).
Absorption and fluorescence spectra were determined with a
Hitachi U-3300 spectrophotometer and a Hitachi 850 fluorescence
spectrophotometer, respectively. For the curve-fitting analysis, the
data were inputted into a computer and processed according to the
method of French et al. (1972). Second derivative spectra were
used to determine number and peak position of Chi components.
Molecular mass was determined by gel-filtration chromatography with Sephadex G-100. Polypeptide composition was analyzed
by SDS polyacrylamide gel electrophoresis using the buffer system
of Laemmli (1970). Samples were denatured with 6 M urea, 2.5%
Table 1
SDS and 5% mercaptoethanol and applied to gels containing
acrylamide and 0.13% SDS. Isoelectric point was determined with
a Pharmacia Phastsystem and a Pharmacia calibration kit. The
gel used was PhastGel IEF 3-9 that contains ampholite pH 3-9.
Protein was quantified by the microbiuret method (Ellmann
1962) with bovine serum albumin or soybean trypsin inhibitor as
the standard protein. N-terminal amino acid sequence was determined with a ABI472A protein sequencer and the NCBI GenBank
data base was used for homology search.
Chi was extracted with methyl ethyl ketone as described by
Murata et al. (1968) and determined spectrophotometrically (Porra
et al. 1989) or by HPLC. For HPLC, the extract was dehydrated
with anhydrous Na2SO4 and the solvent was evaporated in a
vacuum in the dark. Chi was dissolved in methanol and applied to
a Waters HPLC system equipped with a//Bondasphere C18 (3.9 x
150 mm) column. The mobile phase was 20% iso-propanol and
80% methanol and absorbance of eluents was monitored at 440
nm. Chi a and b that had been extracted from spinach leaves and
purified chromatographically were used as the standard.
Results and Discussion
Yields of the water-soluble Chl-protein varied with
lots of the sprouts obtained. Several lots of the pale green
sprouts employed in the early stage of experiments contained small amounts of the Chl-protein and large quantities of
dark brown substances which strongly interfered with purification of the protein. Treatment of the extracts at 90°C
for 5 min greatly reduced extraneous proteins but not the
colored substances which were difficult to remove completely from the Chl-protein fraction by repeated column chromatography. A batch of the green sprouts, imports from
Australia, were found to contain a large amount of the
Chl-protein (282 nmol protein per kg) that corresponds to
more than 10 times the contents of the Chl-protein in the
materials employed in earlier experiments. The protein
could be readily purified due to small amounts of the dark
brown substances present in the extracts. All the data
presented here were obtained with the Chl-protein prepara-
Properties of water-soluble Chl-proteins from Brassicaceae plants
Brussels sprouts
Red band max.
Chl/protein
673 nm
1
L. virgin icum
661-663 nm"
4"
Chi a/b
>10
1.0-1.9"
Molecular mass
protein
subunit
78kDa
22kDa
78-80 kDa"
20 kDa*
Isoelectric point
° Murata, T. and Ishikawa, C. (1981).
* Murata, T. (1976).
c
Murata et al. (1971).
" Murata, T. and Murata, N. (1971).
4.7
4.2-4.5"
Cauliflower
Wild mustard
674 nm c
673 nm"
—
—
6.0
c
78 kDa c
—
4.6C
8.0 d
93 kDa"
—
A water-soluble Chl-protein from Brussels sprouts
tion isolated from this material.
Table 1 summarizes the properties of the purified Chlprotein. For the comparison, the properties of the watersoluble Chl-proteins from other Brassicaceae plants are
shown. Molecular mass of the Chl-protein determined by
gel nitration chromatography was 78 kDa. A non-heated
sample gave an identical molecular mass. SDS gel electrophoresis of the protein yielded, however, a single band at a
position corresponding to 22 kDa (Fig. 1). This indicates
that the Chl-protein consists of three or four subunits.
A subunit of a comparable size has been reported for
Lepidium Chl-protein (Murata 1976). Treatment of the
Chl-protein with a zero-length crosslinker, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, or different concentration of urea failed to yield a band corresponding a dimer
(or trimer) of the subunit (not shown). The Chl-protein
was an acidic protein with an isoelectric point of 4.7. The
protein was extremely heat-stable; treatment at 90° C for
30 min resulted in neither aggregation nor bleaching of the
protein (not shown).
