Distribution of bound ADP-ribose derivatives in nuclear protein of rat liver nuclei

Vol.

56, No.

2, 1974

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

DISTRIBUTION OF BOUND ADP-RIBOSE DERIVATIVES IN NUCLEAR PROTEIN OF RAT LIVER NUCLEI 1 Dietrich, L.S. and Siebert, G. fiir Biologische Chemie und ErnZhrungswissenschaft, Hohenheim, Stuttgart, Fed. Rep. of Germany

Abteilung Universitlt Received

Ncbvember

28,

1973

SUMMARY--Most of the acid-insoluble radioactivity produced by incubation of rat liver nuclei with [14C]NAD is.rendered soluble by treatment with cold neutral hydroxylamine. The substances released by hydroxylamine have been determined to be (adenosine diphosphoribose)oligomer, adenosine diphosphoribose, 5'-AMP and adenosine, the greatest activity being found in the adenosine diphosphoribose fracti.on. The distribution of hydroxylamine-sensitive radioactive material in the nuclear proteins varies with the fractionation method employed. Regardless of' the method employed, the "histones" contained only small amounts of hydroxylamine-insensitive radioactive material [poly(adenosine diphosphoribose)] INTRODUCTION-.-In fraction nuclei

a previous

of acid-insoluble with

]:14C]NAD,

to understand nuclei, products separation insoluble fractions, products MATERIALS obtained

formed

radioactivity represents

better

we provide

communication,

the fractionation

as well forned

NAD, were

the Radiochemical

(6 Ci/mole)

was from New hnglanl

Chemicals.

Dowex-1-2X

E. Merck, Boehringer Nuclear,

Darmstadt. u. Soehne, Boston,

'On Sabbatical 2ADP-ribose

and thin

For this

= adenosine

soluble into

of the

and several

of the different

employed. ([U-14C]adenine)NAD

Centre,

layer

Boston,

cellulose

GmbH, Mannheim.

Protosol

the University diphosphoribose

was

([a-14C]adenine)ATP

Amersham.

Nuclear,

(167 Ci/mole)

Massachusetts. plates

were

NAD and snake venom phosphodiesterase

from

cell

nature purpose,

macromolecules

was obtained

Massachusetts. Leave

efforts

of NAD with

on the chemical

identification

AND METHODS--Radiochemicals. from

In continued

hydroxylamine

of nuclear

a small of rat-liver

reactions

data

into

as the chromatographic

from

(1).

enzymes --in vitro.

radioactivity

only

incubation

and transfer

some further

from NAD by nuclear

material,

after

poly(ADP-ribose)'

paper

of acid-insoluble

was shown that

formed

the polymerization in this

it

of Miami,

1972-73.

obtained were from

from

obtained

from

New England

Vol.

56, No.

Isolation

2, 1974

of ADP-ribose

sex were

sacrificed

and nuclei

x lo6

and 0.005 cooled

derivatives.

by decapitation

isolated

Rat liver (0.2-2

BIOCHEMICAL

employing

nuceli

(l-10

(6-8

Labeled

or labeled

0.4 M NH20H, pH 7.0, pellet

was combined

added.

The supernatant After

5'-AMP

and ADP-ribose

dilution,

was placed

1.0 M, 6.0 M formic The fractions

from

lyophilized following

were

acid

(100/60/2,

then

reported

to thin

pH 7.5,

using

scintillation in Protosol

also

as carriers

containing

layer

cellulose

(713,

The location

pH 8.2. volume

centrifugation

non-radioactive after

and eluted

with

0.4 M ammonium formate. were

combined,

and developed (66/l/33,

and 0.1 M P04,

in

the

v/v/v),

pH 6.8/(NH4)2S04/non the

and the results

The NH20H-insoluble for

appropriate

stepwise

of the radioactivity

and quantitated

PCA was

was neutralized

perchlorate,

scanner

radioactivity

thin

of the Radioactive

Material

284

made Acid

Soluble

layer

quantitated

material

as previously

(1).

