Phosphocellulose, an affinity chromatographic system for chorismate synthase and the aromatic complex of Neurospora crassa

Vol.

67,

No.

1, 1975

BIOCHEMICAL

AND

PHOSPHOCELLULOSE, SYSTEM

FOR

AN

Division, Oak Ridge Graduate

Received

August

SUMMARY:

Cole

COMMUNICATIONS

THE

AROMATIC

CRASSA*

and

F.

H.

Gaertner

Ridge National Laboratory, and School of Biomedical Sciences,

The University Oak Ridge,

of Tennessee-Oak Tennessee 37830

22,1975

Unlike

and the aromatic phosphate and fact that other

AND

NEUROSPORA

W.

RESEARCH

CHROMATOGRAPHIC

SYNTHASE

OF

K. Biology

AFFINITY

CHORiSMATE

COMPLEX

BIOPHYSICAL

other

enzymes

of the

aromatic

multienzyme

system,

synthase

chorismate

complex of Neurospora crassa were found to bind to a column of cellulose to elute at a relatively high concentration of phosphate (-0.2 M). The enzymes with similar ionic properties failed to bind to phosphocellulose

suggests that the binding of the former two enzyme systems is due to a specific affinity for This conclusion is not only supported by the fact that these same enzymes did phosphate. bind to a column of carboxymethyl cellulose, but also is consistent with the nature of the catalytic reactions of the enzymes. Both the shikimate kinase enzyme of the aromatic complex and chorismate synthase would be expected to have active sites which accomodate a phosphate moiety. We anticipate that other enzymes which involve phospho-substrates will also be amendable to this procedure. INTRODUCTION In previous observed

that

studies

11

of

phosphoenolpyruvate -

gradient

the

synthase Recently,

protease

efficient

* Research with the

o very

this and

bind

eluate

protease natural

some

nearly

of the

is inactive inhibitor

sponsored by the Energy Union Carbide Corporation.

C’op.vright o 1975 bv Acudemic Press, ITIC. All rights of reproduction itI art.v fkn reserved.

conversion

of

was same during

with

complex

in coincidence

we

have and and

a linear and

the

elute

phosphate 3-enzyme

(3).

demonstrated

early

crassa,

(DEAE)-cellulose 12 ,M)

aromatic

molecular

N.

of erythrose-4-phosphate

(0.08-o.

5-enzyme

protease

system

to diethylaminoethyl range

the

active has

specific

the

concentration

complex

coincidently,

Fortunately,

L-tryptophan

same

multienzyme

catalyzing

In particular,

( 1, 2 ).

anthranilate

aromatic

13 enzymes to

in approximately

on the

in N. properties

stages

crassa

(4).

as the

aromatic

of purification

due

The enzymes.

to a highly

(4).

Research

and

170

Development

Administration

under

contract

not

Vol.

67,

No.

In our found

that

slightly

own

the

could

on this does

again

some lead

studies

protease

(which,

However,

BIOCHEMICAL

1, 1975

protease

not

bind

protects

activity

to artifactual

and

BIOPHYSICAL

its

may

results

in studies

of the

above

RESEARCH

inhibitor

the

COMMUNICATIONS

(unpublished

to DEAE-cellulose

fortuitously,

proteolytic

AND

and

aromatic

that

the

at

later

concerning

the

from

stages

structure

we

inhibitor

enzymes

be unmasked

data),

have

binds

only

proteolytic

attack).

in purification and

which

functions

of the

aromatic

enzymes. As a consequence eliminate of the

the wide

protease

from

difference

in the Since

chromatography. column),

we

them

Here

report

synthase

indicate

that

enzymes

as well AND

grown,

G-25

with

for

extraction

similar

state

that

the

aromatic

protease

bind

tightly

advantage

in

column bind

free

of the

to

of the

aromatic

ion-exchange

enzyme

such

as

DEAE-cellulose, protease. components,

to phosphocellulose.

as a affinity

to

(an anion-exchange

enzymes

two

attempted

by taking

DEAE-cellulose

phosphocellulose,

serve

types

purification and

have

to a cation-exchange

the

complex, may

to

tightly

the

result

aromatic

Organism

with

and

Our

chromatographic

system

results

for

these

of enzymes.

Preparation

sulfate,

&potassium and

and

All

0.25

M

several HCl,

and

with

(pH

procedures Cellulose

allowed

with

171

4

x

10

out

phosphate

for

x

-4

crassa

sulfate,

(strain

of

previously

(1,

Mdithiothreitol at

O-5”

(Whatman

5).

and

10e4

C. Column

Chromedia

water,

filtered

on a Buchner

30 min

at

C.

