Enzyme-bound early product of purified poly(ADP-ribose) polymerase

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ENZYME-BOUND EARLY PRODUCT OF PURIFIED POLY(ADP-RIBOSE) Koichiro

Yoshihara, Tomotaka Hashida, Hiroko Yasuharu Tanaka and Hajime Ohgushi

Department

Received

POLYMERASE

of Biochemistry, Kashihara, Nara,

Nara Medical Japan.

Yoshihara,

University,

June 16,1977 SUMMARY

Bovine thymus poly(ADP-ribose) polymerase with a purity of 99% on a SDS-polyacrylamide gel electrophoresis was able to initiate poly(ADP-ribose) synthesis without adding any exogenous acceptor protein to the reaction system. Analyses of the early reaction product synthesized without exogenous acceptor protein revealed that the product was oligo(ADP-ribose) with a mean chain length of 2.6 and was bound tightly to the enzyme protein. When the radioactive early reaction product was chased by incubating further with cold NAD+, ADP-ribose AMP-residue of the oligo(ADP-ribunit was found to be added to the terminal ose) attached to the enzyme. The stability of the early reaction product in high concentration of salt, strong acid, sodium dodecyl sulfate, and urea strongly suggests a covalent nature of the binding of oligo(ADP-ribose) to the enzyme. An enzyme which a homopolymer (l-3). gators(

of several

histones(

in cell

mechanism

investigated.

sence

its

the

ed that ribose

of

units

are

recent

protein

synthesis

of this

ADP-ribosylated

polymer

Copyright All rights

0

1977

of reproduction

SDS; sodium

by Academic in any

Press,

form

dodecyl

Inc. resenvd.

enzymes(l5,16). is ),

) has been reported. reaction

field

is

revealed

not yet that

chain,

protein-bound

we

ful-

the pre-

the enzyme reaction(26-28 initially

the

polymerase,

). suggest-

and additional

ADP-

ADP-ribose,

polymer(29). is

initiated

a purified enzyme, free of other nuclear an exogenous acceptor protein absolutely Abbreviations: ribosyl)-5'-AMP.

for

upto includ-

of DNA synthesis(lZ-21

of poly(ADP-ribose)

transferredto

a monomer

poly(ADP-ribose)

in this

investi-

proteins

and nuclear

polymerase

advances

of the

from

differentiation(25

a prerequisite is

successively

in an elongation

If the

is

ranging

control

of poly(ADP-ribose)

and elongation

some nuclear

resulting

in cell

system

initiation

in the

by other

to chromosomal ),

the enzyme,

involvement

However,

of DNA in the

As for

sizes,

is bound

proteins(l3,14

proliferation(ZZ-24)and

The precise ly

of various

role

understood,

has been determined

units,

), nonhistone

the biological

fully

moiety of NAD+ into poly(ADP-ribose), is found in mammalian cell nuclei

units,

polymer

ADP-ribose

7-12

Although not

ADP-ribose ADP-ribose

The structure of this 4-6). Poly(ADP-ribose)

a polymer ing

polymerizes

of repeating

sulfate,

exclusively

proteins, for the

as stated

above,

may be expected to require reaction. In spite of exten-

phosphoribosyl-AMP;

2'-(5"-phospho-

1281 ISSN

0006-2

91 ‘Y

Vol. 78, No. 4, 1977

sive for

purification,

however,

exogenous Recently

BIOCHEMICAL

poly(ADP-ribose)

to homogeneity(32).

In the present

polymerase

any exogenous (ADP-ribose)

the enzyme did

not show an absolute

requirement

acceptor(30,31).

we purified

(ADP-ribose)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acceptor bound

is able protein

polymerase study

we will

to initiate and that

from bovine show that

a purified

poly(ADP-ribose) the early

to the enzyme protein

product

by a covalent

almost poly-

synthesis

reaction

probably

thymus

without

is an oligolinkage.