Characterization

(4).

and the

of 6% ice-cold

plates

spectrometry.

and Morris

The NH20H-soluble

radioactivity

a chromatographic

histones

in 8 ml of ice-cold

NH40H/H20 v/v),

into

Potter

containing

step

5 ml

the

was centrifuged

column

acid

with

fractionated

and the sample,

formate

M KC1

was immediately

employing

incubated

potassium

and 6.0 formic

v/w/v).

was determined

dissolved

after

0.025

then washed

of Grunicke,

isobutyrate/concentrated

propanol

liquid

added

each elution

M NH4 acetate,

employing

were

buffer,

insoluble

on a Dowex-l-LX

EtOH/l.O

plates

of the

containing

The mixture

The material

obtained

(2).

10-3 M [14C]NAD

fractionated

were

Tris-Cl

removed

of the wash approached

the wash and an equal

material

and applied solvents:

1 hour.

with

were

were

Nuclei

of mixed

the livers

and Potter

pH 8.2,

nuclei

proteins

once with

removal

anesthesia,

at 25O.

COMMUNICATIONS

(250 grams)

incubated

the procedure

nuclear

with

rats

the radioactivity

at 0' for

material

KOH.

10 min.

proteins

RESEARCH

of Blobel

were

The labeled

using

washed

ether

buffer,

and Wang (3).

fractions

nuclei

for

Nuclear

and nonhistone

with

after

until

times).

of Kostraba

resulting

SIV-50

mg protein)

bath.

of the TKM buffer

procedure

Albino

cpm) in 3 ml of a Tris-Cl

to O" in an ice

background

BIOPHYSKAL

the procedure

M MgC12 (TKM buffer)

portions

AND

by Treatment

was

Vol . 56 I No. :!, 1974

with

NH2OH.

column,

BIOCHEMICAL

In all

the

radioactivity .In addition

demonstrated

.Labeled suspiected

ADP-ribose

the procedure procedure

toluene

as well

as the ion

with

carrier

adenosine,

5'-AMP

digestion

as a reaction

of being

as products

oligomer

Similar

of ADP-ribose

polymer

of Shima

et al.

(5).

was determined

Protein

was assayed

employing

bovine

applicable,

as an internal

of the

2'-phosphoribosyl

of the reaction.

length

where

and

treatment

demonstrated

of chain

and,

exchange

of the ADP-ribose

product.

ADP-ribose

(6) with

techniques

utilized

phosphodiesterase

of 'Lowry et al.

Radioactivity

systems

migrated

5'-AMP

and 5'-AMP

Analysis

layer

always

ADP-ribose.

material

thin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was carried

liquid

cpm were

the

as a standard.

scintillation

converted

employing

employing

serum albumin

standard

out

spectrometric

to dpm employing

[14C]

standard.

RESULTS AND DISCUSSION-Composition

of Radioactive

The distribution by cold

neutral

be accounted

for

were

acid

that

ca.

5'-AMP since

are

insoluble

and adenosine incubations

a pathway

which

be determined

and intriguing metabolism

is

treated

with

is most

interesting.

identical

by the data

and 5'-AMP

from

observation of ADP-ribose.

found

cold

neutral

catabolic

products

NAD is

metabolism

and might

in

indicate

If on the other

285

5'-AMP

pathway, multiple

hand,

fraction. since

the form

to be the

Whether

these

they

is not

of bound true

precursor

in no

the nuclear-bound

metabolism

the formation

an independent

can

The observation

for

of ADP-ribose

If

here.

The label

[ 14 C]ATP resulted

nuclei.

NH2OH.

solubilized

covalently NH20H.

with

of ADP-ribose

presented

1.

[14C]NAD appears

liver

is

in the ADP-ribose

can be accounted

rat

with

ADP-ribose,

material

conditions into

independent

which

shown in Table

to nuclear

consumed

are

is

being

bound

of radioactivity and 5'-AMP

by Treatment

(ADP-ribose)oligomer,

[14C]NAD

under

adenosine

adenosine

probably

Soluble

radioactivity

nuclei

of the activity

until

25% of the

incorporation

liver

by 4 compounds:

most

compounds

made Acid

of the nuclear-bound

NH20H in rat

and adenosine, These

Material

or represent

known and cannot

of nuclear-bound this

functions bound

would

present

a new

in nuclear compounds

are

products

BIOCHEMICAL

Vol. 56, No. 2, 1974

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 Distribution Material

of Radioactivity

Extracted

by Cold

Compound

in Acid-Soluble Neutral

Hydroxylamine % Fraction

dpm

(ADP-ribose)oligomer

18,000

19

ADP-ribose

50,500

55

5,900

6

18,200

20

5'-AMP Adenosine

92,600 Nuclei placed

incubated for on column.

