Macromolecular derivatives of NAD+ in heart nuclei: Poly adenosine diphosphoribose and adenosine diphosphoribose proteins

BIOCHEMICAL

Vol. 71, No. 1,1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MACROMOLECULAR DERIVATIVES OF NAD+ IN HEART NUCLEI: POLYADENbSINEDIPHOSPHORIBOSE AND ADENOSINE DIPHOSPHORIBOSE PROTEINS Ari M. Ferro* and Ernest Kun** Research Institute, Departments of Pharmacology, Biochemistry and University of California, San Francisco Medical Center, San Francisco, California 94143

Cardiovascular Biophysics,

Received

May 6,1976

Summary: Rates of poly (ADP-R) formation from NAD+ were determined in isolated pigeon heart and liver nuclei. In heart nuclei Km for NAD+ was 330 PM. On a DNA basis rates were more than twice in heart nuclei than in liver nuclei. The polymer poly (ADP-R) was identified in both nuclear systems by isolation, digestion with snake venom phosphodiesterase and chromatographic separation of phosphoribosyl-AMP and AMP. ADP-R binds to macromolecular nuclear components to form ADP-R derivatives, which upon digestion with snake venom phosphodiesterase yield only AMP, distinguishing these ADP-R compounds from poly (ADP-R). Thenucleicacid-like identified

polymeric

by enzymatic

degradation

with

to 2'-(5"-phosphoribosyl)-5'-AMP cell

nuclei

enzymatic

the acid

precipitable

of chromatin animal therefore

cells

derivatives

of

this

we compared

goal

of heart

capacity

generation difference of the

poly

difference of rates

same species.

this

Assessment

the

rates

mitosis a far

greater

in mitotic of poly Besides

mitotic potential

(ADP-R) poly

potential of the

formation (ADP-R)

in nuclei

*Supported by Graduate Training Grant HLO-5251 **Recipient of the Research Career Award of the Abbreviations: poly (ADP-R) = poly (adenosine ADP-R = adenosine diphosphoribose NP-40 = nonidet P40, detergent DTT = dithiothreitol

Copyright All righrs

0 1976

by Academic Press, Inc. in any form reserved.

of reproduction

for

150

from

in nuclei have a

of liver

hepatocytes.

re-

It was assumed

may be reflected from

NAD+, we also

NIHL USPHS

of the

diphosphoribose) product

Pursuing

cardiocytes

isolated

of Shell

Co.

of

in a variety

(ADP-R)

phenomenon

(7);

of macromole-

functions.

two organs

formed

role

poly

of

mitochondria derivative

substances

known that

the wellknown

some form

in a variety from

biological

physiological

It is

of the

macromolecular

and identified

pigeons. whereas

only

3.1.4.1) and liver

of counting

NAD+ with

is present

of these

different

of syntheses

(8),

of labelled

of the

identification

have greatly

basis

consists

was isolated

is the

(E.C. of thymus

On the which

enzyme

protein

(ADP-R)

NAD+ requires

of 6 week old for

indicates

this

that

(5,6).

incubation

that

has been

preparations

(ADP-R) synthase,

after

(ADP-R)

venom phosphodiesterase

HeLa cells

poly

i t appears

which

and liver

limited

of

the

seems unlikely.

tissues

snake

an ADP-ribosylated

the assumption

of animal

that

for

radioactivity

Recently

NAD+ in animal cular

and nuclei assay

preparation,

cells.

of NAD+, poly

and AMP in chromatin

(1,2,3,cf.4)

customary

product

both find

in a tissues that

ADP-R

Vol.71,No.1,1976

itself

can bind

existence

covalently

of a family

experimentally (ADP-R)

BIOCHEMICAL

AND BIOPHYSICAL

to macromolecular

of hitherto

demonstrated

in the

unknown

components macromolecular

same chromatin

RESEARCH COMMUNICATIONS

of chromatin. metabolites

preparations

where

The of NAD+ is poly

is synthesized.