MATERIALS AND METHODS Chemicals and enzymes. [Adenine-2,8-3H]NAD+ was purchased from New England Nuclear, Boston, USA. ADP-ribose, NAD+(grade 5), calf thymus whole histones (type 2), calf thymus DNA(type 1) and trypsin were products of Sigma. DNase I and snake venom phosphodiesterase were from Worthington Biochemical Corp., New Jersey, USA and pronase was the product of Calbiochem, San Diego, USA. Assay for protein and DNA. Protein and DNA were estimated by the methods of Lowry --et a1.(33) and Burton(34), respectively. Assay of poly(ADP-ribose) polymerase. The standard reaction mixture contained 25 mM Tris-HCl buffer,pH 8.0, 10 mM MgC12, 0.5 mM dithiothreitol, 10 YM [adenine-2,8-3H]NADf(25,000 cpm/nmole), 2 pg each of calf thymus DNA and calf thymus whole histones, and an appropriate amount of the enzyme in a total volume of 0.2 ml. The mixture was incubated at 25°C for 10 min. Other procedures were performed as described previously(35). One unit of the enzyme activity was defined as equivalent to one pmole of ADP-ribose incorporated into acidinsoluble material per min under described condition. For the preparation of the early reaction product, some ingredients of the reaction mixture, scale of the reaction and incubation time were changed as indicated in the experimental results. SDS-polyacrylamide gel electrophoresis. The gel electrophoresis and staining of protein band with amido black were performed according to Hayashi and Oba (36 ), with a slight modification of the method of Weber and Osborn(37). The gel was cut into 1.5 mm thickness and each disk of the gel was digested in 0.5 ml of 0.1 N NaOH in a counting vial at 37°C overnight and neutralized with 0.7 N HCl. After standing at 37°C for 2 hours,each sample was mixed with 6 ml of a xylene-based scintillator(O.7% diphenyloxazol, 0.003% POPOP and 25% triton X-114) and the radioactivity was counted by a liquid scintillation spectrophotometer. Preparation of poly(ADP-ribose) polymerase. Poly(ADP-ribose) polymerase was extracted from bovine thymus gland and purified to homogeneity by selective precipitation with ammonium sulfate and column chromatographies on DNA-cellulose, hydroxylapatite and sephadex G-200, successively. About 1,300-fold purification was achieved from the initial extract with a recovery of lo-20%. The molecular weight and the purity were estimated to be 130,000 and 99%, respectively, on analyses with SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 0.12-0.18 unit per mg. The maximum velocity estimated from the Lineweaver-Burk plot was 0.9-1.0 pmoles per min per mg protein. The details of the purification will appear elsewhere(32). RESULTS AND DISCUSSION Recently

we purified poly(ADP-ribose) polymerase from bovine thymus The purity of the enzyme was estimated to be around

homogeneity(32).

SDS-polyacrylamide the reaction. enzyme reaction.

gel As will

The enzyme

electrophoresis. be shown below,

Therefore,

DNA does

DNA seems to be required

1282

absolutely

not accept for

required ADP-ribose

almost to 99% by DNA for in the

the enzyme activation

Vol.78,No.4,

in this

1977

Nistones

system.

an optimal vate

BIOCHEMICAL

the

However,

reaction(28),

during

the purification(32)

out

adding

not

necessarily

correct,

These

the

role

possibilities or the

distinguish

are

and its were

tion

of poly(ADP-ribose)

1 JIM and the action

omitted

thus

l.,

as a single

peak

which

radioactivity

was found

sult

suggests

A possibility

that

which

coincided

Another is also

zyme

oligo(ADP-ribose)

elongating In order

further,

that

with

to confirm

trophoresis.

