Purification and Some Properties of Chalcone Synthase from a Carrot ...

J. Biochem. 98, 9-17 (1985)

Purification a Carrot

and Some Suspension

Synthesis

Properties Culture

Induced

and Preparation

Yoshihiro Yoshiyuki Ushio

Synthase

Antiserum

OZEKI,* Katsuhiro SAKANO,* Atsushi TANAKA,** Hiroshi NOGUCHI,***

*Department

and Tatsuo

KOMAMINE,*,z

SUZUKI****

of Botany

, Faculty of Science, The University Tokyo 113, **Laboratory of Pesticide

Bunkyo-ku,

from

for Anthocyanin

of Its Specific

SANKAWA,***

Hongo,

of Chalcone

of Tokyo, Resistance,

National Institute of Agro-Environmental Sciences, Tsukuba, Ibaraki 305, ***Department of Phytochemistry , Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, and ****Biomedical Laboratory , The Kitasato Institute Hospital, Shirogane, Minato-ku, Tokyo 108

Received

for publication,

Chalcone resis

synthase from

induced

cell by

acetic

acid

enzyme and mary

(2,4-D)

of

Km

those

Abbreviations: ME,

Tris-buffered

Vol.

98,

No.

chalcone

properties that

from

and

of

carrot

pH

of

synthase

parsley

antiserum

CHS, PBS,

be sent.

Present

address:

carrot

reaction 5.7

The

pri

was

8.0.

,ƒÊM and

18 ƒÊM,

discussed

in

com

previously.

was obtained

trom

was examined

against

Biological

the

chalcone

Institute,

mice bred

by Western synthase

was

Faculty

of Science,

HRPO,

horse

Tohoku

Uni

980. chalcone

synthase;

2,4-D,

MW,

molecular

weight;

phosphate-buffered

saline;

2,4-dichlorophenoxyacetic PAGE, SDS,

polyacrylamide sodium

saline.

1, 1985

the

were

reported

specificity

should

from

for

electrophoresis,

were

the

of the

of

malonyl-CoA

chalcone

was

80,000-85,000

electrophoresis.

optimum

Crossreactivity

high

synthase

slab-gel

free conditions.

Miyagi

chalcone

cultures

of

polyacrylamide

SDS

the and

cell

disc-gel

by

electropho

synthesis

2,4-dichlorophenoxy

weight

against

2-mercaptoethanol;

ammonia-lyase;

and

gel

anthocyanin

containing

molecular

subunit

4-coumaroyl-CoA

of

medium

pathogen

correspondence

Sendai,

filtration the

polyacrylamide

which

specific

blotting, and demonstrated.

versity,

A

in

Antiserum under

1 To whom

gel

for

a

it.

by

carrot

from

lacking

was

These to

homogeneity of

cells

by

product

respectively. parison

one

40,600

for

to

cultures

determined

values

7, 1984

purified

the to

about

reaction

The

was suspension

transferring

was one

November

9

acid;

gel electrophoresis;

laurylsulfate;

SPF,

specific

radish

peroxidase;

PAL,

phenylalanine

pathogen

free;

TBS,

10

Investigation of secondary metabolism in plant cells has recently progressed and many enzymes involved have been identified and some purified. In particular, the flavonoid pathway has been ac tively studied for the last ten years. Hahlbrock and his colleagues have purified various enzymes of the pathway and prepared rabbit antisera against them (1-3). They also succeeded in preparing cDNA for some of their enzymes (4, 5). Using the antisera and cDNAs, they attempted to deter mine how expression of the enzyme activities is regulated (2-9).