Materials and Methods. Isolation of purified nuclei from liver and cardiac tissues of 6 week old pigeons (250 g body weight) and assays of DNA and protein were carried out by standard techniques (9). Higher yields of cardiac nuclei were occasionally obtained by substitution of the high density centrifugation step with seve 1 centrifugal washings at 700 x g with 0.25 M sucrose-l mM Mg$12. The source of 'a C labelled (uniformly in the adenine portion 253 Ci/mole) NAD was Amersham-Sear1 Corp. and the radiochemical counting techniques were the same as published previously (7). Labelled ADP-R was prepared from labelled NAD+ by hydrolysis with calf spleen glycohydrolase (E.C. 3.2.2.5) and isolated by paper chromatography with a yield of 75% (93% purity). Poly (ADP-R) was isolated by a published method (10) except phenol extraction was employed after pronase digestion. At stage 5 (see Table) the isolated poly (ADP-R) was dissolved in 50 mM Trisacetate (pH 7.2) + 8 mM MgCl and digested with purified (14) snake venom phosphodiesterase (Boehringer) at 2 6 0 pg/ml concentration for 2 h at 37'. The amount of poly (ADP-R) used for digestion was 4.5 nmoles (32,500 CPM) from liver and 7.8 nmoles (24,000 CPM) from heart nuclei. The entire incubation mixture was applied to Whatman No. 1 chromatographic paper, washed with 80% ethanol and developed in isobutyric acid-30% NH3-H20, 66:1:33 (v/v). Authentic nucleotides were cochromatographed with samples. Treatment with nucleases prior to snake venom phosphodiesterase was found unnecessary because non-radioactive breakdown products of DNA did not interfere with identification of radioactive nucleotides derived from p0ly (ADP-R).

4OOr

0

0

40

60 Minutes

I20

Fig. 1: Time course of poly (ADP-R) formation from NAD+ (3.0 mM) incubation system (100 ~1) contained 30 mM MgC12, 4 mM KF, 60 mM KCITh;?oO mM Tris-Cl (pH 8.0), 0.5 mM EDTA (Na)2, 2 mM DTT and 14C-NAD+ 8 x 105 CbM. In the liver system 2.0 mg protein (Lowry) and 0.4 mg DNA (Burton), and in the heart system 0.4 mg protein and 0.1 mg DNA were present. t = 37'. Acid precipitable radioactivity was determined by the glass fiber filter technique using 6% HC104 as protein precipitating agent. Fig. 2: Saturation of poly (ADP-R) synthase of heart nuclei by NAD+. v = nmoles NAD+ converted to poly (ADP-R) in 3 minutes at 37" H 8 0 , K,,, = 336d4. Conditions for the assay are described in legend of !ig.'l.

151

Vol. 71, No. 1,1976

Results expressed twice

and Discussion.

as moles

in heart

of heart

Extremely recently which

That

in liver to liver

of the

acid

(ADP-R)

nuclei

(ADP-R)

synthase

precipitable

unknown. to Km values

synthase

activities

systems of rat

for

this

radioactive by its

NAD+ (3 mM) as

enzyme

enzyme content

or to

As shown in Fig.

'2, the Km

reported

liver

for

rat

(11).

heart

of NAD+ used by this

Km found

from

is due to higher

have no quantitative

was proved

formation

(ADP-R) per mg DNA were more than Whether the increased capacity 1).

(Fig.

is at present

poly

(ADP-R)

results

(ADP-R)

to poly

nuclei

The concentration

1/210th

of poly

is 330 PM, similar

and other

low poly

the reported poly

nuclei

12)

(13). is

than

as compared

heart

cf.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NAD+ converted

in enzyme regulation

of NAD+ for $i,

Rates

x low9

nuclei

nuclei

differences (250

BIOCHEMICAL

nuclei

author

were

(13)

reported

was 1.2 PM,

in rat

tissues

(12);

consequently

(Figs.

