Isolation and partial characterization of the ADP-ribosylated nuclear proteins from Ehrlich ascites tumor cells

Vol. 91, No. 4, 1979 December

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

28,1979

ISOLATION

Pages

AND PARTIAL Peter

Institut Received

Adamietz,

fiir

October

CHARACTERIZATION OF THE ADP-RIBOSYLATED FROM EHRLICH ASCITES TUMOR CELLS Karin

Physiologische

23,

Klapproth Chemie,

and Helmuth

Universitat

1232-1238

NUCLEAR PROTEINS

Hilz

Hamburg,

Germany

1979

SUMMARY The (ADP-ribose)n protein conjugates formed by incubation of Ehrlich ascites tumor cell nuclei with 1 mM (3H)NAD were isolated by chromatography on boronate cellulose columns with a yield of >85%. Possible contamination by glycoproteins was excluded by rechromatography after specific release of the (ADPribose), residues from their acceptors. Dodecyl sulfate gel electrophoresis revealed numerous protein bands which coincided with the (3H)ADP-ribose bands obtained by fluorography of the gels. 40% of the acceptor proteins were identified as the nucleosomal core histones. Most of these histones, however, appeared in the non-histone fraction because of extensive modification by poly(ADP-ribose). Drastic changes in properties were also seen in the true non-histone proteins which comprised 60% of the total conjugated protein. Besides several prominent acceptor proteins (Mr = 12,000; 31,000; 125,000) numerous proteins were detected indicating a considerable heterogeneity of non-histone acceptors. INTRODUCTION Covalent

modification

moiety

from

(ADPR), ion

has been (for

jugates

gates

implied

in the

see 1, 2). extract)

reports

from

EAT cells

for

leading

So far, of rat

liver

isolation

histones

partial

of all

major

of the

The re-

of gene express-

(ADPR), except

protein

con-

of a selected

(3).

of the nuclear

(ADPR)n

characterization. changes

and polymeric cells.

forms

has been described nuclei

of the ADPR

monomeric

eukaryotic

no isolation

and non-histone

to drastic

with all

transfer

proteins

protein

The procedure serve

conjuallow-

as acceptors

in the properties

of the modi-

Preparation of nuclei: Nuclei of Ehrlich ascites tumor cells transplantation into Balb/c mice) were prepared as described modified by inclusion of 1 mM phenylmethyl sulfonyl fluoride buffers following lysis.

(6 days after previously (4), (PMSF) in all

fied

groups

conjugates

regulation

and their that

by enzymic

in nearly

proteins

the first

ed to demonstrate, (ADPR),

forming

of non-modified (HCl

paper

proteins

has been detected

reviews free

subfraction This

NAD to proteins

residues

action

of nuclear

proteins.

MATERIALS

AND METHODS

ABBREVIATIONS: EAT cells = Ehrlich ascites tumor cells; ADPR = adenosine diphosphate ribose; (ADPR), = oligomer and polymer of ADPR with n units 0006-291X/79/241232-07$01.00/0 Copyrighr Ail rights

@ 1979

by Academic Press. Inc. in any form reserved.