With the use of suspension cultures of parsley (10), Haplopappus gracilis (11), Phaseolus vulgaris (12), and Daucus carota (13), phenylalanine am monia-lyase (PAL) and chalcone synthase (CHS) have been found to be the key enzymes in flavo noid metabolism; PAL regulates the metabolic flow from primary metabolism to the phenylpro panoid pathway and CHS regulates the switching step from the phenylpropanoid pathway to flavo noid metabolism. Both of them were induced by ultra-violet light in suspension cultures of parsley (10) and Haplopappus gracilis (11), and by an eli citor in a Phaseolus vulgaris suspension culture (12), and suppressed by gibberellic acid in a carrot suspension culture (13), resulting in induction or reduction of the flavonoid pathway. These studies also revealed that CHS, rather than PAL, is more important in flavonoid metabolism, as reported by the authors mentioned above. Nevertheless, only two groups have reported CHS purification (1, 14, 15) and detailed properties have only been reported for the enzyme from a parsley suspension culture, in which CHS was induced by UV irradia tion (1, 14, 16-18). The main difficulty in CHS pu rification may be due to the extreme instability of CHS as shown by Kreuzaler and Hahlbrock (14). As the first step in elucidation of the regula tory mechanisms of induction of anthocyanin syn thesis by auxins (19, 20), we found that the key en zyme in that induction was CHS in this system (Ozeki, submitted). The purification of CHS and the preparation of antiserum against CHS were at tempted next to investigate the mechanisms of the induction of anthocyanin synthesis. In this paper, we report the purification of CHS to a very high degree from our carrot suspension culture and the preparation of a highly specific and pure antiserum against the CHS from mice under specific pathogen

Y . OZEKI

et al.

free (SPF) conditions. Their properties will be discussed in comparison with those reported for the parsley enzyme, in which CHS was induced by UV irradiation. MATERIALS

AND

METHODS

Buffers and Chemicals-The buffers used were; (A) 100 mM KP1, pH 8.0, 4.2 mM ME. (B) 10 mM KP1, pH 5.7, 4.2 mm ME, 1 mm EDTA. (C) 50 mm KP1, pH 5.7, 4.2 mm ME, 1 mm EDTA. (D) 300 mm KP1, pH 5.7, 4.2 mm ME, 1 mm EDTA. (E) 50 mm KP1, pH 7.25, 4.2 mm ME. (F) 600 mm KP1, pH 7.25, 4.2 mm ME. (G) 25 mm Tris acetate, pH 8.3, 1.4 mm ME, 0.2 mm EDTA. (H) 1 : 13 diluted Polybuffer-96 (Pharmacia Fine Chemicals AB, Uppsala), pH 5.75, 1.4 mm ME, 0.2 mm EDTA. (I) 10 mm NaP1, pH 7.4, 140 mm NaCl (P1-buffered saline, PBS). (J) 25 mm Tris, 192 mm glycine, pH 8.3, 20 % methanol, 0.03 SDS. (K) 20 mM Tris-HCl, pH 7.4, 140 mM NaCl (Tris-buffered saline, TBS), 0.05 % Nonidet P-40, 0.02% SDS, 1.5% gelatin. (L) TBS containing 0.5 % Nonidet P-40, 0.2 % SDS, 0.2 % gelatin. 4-Coumaroyl-CoA was prepared according to the method of Stockigt and Zenk (21). [2-14C] Malonyl-CoA (46.1 mCi/mmol) was obtained from New England Nuclear, Boston, Mass., and diluted with cold malonyl-CoA purchased from Sigma Chemical Co., St. Louis, Mo. Naringenin (4',5,7 trihydroxyflavanone) was purchased from Nakarai Chemicals, Ltd., Kyoto, and purified by charcoal treatment and recrystallization from aqueous meth anol. Chalcone (4,2',4',6'-tetrahydroxychalcone) was synthesized from naringenin according to the method of Moustafa and Wong (22). Rabbit anti-mouse IgG serum was purchased from Medical Biological Laboratories, Nagoya. Horse radish peroxidase (HRPO) conjugated goat IgG against rabbit IgG and colored materials were purchased from Bio-Rad Laboratories, California. Molecular weight marker proteins were obtained from Boehringer-Mannheim 1, Mannheim. Cell Daucus

Cultures-Suspension-cultured carota

according

to

ported were and cultured

cv. the

(19). sieved cells

modified

In

brief,

in

the

size

150

ml

81

of

re

subculture,

31 ƒÊm

modified

of

cultured

previously

every

and

range of

were

method for

through

in

cells

"Kurodagosun"

screens

31-81 ƒÊm Lin

cells

nylon were and

J.

sub

Staba's

Biochem.

PURIFICATION

(23)

OF

medium

2,4-D

containing

(24)

these

in

500

and

cells

sity

10-6m

4-5

days

after

the

cultured

mediately

stable

for

Purification was chilled an

in

was

in

extracted

by

at

4•Ž.