1, 2) was predominantly

meaning. material

isolation

(Table)

and identification

(Fig.

3).

TABLE Liver

Stages of purification (see Ref. 10) 1.

First TCA precipitate After NaOH treatment After pronase After phenol extraction After ethanol precipitation

2. 3. . 5.

Heart

108

250

Zf

210 210

83

210

75

167

acetate

The incubation system (2.3 ml). contained the same molar concentrations of ingredients as shown in legend to Fig. 1. The amount of nuclei per system was 61 mg protein, 12 mg DNA for liver and 43 mg protein, 2.4 mg DNA for heart. Results are expressed as nmoles of ADP-R residues per mg DNA.

As shown snake

in Fig.

3, digestion

venom phosphodiesterase

of AMP, identified results

identify

also

reported

poly

(14)

phosphoribosyl-AMP

yields

(ADP-R)

by the ADP-R and AMP markers.

venom enzyme does not act by nicotinamide

of isolated

poly (4.3

(ADP-R)

synthesized

mM inhibits

by others

It

on phosphoribosyl-AMP,

(1.2,3,15)

is

but

by both

152

poly

5 with

and small known that

hydrolyzes

nuclear

77%) and by thymidine identifies

at stage

the

ADP-R.

preparations. (4.3

(ADP-R)

mM inhibits synthase.

purified amounts snake These Inhibition 78%) as

BIOCHEMICAL

Vol.71,No.1,1976

AND BIOPHYSI~AL

RESEARCH COMMUNICATIONS

0) ADP-R nn

6

AMP

A n

“A~P-R

rim

A n

AMP

2

0

k--0

21.5cm

4 s

L----22.5cm 0

----A S

Fig. 3: Identification of poly (ADP-R)_. A = adenosine, 0 = origin of-chromatogram, s = solvent front, a) liver nuclei, b) heart nuclei. The average chain length of poly (ADP-R) was 14 for the liver and 16 for the heart. Incubation conditions added

NAD+, yields

precipitable (see

of a nuclear

as used for

covalently

covalently

radioactive

Methods)

the bound

shown in Figs.

the

preparation biosynthesis bound

material

of heart of poly

with

ADP-R (6.6

(ADP-R),

except

ADP-ribosylated

was digested

macromolecules. with

snake

mM) under in

similar

the absence When the

venom phosphodiesterase

only

radioactive nucleotide released was AMP, which identifies Binding of ADP-R to heart nuclear preparations is ADP-ribose.

4 and 5.

Fig. 4: Covalent binding of ADP-R to pigeon heart nuclear preparation as a The reaction system calculated for 1 rnT function of protein concentration. volume contained 16 mM Na phosphate (pH 8.0) 6.6 mM ADP-R (8.3 x 106 CPM) and increasing amounts of heart nuclei (31 mg protein and 1.7 mg DNA per ml) in Time of incubation 20 min. at 37". 50 mM Tris-Cl,1 mM DTT,l% NP-40. Fig. 5: Covalent binding of ADP-R as a function of ADP-R concentration. Conditions are the same as described in legend of Fig., 4. The concentration of protein was 13 mg/ml, DNA = 0.7 mg/ml. 153

of

Vol.71,No.1,1976

BIOCHEMICAL

When the amounts on a DNA basis same order products

of macromolecular

not

(nmoles/mg

specific

covalently

be described

for

bound

poly

formed

(ADP-R)

DNA) as poly

heart

nuclei;

ADP-R.

from ADP-R are

the values

(ADP-R).

they

are

of the

The macromolecular

represent

The molecular

found

calculated

a mixture

structure

ADP-R

of proteins

of these

substances

elsewhere.

ACKNOWLEDGMENT This research was supported REFERENCES 1. Chambon, Biochem.

products

5) as was done for

of magnitude are

containing will

(fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by the NIH program

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grant

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3.

Sugimura, T. in Progress in Nucleic Acid Research (eds. Davidson, J.N. and Cohn, W.E., Acad. Press, 1973.

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