of reproducrion

1232

Vol. 91, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Labeling of (3H)(ADPR)n residues: EAT nuclei (~26 mg DNA) were incubated at 25" with 1 mM ('H)NAD (29.6xlOo dpm/umol), 50 mM Tris - HCl pH = 8.2, 45 mM KCl, 5 mM MgCIP, 1 mM PMSF and 5 mM dithiothreitol (DTT) in a final volume of 6 ml. After 15 min the reaction was stopped by the addition of an equal volume of 10% perchloric acid and washed twice with 10 ml of 5% perchloric acid. Boronate cellulose chromatography: The perchloric acid precipitate (100 mg protein) was solubilized in 20 ml 50 mM sodium phosphate pH 6.5 - 6 M guanidine hydrochloride - 5 mM DTT, and centrifuged for 18 h at 210000g and 4°C. The DNA-free supernatant was adjusted to pH 8.2 with morpholine ( 50 mM final concentration) and applied to a boronate cellulose (E. Merck, Darmstadt) column ( 1.3 x 25 cm). The resin was washed (40 ml/h) protein free (< 1 ug/ml) with 50 mM morpholine pH 8.2, 6 M guanidine hydrochloride, 1 mM DTT. The retained material was eluted with 0.2 M sodium phosphate pH 5.5, 6 M guanidine hydrochloride, 1 mM DTT (cf. 5). Gel electrouhoresis: a) The dodecyl sulfate system of Laemmli ( 6) was modified by inclusion of 6 M urea in sample and gel buffers. 100 ul samples containing 5-80 ug protein were put onto 14x9x0.15 cm flat gels consisting of 7.5- 15% polyacrylamide (exponential gradients). 18 V/cm were applied for 3 h. b) The 0.9 M acetic acid / 2.5 M urea system of Panyim and Chalkley ( 7) was used on 15% polyacrylamide flat gels (cf. a) without and c) with the inclusion of 0.4% Triton X 100 ( 8). Separation was terminated after 6 h at 15 V/cm. Separation of histones and non-histone proteins was performed according to Levy et al. (9) using BioRex 70 chromatography. Release of (3H)(ADPR)n from protein: a) (ADPR)n protein conjugates (1 mg/ml protein) were incubated for 40 min at 37°C with 0.2 M NaOH. Protein was preb) (ADPR)n attachcipitated with 20% trichloroacetic acid before analysis. ed to 1 mg protein was digested with 10 pg purified phosphodiesterase I during 4 h at 37°C in the presence of 50 mM Tris . HCl pH 8.4 - 4 mM urea in a total volume of 1 ml. Purification of phosohodiesterase I (E. Merck, Darmstadt) was achieved by affinity chromatography on Reactive Blue Sepharose (Sigma, Miinchen) similar to (10) (unpublished experiments). Fluoroqraphy: Gels were processed as described in (11) and exposed to Kodak royal X-omat film for 10 days at -80°C. Protein determination was performed act. to Lowry (12). RESULTS AND DISCUSSION 1.

Purification

Nuclear cell

of (ADPR)n protein -

acceptor nuclei

chloric

proteins

with

acid

were

ADP-ribosylated

1 mM (3H)NAD

which

conjugates

(4 ).

at the same time

ated

conjugates

(13).

than

95% of the

protein

dine

hydrochloride

and freed

The reaction extracted

The perchloric bound

acid

(ADPR), from

by a short

was stopped

histone insoluble

residues

incubation with

HI and its material

of EAT 5% perADP-ribosyl-

containing

was solubilized

more

in 6 M guani-

DNA by ultracentrifugation.

The supernatant

was subjected to boronate cellulose chromatography (cf.5,3), Most of the protein eluted from the column without retention. Only a small fraction was retained together

with

nearly

washing,

the

labeled

linkage purification

to boronic

all

of the

(ADPR), acid

procedure

3H-labeled

protein

by lowering are

summarized

(ADPR),

conjugates the

pH. in table

1233

were

residues. released

The quantitative 1.

After from

extensive the covalent

aspects

It shows that

of the

purification

Vol. 91, No. 4, 1979

Table

1:

BIOCHEMICAL

Purification

AND BIOPHYSICAL

of (ADPR), protein

conjugates

formed in EAT cell

Protein

(3H)ADPR Residues [dpmx 10m61 [%I

Fraction

RESEARCH COMMUNICATIONS

(~H)ADPR

mg protein

Imgl

[Xl

113

100

.30

95

.30

HC104 Insoluble

33.8

100

DNA Supernatant

32.1

95

107.2

28.8

85.2

4.3

3.8

2.0

5.8

96.8

85.6

Boronate

nuclei.

Column

- binding

fraction

- non-binding

fr.

ADPR residues and processed

were labeled as described

by the boronate

column

by incubation in Methods.

was about

25 fold

6.7 .02

of nuclei

(26 ug DNA) with

as judged

by the

(3H)NAD

increase

in specific

labeling. A small

subfraction

column

and appeared

peak.