17,000 •~

g

for

were

absorbed

with

buffer

lected

15 to

was

the

at

h.

Sephadex

loaded

on

then

with

gradient

cm ƒÕ •~

The

buffer

passage

two

cm),

hydroxyapatite kyo) buffer buffer

E. E,

then

(2.6 The and

Vol. 98, No.

1, 1985

20

was the

cm)

with was

eluted

(w/v)

these

con

lost

over

one

a

with of in

the

POPOP tion

thin

acetic

the

and

radioactivity of

organic

cocktail

the

above

and

of

the

was

dissolved

in

spotted

on Co.,

in PPO with

in

naringenin

and

light them and

were

was 0.25

a liquid

purification

directly

cen

layer

(Funakoshi

chromatography

was

5

chromatography

spots

5 g

toluene

layer

layer as

of

Of thin

and

the

vigorous

ultra-violet

solution

of

was

the

a

by both

desk-top

residue

under

1 liter

a

organic

plates

off

in

in

After

acid,

for

stopped

the

layer

Japan).

mCi/mmol)

with

methanol

of

incubation

containing

of

The of

(25

chalcone

150ƒÊl

The

4-coumaroyl

was

centrifugation

counter.

cedure

5ƒÊg

slightly

composed

after

reaction

up.

being

CoA

ethylacetate

for

reported

products.

nmol

Usually,

detected

in

ME, 1

8.0.

dried

Tokyo,

sured

those

was

[2-14C]malonyl

2 min,

(v/v)

conditions

as

(100 ƒÊl)

nmol

amount

scraped

To

by

14

After

small

30%

(3.5

ml

mixture

and

and

same

of

200 ƒÊl

for

assay

the

(14),

naringenin

Ltd.,

by

Ltd.,

100

30%

Under

separation

30•Ž,

chalcone

on

equilibrated

washed

enzyme

E

loaded

eluted against

ultra-filtration

was

Hahlbrock

Avicel-cellulose

buffer

buffer

Co.,

a

with

pooled

columns

80•Ž.

the

pH

of

taken

the

were

G25

was

to

and

nmol

at

trifuge

B and

and

enzyme,

agitation.

was

eluted C

1.5

and

ƒÊg

(3 cm ƒÕ

were

Chemical

cm ƒÕ •~

column then

with

they

(Nippon

column

buffer fractions

CoA,

addition

equili

buffer

by added

Assay-The

KPl,

10 min

for

After

was

Sephadex

and

ml

CHS

replaced

to

four

cm)

B.

200

from

active

was

through 35

C,

formed

IA).

the

buffer

10 ƒÊmol

the

added

column

buffer

with

assay

solution

CL-6B

washed

55

standard

through 30

Kreuzaler for

stirring

protein

was

containing

dialyzed

activity

essentially

modified

centrifugation,

cm ƒÕ •~

The

with

ml

with

desalted

(4.5

B.

been 258

(Fig.

and

and

by

centrifugation was

at

was eluate

column

CHS

was

was

the

Tokyo)

the

A

concentrated

Activity

were

Solid

After

by

by

by

to

slowly.

(NH4)2S04

columns buffer

had

linear

D

A

removed

extract

saturation

equilibrated

column

6 h

a CM-Sepharose

cm)

crude

collected

buffer

G25 with

80%.

CHS CHS

col

min.

was chro

Ltd.,

and

eluate

of

buffer

(NH4)2SO4

The

saturation,

storage

with eluate

buffer

Glycerol

30-40%

cm) the

month.

was

15

of

then

on

Chemicals

saturation,

column.

1 ml. by

85%

The

was

column

purified

Co.,

solid

the

85 %

and

ditions,

then

supernatant

was

for

removed g,

were

in

brated

x 17

to

Proteins

dissolved

a

17,000 •~

supernatant

12

resin

for

A

Mfg.

after

10 ml

A.