As shown

2,

a larger

cubating

it

activity

shifted

broadening the mobility polymer.

further

with toward

cold origin

In order

to confirm

material length

binding part

of the

not contain

complex to other

bound

with

to the

1283

this

conclusion,

en-

and the chain

later. product

and the

acid-insoluble

gel

enzyme elec-

radio-

the peak of the

a simultaneous

The results can be interpreted by the elongation of the bound

and extend

of

acceptor

as the enzyme protein, as product was chased by in-

as above,

and became broader

the

unpublished

by SDS-polyacrylamide of the

NAD' and analyzed

be an en-

in

of 2.6,

be shown

same position early reaction

of the enzyme band(Fig.2). of the enzyme is affected

did

14C]NADt(an

chain

The re-

to the enzyme.

experiment

is transfered

as will the

was found again at the exactly sharp peak. When the labeled

was found

of DNA.

product

is an intermediate

was analyzed

activity a single

re-

chromato-

applied

the material

radioactive

an average

product

in Fig.

that

ADP-ribose

and characterize reaction

The early

column

in the enzyme peak might

the material

incubation,

to

the region

by [carbonyl-

the

was decreased

No acid-insoluble

reaction

from a similar

which

because

on a prolonged

the early

material

from

unlikely

including

the sys-

the elonga-

set).

G-200

to

enzyme system

To simplify

time(20

ac-

In order

and to limit

radioactivity

by the fact

possibility

of this

of [~H]NAD+

are

without

product

as follows.

early

does

of many

considerations system.

the enzyme peak.

was replaced

in spite

synthesized

by sephadex

area

even with-

enzyme system

in this

mixture, a short

of the

of NAD+ as judged

molecules, is

with

binding

enzyme and ADP-ribose, is

for

in the other

[adenine-2,8-3H]NADt

result). the

analyzed

acid-insoluble

was excluded

moiety

reaction

was analyzed

the total

the

is

the concentration

the radioactive

zyme-NAD+complex, nicotinamide

purified

of the

the enzyme

activated

If these

reaction

was performed

in Fig.

strongly

were

from the

obtained

gra WY. As shown

our

when

activator from

of ADP-ribose

as an acceptor, an early

chain,

incubation

product

a potent separated

poly(ADP-ribose)

serves

properties

histones

that

further

DNA was used to acti-

the enzyme was fully

indicate

possibilities,

tem,

d(T),

DNA fraction

as an acceptor

either

two-fold

thymus

of histones(7-12,26-29).

enzyme itself

these

was prepared

facts

histones

about

calf

d(A)-poly

thymus

was used,

require for

dissolved

when poly

or a calf

histones.

suggestions

the reaction

of freshly

enzyme

ceptor

stimulated

concentration system.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

shift

radioand a

as that ADP-ribose

two samples,

the

Vol. 78, No. 4, 1977

BIOCHEMICAL

IO

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20

30

FRACTION

40

;; 22

50

NUMBER

Fig. 1. Sephadex G-200 column chromatography of early reaction product. Poly(AOP-ribose) polymerase(60 Ag) was incubated in the reaction mixture principally as described in methods except histones were omitted from the mixture and the amount of calf thymus DNA and the concentration of substrate were changed to contain 200~1g of DNA and 1 JJM [adenine-2,8-3H]NAD(125 cpm/ pmole), respectively, in a total volume of 2.0 ml. After the incubation at 25 "C for 20 seconds the reaction was terminated by the addition of 2 ml of STB (standard buffer for enzyme preparation, containing 50 mM Tris-HCl buffer, pH 7.4, 1 mM NaN3, 1 mM EOTA, 1 mM glutathione, 0.5 mM dithiothreitol) containing 2 M KCl, and 600 pg poly(ADP-ribose) polymerase was added to the mixture. The sample was put on a sephadex G-200 column(250 ml, 2.5X50 cm) preequilibrated with STB containing 1 M KC1 and eluted with the same buffer. Poly(ADP-ribose) polymerase activity and acid-insoluble radioactivity were measured and indicated by black and white columns, respectively. The position of molecular markers were determined by a parallel run of the column and indiDNA was measured by the absorbance at 260 nm and indicated cated by arrows. by a dotted line.

one obtained chase

from

experiment,

by a paper in Table as 5'-AMP

20 set-incubation were

treated

chromatography 1, about

and phosphoribosyl-AMP of AOP-ribose

After

the chase,

pare

the counts in the

in 5'-AMP

and phosphoribosyl-AMP, results

ADP-ribose indicate

that

of the oligo(ADP-ribose). duct,

calculated

of product

described in 5'-AMP

A and B in Table

of phosphoribosyl-AMP.