This

it

with

for

possible

released

from

zymic

digestion

with

the

on boronate

table

2.

6-8%

still

was incomplete

ciated

with

ment),

contamination

comprised

about

protein-bound

conjugates

of n = 20.

diol

by mild

acceptor

in the

to the

which nuclear

run

boronate

treatments

alkali

(4% of the be less

proteins,

e.g.

were

specifi-

treatment

(5),

or by en-

and then

rechroma-

which

had lost

fraction Since (ADPR),

as shown removal

in

of ADPR

remaining

phosphodiesterase than

that

formation.

compounds

proteins off

resin.

, and 5% after must

ester

containing

alkaline

run through

suggesting

boronate

the ADPR residues

The (ADPR),

both

the

to the boronate

in the

venom phosphodiesterase,

(ADPR), - protein

4% of the total (ADPR),

either

by glycoproteins

of isolated protein

after

cis

Therefore

bound

bind

non-ADP-ribosylated

now appeared

with

the proteins

The (ADPR)n

a value

resin

protein

with

with

snake

not

when rechromatographed

retains

cellulose.

to the

Analysis

bind

conjugates

did

unmodified

interfering

generally

purified

affinity

2.

not

contamination

their residues

also

material

the

, was required.

cally

Only

with

features

column

glycoproteins

tographed

did

structural

the boronate

analysis

together

fraction

contained

Since

of (ADPR),-containing

asso-

treat-

4%.

conjugates

were

obtained protein.

by boronate chromatography The mean chain length of the

residues was determined by the method of Reeder (14) giving The conjugates revealed an extreme tendency to aggregate.

1234

Vol. 91, No. 4, 1979

Table

2:

BIOCHEMICAL

Chromatography fore and after

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of (ADPR), protein conjugates on boronate columns bealkaline or enzymic release of the (ADPR), residues.

Boronate Treatment

Chromatography Non-Binding [ug protein] [%I

Binding [ug protein]

WI

-A: 880

100

10

1

NaOH

70

8

780

88

none

780

10

1

720

92

-B: phosphodiesterase

100

6

50

(ADPRJnproteinconjugate samples containing 900 and 800 ug protein, respectively, were treated with alkali or with phosphodiesterase I and subjected to boronate cellulose chromatography according to methods,using 0.7x3.0 cmcolumns.

Therefore,

analytical

of guanidine

procedures

hydrochloride,

required

urea

When gel

electrophoretic

analysis

presence

of 0.1% dodecyl

sulfate

ed (fig.

1).

rent

Staining

molecular

In order residues

fluorography of the

cating

covalent

The varying

3.

label

extent

Nucleosomal

(fig.

same gel

of the

in the

was largely

prevent-

over

radioactively

was performed

seen in these

IB). the

The

indi-

bands.

points

acceptor

appa-

distri-

stain

protein

experiments

proteins

with daltons. (ADPR),

the protein

with

of different

and non-histone

bands 100000

labeled

(fig. with

thereof.

was performed numerous

to far

(ADPR),, residues

ratio

histones

10000

concentrations

or combinations

material

correlates

of ADP-ribosylation

core

of high

1A) revealed with

generally

protein

sulfate,

intact

from about

the association

attachment

(3H)ADPR/

in the

of the

protein

of the

tritium

or dodecyl

presence

and 6 M urea,'aggregation

ranging

to demonstrate

bution

ences

for

weights

the

to differ-

proteins.

as acceptors

of

(ADPR), - residues When the

isolated

in order

to separate

total

(ADPR),

fraction.

conjugates

residues

The bulk

Since

it

teins

progressively

were

the histones

subjected from

to cation

non-histone

and 14% of the total of the

was to be expected alters

conjugates that their

protein

appeared

increasing

were

to represent

ADP-ribosylation

physico-chemical

1235

exchange protein

properties,

chromatography

(9), found

only

6% of the

in the histone

non-histone

proteins.

of the acceptor both

fractions

pro-

Vol.