followed

debris

and

at

about

1 •~ 4 equilibrated

g the

were

the

to

were

of cell

supernatant

the

stirring

steps

10 min in

The

to

proteins

20 min

for

buffer

room

23 in

0 •~

hydrophobic

to

on

buffer

G25

Fine 0 •~

cm

buffer

further

96

Soda

cm);

with

with

(1 cm

eluate

loaded

(NH4)2SO4

powder at

the

washed

was

Soda

(1.6 The

Butyl-Toyopearl

x 3.5

to

a Sephadex

Polybuffer

(Toyo

cm ƒÓ

on

CHS

column

by

was

with

A

removal

17,000 •~

added

with

sedimented

buffer

Dowex

and of

saturation

for

g of

decantation

(NH4)2SO4

of

the

cells

powdered

following

20 min.

centrifugation

(0.8

filtration 1C).

and

(Toyo

(Pharmacia

1D).

matography

mortar

The

phenolics

150

A for

by

liters

1,100•~g

min,

frozen

nitrogen.

After at

(Fig.

removed

was

cm).

Uppsala)

H

-80•Ž.

and

then

procedures

centrifugation

of

pestle

and

2.8

All

performed

at activity

kg a

liquid

with

temperature.

stored

22

as

CHS

ultra-filtration

(Fig.

G

chromatofocusing

AB,

im

buffer

F of

HW-55F gel

A

buffer

fractions

by

Tokyo)

buffer

with

added

in

nitrogen

Ultra-Turrax

the

frozen

enzyme

CHS-Two

pulverized liquid

day

Thus,

and

and the

7th

in

peak

Toyopearl

Ltd.,

cm ƒÓ •~

a

a month.

of

roughly

the

(1.8

started

observed.

collected

nitrogen

than

sucrose

to

with

replaced

The

concentrated

Co., cm)

concentration

113.

subjected

95

the

KPl

Fig.

were

then Mfg.

to 4 %

On was

conditions,

more

transferred

in

activity

den

synthesis

CHS were

oc

(Pharmacia

containing

transfer. of

liquid

these

Ficoll

the

shown

When

discontinuous

but

cells

in

Under

%

anthocyanin

activity

7-day

6-12

creasing

M

Under growth

and

2,4-D

zeatin,

maximum

5 •~ 10-7

synthesized.

Uppsala)

centrifugation

lacking

and

was by

AB,

gradient

and

11

flasks.

undifferentiated

fractionated

medium

sucrose

anthocyanin

Chemicals

SYNTHASE

Erlenmeyer

only

no

were

Fine

2 % ml

conditions,

curred

CHALCONE

mea g

DM

scintilla

steps,

the

was

pro

omitted;

dissolved

in the

radioactivity

was

same mea

12

Y.

Fig.

1.

(C),

and

Elution

patterns

for

chromatofocusing

CM-Sepharose (D). •œ

CL-6B

relative

sured (25). CHS

mixed

with

plete

Freund's

of

was the

Antiserum

dialyzed same adjuvant.

against

against volume

CHS-Puri

buffer of

Two

I

complete to

ten ƒÊg

and or

CHS

incom CHS

given

as

(B),

(cpm);

injected

?? SPF) then

of

hydroxyapatite of

was

Preparation fied

(A),

activities

and

SPF

follows;

after

intramuscular

injections

with

were

HW-55F

into

mice

conditions. 2 weeks, of given;

et a!.

-•

subcutaneously under

adjuvant

Toyopearl

---

OZEKI

were

subcutaneous

1-5,ƒÊg after

(C57BL/6,

Boosters

of

and

CHS

another

mixed 2

J.

weeks,

Biochem.

PURIFICATION 0.5-0.8 ƒÊg

of

venously; tion

OF CHS

in

a

week

after

was

repeated.

injection, prepared

mice

by

buffer

SYNTHASE

I

or

A

the

room

CHALCONE was

week

the

after

the

bled

and

centrifugation

injec

final the

after

intra

same

booster sera

were

coagulation

at

Western-Blotting,

nodetection-Polyacrylamide (PAGE)

of

disc-gel

the

of

12%

using

of

Hedrick

(25,000),

MW

native

MW,

serum

albumin

Proteins

for

were

using

bovine

the

for

stained

4 (32)

serum

catalase

determination

(a, of

the

the

Brilliant

Western-blotting,

SDS-PAGE to

method

was

method

had

briefly shaking

in

anti-CHS was

min

then

being

K

was

washed paper

was

and

the

was

after

the

buffer

L

reacted

the

detected had

and

once

procedures

98,

way

as

5ƒÊ1

IgG.

for

the washed

with

buffer

5 ml After

described of

above,

anti-rabbit

in

three L

IgG

fixed

reagent

performed

and

in

1 h.