Fig. 2 shows the absorption spectrum of the Chl-protein. Because the absorption maximum of Chi a in the red
region is located at 673 nm, we call hereafter the Chl-protein CP673. The occurrence of a small amount of Chi b
was suggested by a small shoulder at 468 nm. The absorption band of Chi a in the blue region was split into three
bands of comparable heights with maxima at 437, 421 and
384 nm. A similar broad blue band with multiple peaks
have been reported for the water-soluble Chl-protein from
cauliflower (Murata et al. 1971) and wild mustard (Murata
and Murata 1971). Illumination of CP673 with strong
white light (3,000//mol photons m" 2 s"') resulted in slow
bleaching of the absorption bands but, unlike the C. album
135
Chl-protein CP668, loss of the 673 nm band was not accompanied by appearance of a new absorption band at a longer
wavelength (not shown).
The amount of Chi bound to CP673 was determined
by extracting Chi with methyl ethyl ketone (Murata et al.
1968). The total extraction of Chi with acetone or ethanol
was difficult due to formation of protein aggregates that
still contained some Chi. Chi was quantified spectrophotometrically or by HPLC and protein was determined with
bovine serum albumin or soybean trypsin inhibitor as the
standard protein. Irrespective of the methods or the proteins used, the molar ratio of Chi to protein was estimated
to be about one. This is the lowest Chi content among the
water-soluble Chl-proteins so far determined and indicates
that there is no one to one stoichiometry between Chi and
the subunit protein. CP673 contained no carotenoid. Although no Chi b was detected by ordinary spectrophotometric assay, the occurrence of a small amount of Chi bwas confirmed by HPLC. The content of Chi b could not
be accurately determined, however, due to appearance of
several small peaks of unidentified Chi derivatives. Even
when all of them were assumed to have derived from Chi b,
the abundance of Chi b was less than 10% of that of Chi a,
indicating binding of Chi b to only a small fraction of
CP673. Thus, the Chl-protein is heterogenous in the pigment composition.
The three-peaked feature of the blue band of Chi a suggests a heterogeneity of CP673 in the mode or site of binding for Chi a. Curve-fitting analysis of the absorption spectrum of CP673 in the red region also resolved three major
Chi a forms with maxima at 665, 674, 683 and a minor Chi
a form at 696 nm (Fig. 3A). A small band at 655 nm is
ascribed to Chi b. The fluorescence band determined at
room temperature was also resolved into four components
with maxima at 669, 677, 687 and 698 nm, which are
kDa
94—
67—
43—
8
0.6
30-
J 0.4
20-
14-
400
500
600
700
800
Wavelength (nm)
Fig. 1 SDS polyacrylamide gel electrophoresis of the Chl-protein CP673. Lane 1, marker proteins; lane 2, CP673.
Fig. 2 Absorption spectrum of CP673. Numbers indicate location of peaks and shoulders in nm.
136
A water-soluble Chl-protein from Brussels sprouts
640
16.5
16.0
15.5
720 740
15.5
15.0
14.5
14.0
Wavenumber (x 103 cm1)
Wavelength (nm)
690
720
660
16.0
Wavelength (nm)
660
680 700
15.0
14.5
14.0
750
13.5
13.5
780
13.0
12.5
3
Wavenumber (x 10 cm"')
Fig. 3 (A) Curve-fitting analysis of the absorption spectrum in
the red region of CP673. Thick solid line, absorption spectrum of
CP673; thin solid line, absorption spectra of Chi a components resolved; dotted line, sum of absorption spectra of Chi a components. (B) Curve-fitting analysis of thefluorescencespectrum of
CP673. Thick solid line, fluorescence spectrum of CP673; thin
solid line, fluorescence spectra of components resolved; dotted
line, sum of fluorescence spectra of resolved components.
1
CP673
5
BnD22
P22
WCI-3
STI
10
I* ~ 1
1
!I
, -
t
i
R E QiV;K;D
L
s
15
i
i
1
i
1
i
.
i
i
i
1
1
|
i
I
t
i
I
1
i
1
i
1
1
I
1
1
1
1
i
. D F
20
l~
N;G;N P;V;K R G A
E1
1
V
1 J?
V|- A
D D D
related to the Chi a components at 665, 674, 683 and
696 nm, respectively, with the Stokes shifts of 2-4 nm
(Fig. 3B). The different Chi a forms are ascribed to different natures or modes of interactions of Chi a with the protein molecule. When the Chi a to protein ratio of about one
is taken into account, a simple explanation would be that
the CP673 preparation is a mixture of the protein molecules that bind one each Chi a molecule at different sites.
At any event, the heterogeneity of CP673 in both composition and binding of Chi casts a strong doubt on the notion
that Chi is an intrinsic constituent of the Chl-protein. An
implication is that CP673 is a carrier of Chi but not a component related to the capture and utilization of light in photosynthesis.
Homology search of the amino acid sequence, however, provided evidence suggesting a role of CP673 in a
physiological event not related to photosynthesis. The Nterminal amino acid sequence of CP673 is shown in Fig. 4.