91,

No.

4, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-

130

KD

-

68

KD

-

43 40

KD KD

-

31

KD

-18.7

KD

-11.8

KD

f

H3

L

HZ8 HZA H4

A

01

A

B

C

D

E

F

G

0

B

2

SDS gel electrophoretic analysis of the (ADPR) protein -11 nuclei - 80 ug of isolated (ADPR) protein"conjugates lo5 dpm) were analized on 7.5 - 15% gradient gel!! as described in A: coomassie blue stain, B: fluorography.

conjugates (5.4 x methods.

Figure 2. SDS gel electrophoretic analysis of histone and nonhistone subfractions. - (ADPR) protein conjugates were separated into a histone and a nonhistone fraction "(cf. methods). Samples with and without alkaline treatment were analyzed on polyacrylamide gradient gels. A: 80 ug (ADPR) protein conB: Histone fraction derived from A) by Bio Rex 70 chr8matography. jugates. C: Histone fraction after treatment with alkali. D: Non-histone fraction derived from A) by Bio Rex 70 chromatography. E: Non-histone fraction after 30 ug of purified core histones isolated from EAT treatment with alkali. F: Molecular weight markers: B-Galactosidase (130 KD), bovine albumin cells. G: (68 KD), ovalbumin (43 KD), aldolase A subunit (40 KD), DNAase I (31 KD), B-lactoglobuline (18.7 KD), cytochrome C (11.8 KD).

were as

analyzed shown

besides

in some

H2A and H4. protein

stain

Identification

again

fig.

after

2 (Band

minor

of

these

0.4% Triton

X 100 (8)

the duced

SDS gel an

even

true

histone

the

expense

the

of a whole

fraction

migrating

histones

(not

comprised

slightly

as

the

behind

by mild

of the

the acetic

system

bands

histones

H2B/

treatment core

proteins

acid/urea

two main

authentic

alkaline

nucleosomal

acceptor

only

Indeed,

the

(fig.

2C).

histones

was

with

shifted

confirmed

(7)

by

elec-

and without

shown). (fig.

electrophoresis fraction.

histone

positions

using

fraction

higher

of the ADPR residues.

detachment

of the ADPR residues

to the exact analysis

non-histone

C),

components

Release

trophoretic

The

specific

2D)

was

system.

amount The

series

of

free

appearance of fine

also

treated

The removal core

with

of the

histones

(fig.

of the core protein

1236

bands

alkali

2E)

histones with

and

(ADPR), than

found

occurred apparent

analyzed

residues

in

proin

mainly molecular

the

at

BIOCHEMICAL

Vol. 91, No. 4, 1979

weights

between

the modified total

10 and 30 KD.

histones

acceptor

dramatically

changed

of poly(ADPR) Additional

the

conjugates

X 100)

relative

in the non-histone data

properties

of the

shifting

the

analyses

together

staining

with

Most

in the non-histone

did

not

removed

by alkali,

25000;

31000;

rying

chain

region, the

belong

35000)

ADPR residues

with

Less

size

Hl being than

and less et al.,

ating

soluble

of the

according

conjugates),

(ADPR),

(Mr = 12000;

conjugates

residues

(ADPR),

in the unaffected

to high-

H2B and H3. protein

of polymeric

+

a definitely

appeared

to the decreasing

protein

acid

residues

high

were

22000;

residues

of va-

molecular

weight

by the removal

resolving

was not

were

power

included

associated

was modified

groups

diol

derivative,

(5)

of

of the

system

in this

with

study.

the HI fraction,

by ADP-ribosylation

(Braeuer

and free

of contamin-

histones

most of these also

terogeneous past

proteins

fraction.

Analyses only

status

presented

ADPR residues separation of the

after

selective

the first

time

into

non-histone

have therefore and their

to the

removal that

value

of ADP-ribosylation.