HRPO

been

were

it

on the

times

lacking at

for

K,

IgG

room

No.

1, 1985

of

this

the

was

of

buffer

the kit, with SDS. tem

pH

not

mination

only

of

230

from

Thus,

also

Folin

reagent

hindered

was Blue

Therefore, enzyme

achieved matography

R-250

by

The purity PAGE

little

loss

of the final and

high

of

deter

proteins

the

Poly with

the

with

front

on

Polybuffer

Co SDS from

This

hydrophobic of

(20

the

absorbance

necessary.

Butyl-Toyopearl with

activity

colored

of

was

was Poly

determined.

at

I, the

CHS

determination

removal

Table

with

slightly

solution

the

pattern be

The 10-fold

procedure,

its

elution

KP1

effective;

enzyme

of

not

than

in

interfered

protein

and

Brilliant

PAGE. the

the could

mm KP1.

6-fold.

the

also

with most

also

this

because

column

buffer

omassie

but

KP1 7.25

more

than

was eluted

pH

mm

shown

inhibited

step was

200-250

was

With

protein

nm. this

As

1.

mm

at

to

step

inhibition)

500

300-400

more

Fig. column

CHS

with

6.5-6.0.

HW in

This

6.9,

while

with

I).

increased at

IB).

pH

increased

(Table

elution

Toyopearl shown

than

chromatofocusing

eluted

at

(Fig.

removed

was

Toyo

hydroxyapatite

at

eluted

CHS

step

on

Butyl-Toyo

CM-Sepharose

and

impurities,

were

CHS

CM-Sepharose

The

are

pH;

CHS sulfate

and

concentration

amounts

by native

perature.

Vol.

with

buffer

L

by

CHS

CHS.

filtration

on

effective

higher

30%

nitrocellulose

anti-mouse

with goat

paper

with

1ƒÊl

buffer

most

purity

on

the

remove

ammonium

hydroxyapatite

affected a

by

gel

from

acids. to

chromatography.

elution

mainly

inactivated

by

chromatofocusing

the

were

catalyzed

chromatographies

and

large

otherwise

chromatofocusing

on

purity

gentle

containing The

to

buffer washed

with

with

once

same

in

was

which

extract

chlorogenic necessary

hydroxyapatite,

and

by

was

reaction

hydrophobic

much

and

was

crude

which

acid

suspen

synthesis

chromatographies

CL-6B,

at

phenolics,

(31)

HW-55F,

carrot

The

sequentially

and

and kit

of

the

impurities

modi

nitrocellulose

soaked

2 h.

of

the

Immu the

gelatin

buffer

hybridized

in

the

times

IgG

HRPO-conjugated paper

same for

slight and

3 h). to

3 %

then

three

time rabbit

J

paper

and

serum

of

buffer

of

with

the

washed

each 5ƒÊl

of

with

to

immuno-assay

sites

K

(29) buffer for

nitrocellulose

5 ml

paper

width

Bio-Rad

blocked

buffer

mouse

All

CL-6B

was

according

according

residual

the

with

was

a 7 cm

the

been

overnight,

Hershey

buffer

performed

After

paper

the

V in

of

manual.

of

purified

Stepwise

transferred sheet

and

tank

was

separated

electrophoretically

Howe

60

were

nitrocellulose

(the

nodetection fied

a

of

modification voltage

proteins

and

immediately

20

was

patterns

Blue

extracted.

compounds inhibited

pearl

MW.

was lot

a

anthocyanin

caffeic

and

39,000;

CHS•\From

which

treatment

55F, For

the

a

anthocyanin,

pearl

R-250.

by

contained

bovine

subunit

Coomassie

CHS

fractionation,

of

(21,500),

that

with

albu

and

in

induced,

these A

of

culture

Dowex

(10%,

RNA-polymerase

165,000)

to

slab-SDS-PAGE

trypsin-inhibitor and

fl, 155,000; ƒÀ',

sion

Denatured

(158,000),

used

mo

Andrews

filtration.

