Purification of thymidylate synthetase from L. casei by affinity chromatography

BIOCHEMICAL

Vol. 49, No. 4, 1972

PURIFICATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF THYMIDYLATE

SYNTHETASE

L. CASE1

FROM

BY AFFINITY

CHROMATOGRAPHY’

Peter

V. Danenberg,

Robert

and Charles HcArdle

HeIdelberger3

Laboratory

Unlversfty

for

September

Cancer

of WIsconsIn,

Wfsconsln,

Received

J. Langenbach‘,

Research Madison,

53706

28,1972

An afftnlty colurm has been constructed by coupling 2’-deoxyc acid through the St-phosphate group to Sepharose via a 6-e-amfnoThls material adsorbs thymfdylate synthetase benzamldohexyl chain. quantftattvely from a crude extract of Lactobacillus casel, allowing purtftcatfon to homogeneity In a sfngle step.

%v ur y

Thymidylate Inhlbftory

drug

synthetase, 5-fluorouracfl

In DNA biosynthesis this

laboratory

(4),

(5,7-11).

Especially

synthetase

Is quite

stud&

with

and perhaps

the object

out by sequences

labile

Ehrllch

and In very

enzyme,

using

Excellent developed

enzyme

research

the purlflcatlon

carclnorm

recently

tumor-

rate-IImItIng

laborious

to develop

(12).

the

now,

low content

we sought

chromatography

mstrtx

of

ascltes

the potent

of considerable

Until

of this

conventional cells, (6).

procedures

thymldylate In order

a purlflcatlon purlflcations afflnlty

In

to facllttate procedure of other

systems

(13).

This work was supported In parts by grants CA 07175 and CRTY-5002 the NatIona Cancer Institute. NatIonal Institutes of Health. Present address: Eppley Institute Nebraska, Omaha, Nebraska.

3

enzyme for

and others.

have been obtalned

based on an agarose 1

has been

from

this

based on affinity enzymes

(l-3).

(2,3,5,6)

enzyme has been carried

our

the target

Anmrican

Cancer

Socfety

Copyn’ght 0 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.

Professor

for

Cancer

of Oncology.

1029

Research,

UnIversIty

from of

Vol. 49, No. 4, 1972

BIOCHEMICAL

The success attaching

of affFn1ty

to mFnFmFze steric

interference

prfnclple,

coupled

to Sepharose

through

chromatography, thymldylate Lactobaclllus the 30-702 Materials

chromatography

or competltlve

a substrate

we have

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

this

with

Fnhlbltor

the substrate

material

The enzyme from

sulfate

enough

(13).

chain.

from

Using

the matrix

this

acid

(dUrd-5’-P)

When used

In colurm

selectively

to adsorb

an amethopterln-reststant

to apparent

precfpltate

In many cases on

2’-deoxyurfdyllc

has the ablllty

case1 was purlfled

far

the enzyme

a e-amlnobentamFdohexy1

synthetase.

anmonlum

depends

homogeneity

of the crude

In one step

from

Fysate.

and Methods

6-Amfnocaproic acid, &-nitrobenzoyl chlortde, and cyanogen bromide were obtalned from AIdrIch Chemical Company, Milwaukee, Wls. Sepharose 46-200 was from Sigma Chemical Co., St. Louis, MO. An amsthopterln-reslstant strain of LactobacilFus case1 was kindly provided by Dr. F. H. Huennekens and was grown accordtng slap IR spectra were determlned on --et al. (12). a Beckman IRIO, UV spectra on a Beckman DB-6, and NMR spectra on a PerklnElmr Mode1 R12. The elemental analysFs was done by Spang MIcroanalytica Laboratories, Ann Arbor, Hfch. Paper chromatography was carrFed out using Whatman No. 40 paper, In n-butanol-acettc acid-water (5:2:3). AnalytIcal TLC was performed on Eastman chromatogram sheets using the system chloroformmethanol (4:l). All other reagants were the best grade avallable. 6-e-NItrobenzamFdohexan-l-01

(I).