1237

the

separation

of the

and groups

nucleosomal

properties Drastic

represent

shift-

changes a rather

as performed

to individual

of

histones

conjugates

their

fractions

respect

boro-

of the modifying

fraction. which

nuclear with

for

protein

changed

This inter-

an immobilized

modification

proteins

of selected

et al.

study.

specific

of ADP-ribosylated

the non-histone

limited

in this on the

with

(ADPR),

completely

acceptor

yield

was based

by McCutchan

ADP-ribosylation

conjugates

seen with

in the

Subfractionation and for

by poly

an analysis

introduced

analysis

unequivocally

in high

procedure

present

and applied

(3).

electrophoretic

for

isolation

a reaction

nucleotides

their

conjugates

was a prerequisite

of cis

showed

the

that,

gels

When the

to be rather

(3H)ADPR

(ADPR)n

by Okayama et al.

ing

loss

Hl population

when the

oligo

were

total

was achieved

action

core

class.

in 5% perchloric

2% of the

proteins

nate

urea

in preparation).

Isolation goal

fraction.

of the molecules.

2% of the than

(acid

(60% of the total

of the

appeared

due presumably

increasing

Histone

hand,

in the case

fractionthanhistones

new bands the

25% of the

the non-histone

indicated

that

ADP-ribosylation

especially

systems

controls

histone

The position

length.

that

H2A and H4 comprised histone

indicating

on the other

into

showed

comprised

histones,

in different

prominent

procedure

fraction

core

fraction

to the

several

Lowry

can be deduced

histones

histones

of the ADP-ribosylated

however,

it

appropriate

intensities,

er proportion proteins

by the

From these

electrophoretic

Triton

Quantitation

present

protein.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hein

acceptor

Vol. 91, No. 4, 1979

BIOCHEMICAL

Our data

with

clear

are

compatible

proteins

changes involved

by transfer

in properties in the

the

notion

that

of ADPR residues

and function

regulation

AND BIOPHYSICAL

of various

postsynthetic being

modification

associated

of the affected forms

RESEARCH COMMUNICATIONS

with

chromosomal

of nu-

drastic

proteins

is

of gene expression.

ACKNOWLEDGEMENTS We thank

Frau

supported

E. Spikofski

by the Deutsche

for

propagating

the tumor

cells.

This

work

was

Forschungsgemeinschaft.

REFERENCES 1. 2. 3. 4. 5. 6. L: 9. IO. 11. 12. 13. 14.

Hilz, H. and Stone, P. (1976) Rev. Physiol. Biochem. Exp. Pharmacol. 76, l-59, Springer Verlag, Berlin, New York Hayaishi, OFand Ueda, K. (1977) Ann. Rev. Biochem. 4&, 95-116 Okayama, H., Ueda, K. and Hayaishi, 0. (1978) Proc. Natl. Acad. Sci. USA 75, 1111-1115 Adamietz, P., Bredehorst, R. and Hilz, H. (1978) Biochem. Biophys. Res. Commun. 81, 1377-1383 McCutchan, T.F., Gilham, P.R. aa Still, D. (1975) Nucleic Acid Res. 2, 853-885 Laemmli, U.K. (197U) Nature (Lond.) 227, 680-685 Panyim, S. and Chalkley,'R. (1969) A??fi. Biochem. Biophys. 130, 337 Alfagune, C., Zweidler, A., Mahowald, A. and Cohen, L. (197r J. Biol. Chem. -249, 3729 Levy, S., Simpson, R.T. and Sober, H.A. (1972) Biochemistry 11, 1547-1554 Heyns, W. and DeMoor, P. (1974) Biochim. Biophys. Acta 358, r Bonner, W.M. and Laskey, R.A. (1974) Eur. J. Biochem. 4K83-88 Farr, A.L. and Randal1,T.J. (1951) Lowry, D.H., Rosebrough, N.J., J. Biol. Chem. 193, 265-275 R. and Hilz, H. (1978) Adamietz, P., B*ehorst, Eur. J. Biochem. 5, 317-326 Y. and Hayaishi, 0. (1967) Reeder, R.H., Ueda, K., Honjo, T., Nishizuka, J. Biol. Chem. 242, 3172-3179

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