25”

74A)

fractionated

90 cm column

as described

carried

deionized to stand

on a

2

were

N.

protamine

7),

conrained

times

Wild-type

desalted

phosphate

Chromatography: washed

treated

chromatography acid,

was

of Extract:

extracted,

ammonium

0.05

Phosphocellulose

in

through

we

enzymes

bind

since

surprising

ethylenediaminetetraacetic

resuspended

wash

lyophilized,

Sephadex

mea/g)

not

bind

hand,

other

of the

of saturation

Pl 7.4

does

exchanger

harvested,

buffers

protease

aromatic

conclusions,

METHODS

at 60%

All

of the

it would

and

at an early

affinity

other

the

cation as for

Growth was

the and

this

MATERIALS

the

to simply

we

preparations

that On

expected

chorismate

the

anticipated

phosphocellulose. we

our

observations

The

cellulose

funnel,

M,

Vol.

was

67,

No.

then

1, 1975

washed

by repeatedly

6 times washing

The

2

x

gradient with

on a

80 cm column

0.15

potassium

0.01-o.

gradient 0.15-0.5 mycel

5 times

of 0.01-o.

15 M

eluted (pH

phosphate

at

T500-ml

mycelium

was

or was

applied

(2)

After

linear

equilibrated

6.5).

37 ml/hr

6.5).

and

with

directly

a 1500-ml

a brief

gradient

either

extension

of 0.15

fi

linear of the

to 0.5

gradient

M

Selectacel was

washed

water, x

6.5).

of CM

(pH The

with

0.05

equilibrated

80 cm column

phosphate (pH

and

standard

carboxymethyl

2

NaCI-0.25

to pH cellulose

6.5),

followed

desalted

extract

cellulose

6.5 was

M NaOH,

with

0.01

M -

eluted

with

a linear

by a second from

gradient

of

40 g of lyophilized

used.

Assays: ( 5 ),

as described

previously.

used

potassium

NaOH,

75 g of lyophilized

and

Inc.)

deionized 2

(pH

&

COMMUNICATIONS

used.

Schuell,

A

0.25

of DEAE-cellulose

phosphate

was

with

Chromatography:

with

buffer.

kynureninase

were

and

phosphate

Enzyme

6.5)

RESEARCH

phosphate from

a second

Cellulose

S c hl either

was

80

potassium

(pH

M potassium ium

cm column

phosphate

washed

potassium

x

phosphate,

Carboxymethyl

filtered,

obove

of phosphocellulose

153

treated

M, potassium

described 2

BIOPHYSICAL

water,

0.01

F potassium

mea/g

AND

deionized

extract first

of

(0.78

with it with

desalted

chromotogrophed to a

BIOCHEMICAL

to identify

Anthranilate kynurenine The the

two

synthase formamidase

anthranilate

complex ( 7 ), synthase

( 1 ), and

and

chorismate

aromatic dehydroshikimate

complex

synthase

(6

( 3 ) were reductase

), assayed

assays

complexes.

* We have found it convenient to use a simple. aradient extender between two successive linear gradients. It allows the column to operate without close supervision and eliminates the possibility that the column will run dry. In its simplest form, the extender is a l-liter Erlenmeyer flask with a two-hole rubber stopper which secures two glass tubes (2 mm I.D.) approximately 30 cm in length. One tube contains a shutoff valve 10 cm from the stopper and is slightly longer (8-10 m n) than the second tube. When the system is used to extend a 0.01-o. 15 &potassium phosphate gradient, the flask is filled with 0.15 -M phosphate buffer, stoppered, and inverted into the 0.15 & container of the gradient-maker. The longest of the two tubes is held 3-5 mm off the bottom of the breaker. The distance between the ends of the two tubes determines the volume at which the extension system becomes operational. For example, a difference of 8-10 mm allows the volume in the 0.15 M container of the gradient-maker to lower to - 50 ml before additional buffer enters fromThe inverted Erlenmeyer flask.

172

Vol.

67,

No.

In

DEAE-cellulose

BIOCHEMICAL

1, 1975

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RESULTS

synthase,

and

gradient the

the

at

phosphate bind

tightly

Most

0.2 this (i)

elute

complex

As can

( 2 ). and

and

aromatic

1

be seen

aromatic

exactly

in

are

comp\ex,

same

complex

point

Fig.

widely

in coincidence

1,

chorismate in the

phosphate

on phosphocellulose

separated and

linear

using

the

chorismate

same

synthase

at a concentration

of

AJ phosphate.

tight in addition

binding

does

not

to anthranilate

involve

nonspeciFic

ionic

other

enzymes

synthase,

FRACTION

FIG.