(35,000),

and

(27) of

Purification

using The

chymotrypsinogen

aldolase

were

RESULTS

according

Smith

markers,

(68,000),

(240,000)

(26).

that

by

ovalbumin

min

performed

gel

separated

As

was Davis

and and

Immu

electrophoresis

determined

HW-55F

were

(28)).

was

acrylamide

Toyopearl

proteins

by

(MW)

method

to

proteins

described

weight

and

gel

native as

lecular

I

Protein Determination-Protein was deter mined by Lowry's method (30) with bovine serum albumin as a standard, except for column eluates. Protein in eluates from columns was determined from the absorbance at either 280 nm or 230 nm.

temnerature. Electrophoresis,

a

injected

longer,

were

13

was chro

activity.

sample

SDS-PAGE.

was examined As shown

in

14

TABLE

Y . OZEKI

I.

Purification

of chalcone synthase

from cells of a carrot suspension

Fig. the

Fig. 2. Electrophoretograms of the CHS preparation at the final purification step on native PAGE (left) and SDS-PAGE (right). "T" means total concentration of acrylamide in gel. Fig. 2, a single band grams,

indicating

appeared

on both

the high purity

chromato

of the final sam

ple.

Properties of CHS-The MW of the native enzyme was determined by two methods; 85,000 by native PAGE with various concentrations of acrylamide, and 79,400 from the mobility on gel filtration on Toyopearl HW-55F. The MW deter mined by gel filtration appeared to be smaller than that by PAGE because CHS may be slightly ab sorbed to Toyopearl HW-55F, resulting in a delay

3. assay

Time of

genin; •œ,the

culture (2 kg) .

course

of

purified

the

CHS. • ,

formation

of

products

chalcone; •¢,

in narin

sum.

in elution. The MW PAGE indicates that subunits. The

et al.

isoelectric

of 40,600 obtained CHS is composed

point

of CHS

to be about pH 6.2 from chromatofocusing, though

on SDS of two

was determined

the elution pattern the true pI might

on be

slightly higher than the observed value because the delay in elution caused by the absorption

of of

the

enzyme

protein

on

the chromatofocusing

gel.

The pH optimum of the enzyme reaction was 8.0 in both 100 mm KP, and Tris-HCl, but Tris was slightly inhibitory (a 28 % decrease in activity in Tris-HCl buffer at pH 8.0), as reported by Kreuzaler and Hahlbrock (14). The reaction prod

J.

Biochem.

PURIFICATION

OF

CHALCONE

SYNTHASE

15

Fig. 5. Western-blotting against purified CHS (left) and the crude extract (center) using anti-CHS serum prepared from mice, detected with HRPO-conjugated antiserum. The right photograph shows proteins in the crude extract on nitrocellulose paper visualized with Amino Black. Fig.

4.

with

malonyl-CoA

(A)

Double

onyl-CoA

reciprocal as

the

plots

varying

concentrations; • ,

ƒÊ M; •£,

15 ƒÊM; •›•@

slopes

(•›)

and

,

of

fixed

initial

5ƒÊM; •¡, 25 ƒÊM; •œ,

intercepts

50 ƒÊM.

(•œ)

in

velocities

substrate.

Mal

7 ƒÊM; •¢, (B)

10

Replots

of

(A).

ucts were determined by cellulose thin layer chro matography. As shown in Fig. 3, chalcone was the initial product, but, thereafter, it was spon taneously transformed to naringenin at pH 8.0, as reported by other workers (33, 34). The

kinetics

studied

with

tions the

of

by

in

the

same

4A. of

Fig.

procedure,

double The

the

4B. for

concentra

intercepts

lines The

and

18 ƒÊM

in Km

for

(1/

this

figure

values

thus and,

malonyl-CoA.

against

CHS-The

purified CHS was injected into mice according to the procedure described in " MATERIALS AND METHODS " and antiserum was obtained. Cross reactivity of the antiserum was checked by West ern-blotting (Fig. 5). The antiserum reacted with CHS specifically in the crude extract. There was

Vol.

98,

No.

1, 1985

in crossreactivity

between the com

plete and incomplete Freund's adjuvants used to raise the antibody, but slightly higher reactivity was obtained with the former (data not shown). A single band was observed on the nitrocellu lose paper to which the crude extract was absorbed (Fig. 5, center) and its position was the same as that of purified CHS (Fig. 5, left). These results indicated that CHS was a homogeneous protein and the purified CHS was the same as that in the crude extract electrophoretically, i.e. there was no modification during the purification procedure.

reciprocal

4-coumaronyl-CoA

of Antiserum

were

malonyl-CoA, as

Fig.