No evidence was obtained indicating that the protein consists of heterogeneous subunit proteins. Although CP673
carrys both Chi a and b, no homology was found between
the Chl-protein and light-harvesting Chi a/b proteins that
are encoded by a family of nuclear genes and function in
the capture of light in the two photosystems (Jansson
1994). Thus, the water-soluble Chi a/6-protein is neither a
member nor a denaturation product of the Chi a/b-proteins of photosynthesis.
CP673 was found to be highly homologous with two
proteins from Brassicaceae plants in the N-terminal amino
acid sequence. The 31 amino acid sequence of CP673
perfectly matches that of a 22 kDa protein of rapeseed except for a single replacement of Gly (27) by Ala (Reviron et
al. 1992). The rapeseed protein is related to water-stress because the protein was negligible in leaves of well-watered
plants, appeared in leaves of plants that had been subjected
to drought stress and rapidly disappeared on rehydration.
The N-terminal sequence of CP673 is also identical with
that of a salt or drought-induced 22 kDa protein in radish
leaves in 25 out 31 amino acids when deletion of 3 amino
acids is assumed (Lopez et al. 1994). The rapeseed protein
L!- N E!- —
i
25
fJFJ I ; Q
i
i
i
i
•
i
1
1
i
i
O 1 —'—i—i —
~
1
1
1
i
i
II
i
L.'S N - G
1
I
i
30
G G G L V
A
• •
•
V - T E -—N
i
1
N - G Tj- - ; Y ; L ; L
1
P A K • • S N
—H
A
—
—
—
I E
D I • • T A . F
I R
I . . WA H
i
Ti--!Y!-!L
s
Fig. 4 N-terminal amino acid sequences of CP673, drought-induced proteins and Kiinitz type protease inhibitors. BnD22, rapeseed
drought-induced 22 kDa protein (Reviron et al. 1992); P22, radish drought-induced 22 kDa protein (Lopez et al. 1994); WCI-3 , winged
bean Kiinitz type chymotrypsin inhibitor (Shibata et al. 1988); STI, soybean Kiinitz type trypsin inhibitor (Kim et al. 1985). — , amino
acid identical to that of CP673; • , amino acid deleted. The signature motif of Kiinitz type protease inhibitor is enclosed with dashed line.
A water-soluble Chl-protein from Brussels sprouts
is an acidic protein with an isoelectric point of 5.1 (Reviron
et al. 1992) and the molecular mass (22 kDa) of the two
proteins determined by SDS gel electrophoresis coincides
with that of the subunit of CP673. We suggest, therefore,
that the water-soluble Chl-protein isolated from Brussels
sprouts is a member of the drought-induced proteins in
Brassicaceae plants.
The rapeseed and radish proteins were postulated to
function as an inhibitor of proteases since their amino acid
sequences contain the signature motif of the Kiinitz family
of protease inhibitors (Reviron et al. 1992, Lopez et al.
1994). This possibility could not be examined, however, because the proteins have so far been isolated only in denatured state by SDS gel electrophoresis. The present study
that suggests that the native form of the stress proteins is a
78 kDa Chl-carrying protein is, therefore, an important
step foreword in investigation into the physiological function of the drought-induced proteins. In fact, the N-terminal sequence of CP673 contained the signature motif of
the Kiinitz-type inhibitor family [Val-x-Asp-(x)2-Gly-(x)2Val-(x)5-Tyr-x-Val], where Val may be replaced by Leu, He
or Met (Bairoch 1991). Thus, the water-soluble Chl-protein
enables us to study a protease-inhibitor activity of the
Brassicaceae proteins with the signature motif for the first
time. Preliminary experiments showed that CP673 inhibits
neither pancreatic trypsin nor chymotrypsin (not shown).
In conclusion, the results obtained here indicate that
the water-soluble Chi a/fc-protein CP673 isolated from
Brussels sprouts is not a functional constituent of photosynthesis. There is a possibility that CP673 serves as a carrier
of Chi. At present, however, the most attractive hypothesis
is that the Chl-protein is an inhibitor of a specific plant protease^) and plays a role in regulation of protein breakdown
in water-stressed leaves. Experiments to study accumulation and function of CP673 in leaves under various stress
conditions are in progress.
Finally, it is to be mentioned that a conclusion similar
to ours was independently obtained by Nishio and Satoh,
Department of Biomolecular Science, Toho University,
who isolated and determined the N-terminal amino acid
sequence of a water-soluble Chl-protein from cauliflower
(personal communication).
The authors thank Drs. K. Nakayama and T. Imai for their
aids in the curve-fitting analysis and the N-terminal sequence determination, respectionaly, and Mr. J. Yamazaki for technical assistant.
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A water-soluble Chl-protein from Brussels sprouts
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(Received September 17, 1996; Accepted November 18, 1996)