are

residues

a structure

known to be derived

of poly(ADP-ribose),

was recovered

indicating

the product

by other with

decreased(com-

a concomitant

The two compounds, from

the AMP-terminal

respectively(29).

is

investigators

is remarkably l),

the

As summarized

of each sample

the digestion,

from

and analyzed

to Yamada and Sugimura(38).

the radioactivity

radioactivity

NAD+ and the other

venom phosphodiesterase

radioactivity after

with

radioactive

snake

according

(4-6).

other

with

98% of the total

a homopolymer

increase

with

5'-AMP

and the Therefore,

the

the addition of ADP-ribose is occurring to the AMP-terminal The average chain length of the early reaction pro-

by the method

of Fujimura

1284

and Sugimura(39),

was 2.6.

This

BIOCHEMICAL

Vol. 78, No. 4, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20

IO

30

40

SO

FRACTION AUllBEA

gel electrophoresis of early reaction products. Fig. 2. SDS-polyacrylamide Poly(ADP-ribose) polymerase was incubated in two tubes( tube A and B) in the legend to Fig. 1. except that the incubation condition as described in the of tube B was continued for additional 1 min after 0.5 pmole of cold NAD was added to the initial reaction mixture at 20 set, while the incubation of tube terminated by the addition of A was terminated at 20 sec. The reaction was Acid-insoluble material was collected by 0.5 ml of 40% trichloroacetic acid. centrifuging the sample at 40,000 g for 20 min, washed twice with 2 ml portion of ethanol/l50 mM Tris-HCl buffer(pH 7.4); Z/l(v/v) and dissolved in 0.2 ml of 1% SDS, 25% glycerol, 10 mM sodium phosphate buffer(pH 7.2). After storage at 0°C overnight, 50 ~'1 of 0.05% bromophenol blue was added to the mixtures on SDS-polyacryland two 50 ~1 aliquots of each sample were analyzed One of the two gels from each samamide gel columns as described in methods. ple was cut into 1.5 mm thickness and the radioactivity was measured. The other one was stained with amido black. Open and closed circles indicate the radioactivity of product A and B, respectively. A schematic illustration of the stained gels is shown on the top of this figure.

length

of oligo(ADP-ribose)

is not

acid-precipitable

without

binding

to other

macromolecules. All

these

of the the

results

reaction

is

precipitation

sephadex

fect

G-ZOO in the

bound

the oligo(AOP-ribose) to the

enzyme.

8% trichloroacetic presence

at 20-22°C.

the stability

(ADP-ribose)

that

too.

gel

A treatment These

to the enzyme Okayama --et al.

acid,

formed

The binding during

results

column of the strongly

in the product suggest

in an early was stable

buffer, presence with

step

during

the chromatography

of 1 M KC1 in the elution

on a polyacrylamide

hours

Recently

tightly with

electrophoresis several

indicate

on

or during

the

of 0.1% SDS for

5 M urea

the binding

did

not

af-

of oligo-

is covalent. (30 ) reported

that

1285

when the react ion product

of a poly

Vol. 78, No. 4, 1977

Table

1.

BIOCHEMICAL

Hydrolysis diesterase.

of early

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reaction

products

with

total radioactivity analyzed(cpm)

phosphoribosylAMP(cpm)

A

102,000

61,800

38,300

B

147,000

141,000

4,200

product

snake

5'-AMP(cpm)

venom phospho-

average chain length(ADP-ribose unit) 2.6 ---

Product A and B were prepared under the conditions described in the legend ta Fig. 1 and Fig. 2. The acid-insoluble materials were dissolved in 0.2 ml of 0.7 N NaOH, incubated at 25'C for 30 min and neutralized by the addition of 20 ~1 of N HCJ. To each sample, 20 ~1 of M Tris-HCl buffer, pH 8.0, 2 ~1 of M MgCJ2, 10 ~1 of 10 mM 5'-AMP, and 20 pg of snake venom phosphodiesterase were added and the mixtures were incubated at 37°C. After the incubation for 1 hour, additional 30 yg of snake venom phosphodiesterase was added to each sample, and the mixtures were incubated further for two hours. Radioactive phosphoribosyl-AMP and 5'-AMP in the digested products were separated by a paper chromatography according to Yamada and Sugimura(38). Product A; early reaction product, product B; early reaction product chased with cold NAD.