synthase the

complex the

elute

anthranilate

at approximately

unexpectedly,

to phosphocellulose

That

the

12 M phosphate

gradient.

that

complex

synthase

approximately

facts

aromatic

0.10-o.

anthranilate

chromatography,

attractions with

may similar

be seen ionic

by the

properties

NUMBER

Phosphocellulose chromatogram of an extract of N. crassa 74A prepared as described in “Methods”. In this case the ammonium sulfate fraction was chromatographed first on DEAE-cellulose (see ref. 2, Fig. 3a), then fractions 90 through 140 were pooled, concentrated by ammonium sulfate precipitation at 60% of saturation, and desalted by gel filtration on a 2 x 80 cm In other experiments, column of G-25 prior to chromatography on DEAE-cellulose. with comparable results, the desalted ammonium sulfate fraction was placed directly on phosphocellulose without prior chromatography on DEAE-cellulose. The peak and the anthranilate synthase positions of kynureninase, kynurenine formamidase, Chorismate synthase activity, 0 0; complex are indicated by arrows. aromatic complex, ,,T-.. -: “‘\> . Absorbance at 280 nm is indicated by broken line; phosphate gradient is given by solid line.

173

Vol.

67,

simply the

No.

wash

1, 1975

through

majority

gradient

cellulose

is used, do

not

synthase

the

column,

of protein

phosphate

They

BIOCHEMICAL

bind

and

the

not

stick

0.10-o.

complex

latter

enzymes

the

and 12 l

and

void

kynurenine

chorismate resin,

volume

but

(results

not

elute

and

(ii)

DEAE-cellulose when

synthase

COMMUNICATIONS

formamidase

of the

Moreover,

cation-exchange in the

RESEARCH

region

to phosphocellulose.

aromatic

to this

other

from

BIOPHYSICAL

kynureninase

e.g.l

pooled

did

AND

carboxymethyl

behave

in the

expected

manner.

together

with

anthranifate

shown).

DISCUSSION The whereas

fact

other

that proteins

is acting

as an affinity

complex.

These

both

have

kinase schemes

chorismate

active

reaction

with

similar

in

ionic

which

Fig.

do for

aromatic

The

to phosphocellulose,

suggests

that

this

synthase

and

two

(AROMATIC

enzymes

and

cation the

by virtud.of

phosphate.

these

KINASE

bind

specifically

complex)

reason

SHIKIMATE

not,

complex

chorismate

phosphate

accommodate

by the

aromatic

properties

bind

likely

2.

the

system

potentially

(catalyzed

depicted

and

chromatographic

enzymes sites

synthase

See, the

for

chorismate

elute

from

exchanger

aromatic the

example, synthase phosphoceilulose

fact the

that shikimate

reaction at

COMPLEX1

COOH

CCOH TPNH + n+ FMN

hip

.-, CHORISMATE

FIG.

2

Reactions catalyzed lines emphasize the

by the likelihood

SYNTHASE

aromatic complex of a phosphate

174

and chorismate binding site

synthase. Dashed on each enzyme.

Vol.

67,

No.

exactly

the

possibility in the

BIOCHEMICAL

1, 1975

same

position

of a specific aromatic We

consider

it likely

a specific

that

the

synthase, other

of this affinity

BIOPHYSICAL

gradient

between

chorismate

In support have

phosphate

interaction

pathway,

phosphocellulose. phosphatases

in the

AND

for

the

be only

aromatic should

enzymes notion,

may

it has cellulose

complex not

of this

RESEARCH

be

type

recently

COMMUNICATIONS

coincidental, and

but

the

the

consecutive

enzyme

overlooked. may come

phosphate

bind

in a similar

to our matrix

attention

fashion that

to some

(8).

REFERENCES 1. 2. 3. 4. 5. 6.

Gaertner, Gaertner, Gaertner, Yu, P. Gaertner, Welch,

7. 8.

12, Knox, Dean, 2,

and DeMoss, F. H., and Cole, K. F. H., F. H. (1972) Arch. H., Kula, M. R., and Cole, K. W., F. H., G. R., Cole, K. W., 505418. (1955) Methods K. E. P. D. G., Willetts, S. 344-350.

J. A. (1970) Methods Enzymol. 1_7, 386-401. W. (1973) J. Biol. Chem. 248, 4602-4609. Biochem. Biophys. 151, 277784. Tsai, H. (1973) Eur. TBiochem. 32, 129-135. and Welch, G. R. (1971) J. Bactziol. 108, 902-909 (1974) Arch. Biochem. Biophys. and Gaertner, F. H. Enzymol. R., and

2_, Blanch,

175

246-249. J. E.

(1971)

Anal.

Biochem.