5.7 ƒÊM

Preparation

of

and

slopes

were

reaction

combinations

in

the

replotted

obtained

enzyme

summarized

shown and

were

various

were

as

Vmax)

the

4-coumaroyl-CoA

results

plots

of

no difference

DISCUSSION

Since CHS was identified as the first step enzyme in the flavonoid pathway (14), CHS has been investigated in many laboratories, but only two groups (1, 15) succeeded in purifying it and prop erties were only reported for the enzyme isolated from parsley suspension culture (1, 14, 16-18). The reason for the limited number of reports on the properties of CHS is probably the extreme instability of CHS (16). Carrot CHS was also unstable and rapid purification was required.

16

Y . OZEKI

Therefore, all of the purification steps were per formed within 50-70 h after the enzyme extraction . Gel filtration with Toyopearl HW-55F was useful for a rapid flow rate. During chromatofocusing , addition of EDTA to the buffers was indispensable for a high recovery of the activity. After this , our CHS preparation showed a single band on PAGE and a high specific activity. As regards the parsley CHS, the final specific activity was not reported (1). Kreuzaler and Hahlbrock (14) and Kreuzaler et al. (1) used DEAF-cellulose for the first step of purification. However, DEAE-cellulose could not be used for purification of carrot CHS, because the enzyme was not absorbed to the resin even at low ionic strength. MWs of both the native protein and subunits of CHS from carrot (74,900-85,000 and 40,600, respectively) were almost the same as those of that from parsley (78,000 and 40,000, respectively (1)). The

optimum

hibitory zyme

effect are

enzyme product

Tris to

its

The

substrate, parsley

4-coumaroyl-CoA

a little as and

and

for

as

the

the

initial

been

Km

values

at

saturation

different being

from 1.6 ƒÊM

malonyl-CoA,

en

parsley reaction transfor

reported of

by

others

carrot of those

and

CHS,

the

other of

35 ƒÊM

is small (0.4-0.5 ml/mouse). This drawback can be overcome by using SPF mouse, in which con tamination by other antigens or infection by bac teria is minimized. Thus, we could obtain an antiserum preparation with high specific reactivity against CHS. Using investigation pression The

the specific antiserum obtained of the regulation mechanisms

of CHS

authors

Tazawa,

by 2,4-D

wish

University

of Tokyo, also

and

M.

Kasahara,

ence,

The

tions

and

greatly

indebted and

Department University

technical

gratitude Faculty

their of

of Tokyo,

to

to Prof.

of Science,

for the valuable

are

Dr.

their

of Botany,

authors

Biochemistry,

is in progress.

to express

Department

here, of ex

discussion. Prof.

group,

Botany,

Y.

The Anraku

Laboratory Faculty

for the

M. The

of

of

helpful

Sci

sugges

advice.

REFERENCES

in

carrot

for

the

the

spontaneous

have

determined

reported

8.0

reported

subsequent

apparent

were

about

observed those

naringenin

were

of

Chalcone

and to

(33-35).

of

of

similar (14).

mation

which

pH

et al.

that for

respectively.

Although some differences existed between carrot and parsley CHS, most of the properties of CHS from our carrot suspension culture were simi lar to those of that from parsley (1, 14, 16, 17). These results suggested that CHS from different sources exhibits homogeneity. Furthermore, since CHS which was induced by the removal of 2,4-D from the medium in a carrot suspension culture actually showed the same properties as that in duced by UV irradiation in a parsley suspension culture, CHS was not modified by the different mechanism of induction. However, further experi ments, e.g., comparison of the sequences of the CHS protein or DNA for CHS are required to confirm the homogeneity. Antiserum against CHS was obtained from mice but not from rabbits. The former are more useful than the latter when the amount of antigen available for the preparation of antiserum is lim ited, although the amount of antiserum obtained