6-Amlnocaprolc

acid

(6.6

g;

50 doles)

was sllylated and reduced with Fithlum aluminum hydride accordfng to Vsnkateswaran and Bardos (14) to give 6-amlnohexan-F-01 in 80% yteld. Wlthout further purFfIcatFon, the amino alcohol was taken up In 50 ml of tetrahydrofuran and treated with 7.4 g (40 Moles) of e-nltrobenroyl chlortde and 4.0 g (40 Moles) of trlethylamlne. After one hour at room temperature, the solvent was evaporated and the residue taken up In ethyl acetate. The solution was washed successtvely with 1 N hydrochloric actd, 1 N sodfum hydroxide, and water. The ethyl acetate layer was dried over magnesium sulfate, and the residue obtaFnad after evaporation of the ethyl acetate was crystal I Fzed from chloroform-benzene to give 8.6 g (90%) of 6-e-nF trobenzamldohexan-F-01, m.p. 81-83.; IR (KBr), 1640 cm-1 (mlde C - 0 stretch); NHR (DCCF ),2 1.90 (quartet, 4H, aromatic). m. (CF3HF8N204) Calcd. : e , 58.70; H, 6.77; N, 10.50. Found : C, 59.05; H, 6.61; N, 10.69. 2’-DeoxyurFdFne-5’-(6-~-nltrobenzamFdo)hexy1phosphate

(II).

The

dlsodlum salt of 2’-deoxyurldtne-5’-phosphate (dUrd-5’-P) (0.5 g; 1.43 mMoles) was passed through Dowex 50 (ti) resin In water to convert It to the acid form. The water was removed by evaporation and 3 x 5 ml portlons of dry pyrldine were re-evaporated from the residue. It was then taken up In 50 ml of dry pyrldlne, and treated with 2.0 g (7.5 Moles) of I, along with IO g of dFcycIohexyFcarbodIimFde. After 48 hr at amblent temperature, a paper chromatographic check showed no dUrd-5’-P reemlnlng, but Instead a new product at Rf 0.8 (dUrd-5’-P had an Rf of 0.3). The preclpltated dlcyclohexylurea was removed by flltratlon and the pyrldine

1030

Vol.

49, No.

BIOCHEMICAL

4, 1972

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

evaporated The residue was washed with chloroform to remove --in vacua. excess I, leaving a gummy residue, which was converted to a precipitate by dissolution in methanol and addition of ether. This material had an Rf of 0.0 by TLC (compared to 0.6 for I), and showed one spot at Rf 0.8 by paper chromatography . The new spot was positive to a molybdate spray test for phosphorus. NMR [(CO OD),% 2.20 (quartet, 4H, aromatic), 2.75 (doublet, lH, H-6), 4.80 (doublet, 3 H, H-5), 7.15 (broad singlet, 4H, CH2), 9.1 (broad singlet, 8H, CH2)) showed that urldinyl, e-nttrobenzoyl, and amtnohexyl fragments were present In equal ratios. Phosphorus analysis showed a base-phosphorus ratio of 1: 1. Coup1 Ing to Sepharose. II (1.2 mMoles) was dissolved in methanol and hydrogenated at 40 psi In the presence of 10% palladium on charcoal. When hydrogen absorptfon ceased (1 hr), the catalyst was removed, and the product dlssolved In 50% aqeous dimthylformamide. Activation of Sepharose (15 ml wet volume) and coupltng with the llgand was done at pH 9.0 according to Cuatrecasas et al. (13). This resulted In the attachment -of 200 PMoles of urfdfne per g of Sepharose (dry weight).

Results

The

adsorption

Sepharose

column

30-70%

effectiveness was

anmonium At

activity

through

came

column

was

washed.

to

M,

quantitative

0.1

a single

sharp

thymidylate

demonstrated

sulfate

(1 X 10 cm).

a buffer the

wf th

Sodium

with

when

sulfate

(Fig.

this

by treating

acid,

has recently

which

In the presence that

single

the

peak

the

enzyme

(Fig.

but

M,

the

of Laermnli

been shown

1031

no

with

which

was

increased

achieved

no

adsorption

made

by

treating

coupling

enzyme the

in of

step.

of the peak of

showed

thymidyfate

only

one band

synthetase

was

[‘4~~-5-fluoro-2’-deoxyurIdyffc

to bind

on the gel

band.

(15)

pH 7.1,

was

lfgand

gel electrophoresfs

crude

on a cofurm

was

colunn the

the

volume

There

deleting

the enzyme with

radtoactlvfty

0.05

actfvlty

I).

a control

of methylenetetrahydrofofatc of

to

concentration

buffer

band was indeed

established

up

case1

C.

the

to

disc

lysed

of

tailfng

bromide,

by the method

That

the

of

regardless

of

activity

cyanogen

dodecyl

no

a quantity

of

column,

enzyme activity 2).

of

recovery

6-e-aminobenzamidohexyl-dUrd-5’-P

placing

concentratton

However,

peak

the

by

precipitate

synthetase

Sepharose

of

covalently (16,17). corresponded

to the

enzym

It was found to the observed

Vol. 49, No. 4, 1972

BIOCHEMICAL

.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-0.02MpO.lMPHOSPHATE

_ PHOSPHATE

1.50-

1.25 -

0 i.ooI d d

0.75-

II I

0.50 t 0.25

..1

I !