of ADP-ribose yet

or poly(ADP-ribose)

another

system

ADP-ribose would

for

that

these

strengthened fraction from

rats

that

Distribution

liver

nuclei

that

observations

that

with

Material

as a Function have been

is

of the radioactivity

release

by NH20H.

32Pi

however,

in

dpm

that

there

degradation then

of

the 5'-PO4 The

as a product.

of an --in vitro

system

--in vivo

in the

liver

is

is acid

nuclei

soluble

made

(7). Soluble

("histones")

in Table

2 and extended or "nonhiatone"

one employs

by NH20H Treatment

Employed.

[14C]NAD with

to proteins

("histone"

during

adenosine

observed

91,000

conclude

of acid-extracted

made Acid

incubated

confirmed

not all

If,

5'-AMP

one must

in the 2' position bound

of the Procedure

is bound are

nuclear

at 25O.

to protein

are not artifacts

injected

of Radioactive

then

were

the NH20H treatment

had been

Proteins

linkage

leaving

observations

from

10s3 M [14C]NAD

the AMP moiety

this

hydrolysis

of the radioactivity These

transfers If

with

degradation,

by the observation obtained

Nuclear

that

derivatives.

be free

premise

10 minutes

the procedure

in

When one extracts

dilute

mineral

extracted

by acid

to show that bound)

acid,

is

most

sensitive

of Kostraba

rat most

(6,9). but to

and Wang (9)3

3J%mploying this procedure the chromatin is first solubilized in 1 M salt and then the DNA-histone complex is precipitated by lowering the salt concentration followed by diluted mineral acid treatment of the DNA-histone complex to extract the histones.

286

Vol. 56, No. :2, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 2 Distribution

of l4 C Solubilized

in Histone

and non-histone

by Cold Components

NH20H Soluble

Neutral

NH20H

of Rat Liver

Material

Nuclei

NH20H Insoluble

dgm

dpm

Histone

92,000

2,703

Non-histone

57,350

7,550

149,350

9,253

Material

Nuclei incubated for 10 minutes with lo-3M ]14C]NAD at 25“. Histones extracted with acid employing procedure of Grunicke et al. (4). Values are the average of 2 experiments.

where

the acid

proteins", compared

soluble

only with

ribose)

a 60% found

("residual

complex

("DN~:') .

derived

from

in the

found that

proteins

are

which

of the DNA-histone

protein"

[poly(ADP-ribose)]

is

is not

found

complex

primarily

histone

It

appears

extraction clear

from

and not

that

from

that

(Table

bound

soluble

during

radioactivity most

of the

This

leads

to acid

soluble

the precipitation

between

the "DNA" and the

("residual

neither

in

the DNA-histone

are bound

the DNA-histone

287

from

of the NH20H insoluble material

the data

1) not

the "histones."

are partitioned

insoluble

(Table

extracted

the "histones",

derivatives

presence

proteins

53% of the radio-

extracted

with

is possible

"acidic

Over 80% of the poly(ADP-

the nuclear

proteins"

the products

in the 1 M salt

,sfter

not

associated

It

are

in the material

that

not histones.

proteins

fractions.

some of the ADP-ribose

of the

approximately

demonstrate

the "acidic

removal

in the "histone"

soluble

and 2) material

data

in

until

found

the acid

The overwhelming

proteins."

is

material)

These

one to suggest

significant.

"acidic

protein")

[14C]NAD

being

the residue

extracted

and Wang procedure,

(NH2OH insensitive

1 M salt

"acidic

when all

the Kostraba

was found

activity

are not

34% of the radioactivity

Employing activity

proteins

complex "histones"

material proteins") is

and

highly nor "acidic

3) 2).

Vol.