1. Kreuzaler, F., Ragg, H., Heller, W., Tesch, R., Hammer, D., & Hahlbrock, K. (1979) Eur. J. Bio chem. 99, 89-96 2. Gardiner, S.E., Schroder, J., Matern, U., Hammer , D., & Hahibrock, K. (1980) J. Biol. Chem. 255, 10752-10757 3. Ragg, H., Kuhn, D.N., & Hahlbrock, K. (1981) J. Biol. Chem. 256, 10061-10065 4. Kreuzaler, F., Ragg, H., Fautz, E., Kuhn, D.N., & Hahlbrock, K. (1983) Proc. Natl. Acad. Sci. U.S. 80,2591-2593 5. Kuhn, D.N., Chappell, J., Boudet, A., & Hahi brock, K. (1984) Proc. Natl. Acad. Sci. U.S. 81 , 1102-1106 6. Reimold, U., Kroger, M., Kreuzaler, F., & Hahi brock, K. (1983) EMBO J. 2,1801-1805 7. Schroder, J., Kreuzaler, F., Schafer, E., & Hahl brock, K. (1979) J. Biol. Client. 254, 57-65 8. Lawton, M.A., Dixon, R.A., Hahlbrock, K., & Lamb, C.J. (1983) Eur. J. Biochem. 129. 593-601 9. Lawton, M.A., Dixon, R.A., Hahibrock , K., & Lamb, C.J. (1983) Eur. J. Biochem. 130, 131-139 10. Hahlbrock, K., Knobloch, K.-H. , Kreuzaler, F., Potts, J., & Wellmann, E. (1976) Eur. J. Biochem . 61, 199-216 11. Wellmann, (1976)

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12. Dixon, R.A., Dey, P.M., Lawton, M.A., & Lamb , C.J. (1983) Plant Phvsiol. 71. 251-256 13. Hinderer, W., Petersen, M., & Seitz, H.U. (1984) Planta 160, 544-549 14. Kreuzaler, F. & Hahibrock, K. (1975) Ear. J. Bio chem. 56, 205-213

J.

Biochem.

PURIFICATION

OF

CHALCONE

SYNTHASE

15. Dooner, H.K. (1983) Mol. Gen. Genet. 189,136-141 16. Kreuzaler, F. & Hahlbrock, K. (1975) Arch. Bio chem. Biophys. 169, 84-90 17. Hrazdina, G., Kreuzaler, F., Hahlbrock, K., & Grisebach, H. (1976) Arch. Biochem. Biophys. 175, 392-399 18. Schiiz, R., Heller, W., & Hahlbrock, K. (1983) J. Biol. Chem. 258, 6730-6734 19. Ozeki, Y. & Komamine, A. (1981) Physiol. Plant. 53,570-577 20. Ozeki, Y. & Komamine, A. (1982) in Plant Tissue Culture 1982. Proc. 5th Intl. Cong. Plant Tissue & Cell Culture (Fujiwara, A., ed.), pp. 355-356, Maruzen, Tokyo

21. Stockigt, J. & Zenk, M.H. (1975) Z. Naturforsch. 30c,352-358 22. Moustafa, E. & Wong, E. (1967) Phytochemistry 6,625-632 23. Lin, M. & Staba, J. (1961) Lloydia 24, 139-145 24. Fujimura, T. & Komamine, A. (1975) Plant Sci. Lett. 5, 359-364

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25. Schroder, J., Heller, W., & Hahlbrock, K. (1979) Plant Sci. Lett. 14, 281-286 26. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404 427 27. Hedrick, J.L. & Smith, A.J. (1968) Arch. Biochem. Biophys. 126,155-164 28. Laemmli, U.K. (1970) Nature 227, 680-685

29. Howe, J.G. & Hershey, J.W.B. (1981) J. Biol. Chem. 256, 12836-12839 30. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) J. Biol. Cheer. 193, 265-275 31. Gross, G.G., Mansell, R.L., & Zenk, M.H. (1975) Biochem. Physiol. Pflanzen 168, 41-51 32. Andrews, P. (1964) Biochem. J. 91, 222-233 33. Heller, W. & Hahlbrock, K. (1980) Arch. Biochem. Biophys. 200, 617-619 34. Spribille, R. & Forkmann, G. (1982) Planta 155, 176-182 35. Sutfeld, R. & Wiermann, R. (1980) Arch. Biochem. Biophys. 201, 64-72