5

IO

15

20

FRACTION NUMBER Elution profile for thymidyiate synthetasc from L. case1 on a ~!~~a~inobenramidohexyi-d&d-5’-P-Sepharose coiurm (1 x 16cm).The 30-708 armnonium sulfate fraction (1.0 ml, corresponding to - 10 mg of protein) was placed on the coiunn, and eiuted with 0.02 M phosphate buffer, pH 7.1, containing 10 mH R-mercaptoethanoi, until the A~80 fell to zero. Then the coiunn was eiuted with 0.1 M phosphate buffer, pH 7.1, containing 10 ti fl-mercaptoethanoi, until ail enryma activity was renovad. A2g0: Enzyme activity: ----.

Fig. 2. obtained

Sodium dodecyi sulfate disc gel from tube no. 25 of Fig. 1.

Surprisingly, dUrd-5’-P

at concentrations

the cofactor bind

with

the enzyme was not

eiectrophoresis

removed

Possibly,

up to iOW4h.

mathyienetetrahydrofolate, to the dUrd-5'-P

coiunm

To determine

maximum binding

capacity,

enzyme preparation

until

1032

than

the column

by

in the absence

thymidyiate

more strongly

the crude

from

of sample

synthetase to dUrd-5’-P

the colunm

enzyme activity

may Itself.

was treated began

to

of

BIOCHEMICAL

Vol. 49, No. 4, 1972

seep through.

Subsequent

2.0 pMoies/min

of enzyme activity

activity

obtained

to the published enryn

(11).

synthetase

(2.4 value

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

elutfon

bound.

protein)

conpares

@4oies/min/mg

Work 1s in progress from

had remained

rHoies/min/mg (2.5

Ehrlich

0.1 M phosphate

protein)

on the purlflcation

ascites

cells

using

this

showed

that

The specific favorably

for

the purified of thymfdyiate

coiunm.

References 1. 2.

z: 5.

6. 5: 1’0: 11. 12. 13. 14. ii: 17.

Cohen, S.S., Flaks, J.G., Bamer, H.D., Loeb, M.R., and Lichtenstefn, J. Proc. Nati. Acad. SC?., U.S., 44, 1004 (1958). Heidelberger, C., Kaidor, G., Hukherjee, K.L., and Danneberg, P.B., Cancer Res., 3 903 (1960). Reyes, P., and Hefdelberger, C., Mol. Pharmscoi., 1, 14 (1965). in The Biochemistry of Folk Acid and Ralated Pteridfnes, Blakeicy, R.L., John Wliey and Sons, Inc., New York (1969), pp. 219-266. F,.‘A.-d I .“,a?.“,

A n.,

I---L--L a.mI~“wuc”,

R J

and Heideiberger, C., J. Biol. Chem., 246, 7110 (1971). Langenbach, R.J., Danenberg, P.V., Fridland, A., Cleiand, W.W., and Heidcibcrger, C., Fed. Proc., z, 419Abs (1972). Mathews, C.K., and Cohen, S.S., J. Biol. Chem.) 238, 367 (1963). Frfedkin, Il., Crawford, E.J., Donovan, E., and Pastore, E. J., J. Blot. Chem., 2f7, 3811 (1962). Blakiey, R.L., J. Blol. Chem., 2 8, 2113 (1963). Crusberg, T.C., Leary, R., and K sliuk, R.L., J. Biol. Chem., 245, 5292 (1970). Dunlap, R.B., Harding, N.G.L., and Huennekens, F-M., Btochcmistry, 10, 88 (1971). Cuatrecaras, P., Wflcheck, H., and Anfinsen, C.B., Proc. Natl. Acad. sci., U.S., 61, 636 (1968). Cuatrecasas, P., J. Bioi. Chem., z, 3059 (1970). Venkateswaran, P.S., and Bardos, T.J., J. Org. Chem., 32, I256 (1967). Laennnii, U.K., Nature, 227, 680 (1970). Langdnbach, R.J., Danenberg, P.V., and Heidelberger, C., Bfochem. Biophys. Res. Cornnun. in press. Santl, D.V., and McHenry, C., Proc. Natl. Acad. Sci., U.S., 69, 1855 (1972). l

0,

-I-

1033