56, No. 2, 1974

BIOCHEMICAL

AND

TABLE 3 14 of C Solubilized

The Distribution

NH20H in Components

Histones Proteins

Residual

Material

COMMUNICATIONS

Neutral

Nuclei NH2OH Insoluble

dpm

dpm

34,100

540

53,500

855

Proteins

9,910

3,825

4,150

1,960

101,660

7,180

"DNA"

Nuclei protein Results

RESEARCH

by Cold

of Rat Liver

NH20H Soluble

Acidic

BIOPHYSICAL

incubated for 10 minutes with 10s3 M [14C]NAD fractionated employing procedure of Kostraba are the average of 2 experiments.

Material

at 25'. Nuclear and Wang (3).

TABLE 4 Distribution

of 14C Extracted

Nuclear

Histone Acidic Residual

Proteins Proteins

"DNA"

Components

Neutral

Rat Liver NH20H

Adenosine =pm

AMP cpm

ADP-ribose Cpm

cl00

410

2422

165

1710

1080

3508

355

758

594

730

405

cl00

642

362

2084

7302

1287

Cl00 --Total

by Cold

from

2468

Nuclei incubated for 10 minutes fractionated employing procedure average of 2 experiments.

(ADP-ribose)Oligomer cpm

with 10-3 M [14C]NAD at 25O. Nuclear protein of Kostraba and Wang (3). Results are the

288

Vol.

56, No.

proteins"

2, 1974

contain

BIOCHEMICAL

more than

Some interesting

these

"histones"

material

conditions

it

and "acidic

mer was found.

in the

Vdrtually

proteins

and "residual

histones

and DNA fractions. data

all

incubation

ledge

of the problem,

biological

these

protein

that

ADP-ribose

proteins"

fractions

(ca 90X),

are

most

primarily

being

in the

found

to be in the acidic detected

to nuclear

at our present

in correlating

4.

in

no adenosine

that

in Table

(13%) being shown

out

in

oligo-

were

bound

shown

present

of the ADP-ribose

little

of products

and point

nuclear

COMMUNICATIONS

compounds

was located

hand,

and adenosine

one must be cautious and/or

of the

and "DNA",

the complexity

[14C]NAD

RESEARCH

of poly(ADP-ribose).

On the other

proteins"

with

activity

was found

the 5'-AMP

demonstrate

after

from

"residual

BIOPHYSICAL

on the distribution

proteins".

histones.

These

amounts

observations

the NH20H solubllized Under

minor

AND

level

in

the

protein of know-

"ADPR polymerase"'

with

location.

ACKNOWLEDGEMENTS-This work was supported by grants from the Alexander von Humboldt-Stiftung, the Fulbright Kommission, the Deutsche Forschungsgemeinschaft, the UniversitBtsbund Hohenheim, and the National Institutes of Health (CA 12907). The authors wish to acknowledge the competent technical assistance of Miss M. Ott.

REFERENCE:S-1.

Dietrich, 354,

2. 3. 4. 5. 6. 7. 8. 9.

L.S.

:L133-1140

and Siebert,

G.,

(1973),

Hoppe-Seyler's

Z. Physiol.

Chem.

*

Blobel, G. and Potter, V.R., (1966), Science 154, 1662-1665. Kostraba, N.C. and Wang, T.Y., (1970). Int. .I. Biochem. I, 327-334. Grunicke, H., Potter, V.R. and Morris, H.P., (1970), Cancer Res. 30, 776-736. A. and Sugimura, T., Shima, T., Hasegawa, S., Fujimura, S., Matsubara, (1969), J. Biol. Chem. 244, 6632-6635. Lowry, O.A., Rosebrough, N.J., Farr, A.L. and Randall, R.J., (1951). J. Biol. Chem. 193, 265-275. Dietrich, L.S., Jaus, H. and Siebert, G., (1973), FEBS Letters, in press. K., Yamamura, H., Takeda, M. and Nishizuka, Y., Ueda, K. Yoshihara, Hayaishi, 0. (1969), Cold Spring Harbor Symp. Quant. Biol. 37, 781-786. Burzio, L. and Koide, S.S., (1971), Biochem. Biophys. Res. Comm. 42, 1185-1190.

289

the