(ADP-ribose) amide

polymerase

gel

electrophoresis,

associated they

with

presumed

on the gel

however,

(ADP-ribose) nature

remain early

(data

is not

reaction

product

lated

from

ratio

was about

0.1. before.

is directly

5'-AMP

and furthermore,

the average

pronase

or

the differences

bound

to the

acceptor

reaction

in table

found

that oligoenzyme,

with

non-

of the

reaction

when a small

amount

of the

chain

1286

was quite

limited

enzyme was incu-

a relatively long time, chain exceeded far that length(12-13

was calcu-

product

1, the product-enzyme

extent

bated in the standard reaction mixture for number of the synthesized poly(ADP-ribose) me,

stable of the

by trypsin

Though

a macromolecular

the observed

However,

fairly

our results suggest

of the earJy

of the terminal Thus

is

complex.

of oJigo(ADP-ribose)

the counts

one as expected

that

in this

fraction

proteinases.

electrophoresis

hydrolyzed

yet,

which

of this with

complex gel

to be

nature,

The mobility

shown).

to be cJarified

was found

of the product

was easily

by SDS-polyacryl

non-protein

upon a SDS-polyacrylamide

is involved

When the number

with

of ADP-ribose.

at least the possibility

excluding protein

in our

was analyzed

oligo(ADP-ribose)

by the treatment

material

two systems

liver

material

the complex

an acid-soluble

rat

the enzyme-oJigo(ADP-ribose)

as a unit

In addition,

in these

unidentified

to be an acceptor

and separable product.

from

the synthesized

a still

was not affected

In our system,

into

preparation

ADP-ribose

the total of the enunits)

remain-

0lOCHEMlCAl

Vol. 78, No. 4, 1977

ed almost crease

constant

during

in the total

The polymer

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

incubation

acid-insoluble

synthesized

counts

during

these

enzyme when the product

was analyzed

(unpublished

results).

Thus,

is

to some extent,

increased

new chain

the formation

the reaction,

is

that

a poly(ADP-ribose)

length

gel

from

the

electrophoresis

of the enzyme-bound the chain

polymer

and reinitiate

a

is

units

complex

protein

from

synthesized we do not

units

are

by a sequential, site

to the

have enough

step

the

intramolecular acceptor

site

evidences

of

enzyme

transferred The other

two enzyme molecules.

the catalytic

At present,

the early

The one is that

and ADP-ribose

between

in

one trans-

in the same

to distinguish

two possibilities.

As for dences

the role

of histones

have indicated nuclei

we could

that

or chromatin

direct

effect and the

as stated

above.

still

clarified.

not

A possibility these

(ADP-ribose)

they

synthesis

With

modifications of this

purified

abolished found

referred

purified

is that

enzyme, added

enzyme reaction in some conditions

in these

evi-

two sets

some factor

of however, The

histones.

seemed to be inof the

reaction,

of observations indispensable

is for

the

may be missing from our enzyme preparation Kristensen and Holtlund reported manuscript,

of poly(ADP-ribose) also

our

accumulated

on an incubation

of exogenously

on the

The discrepancy

purification in which

NAD*(7-10).

was almost

synthesis,

are ADP-ribosylated

ADP-ribosylation

the preparation

a partial

histones

of histones effect

enzyme to perform During

in poly(ADP-ribose)

with

not demonstrate

stimulatory

cells,

chain

results).

was separated

by SDS-polyacrylamide

may be conceivable.

and intermolecularly

enzyme molecule.

cell

incubation

of oligo(ADP-ribose)-enzyme

as an acceptor

of ADP-ribose

these

long-term the

of the in-

incorporated(unpublished

the enzyme may release

two mechanisms

can serve

sequentially fer

after

5 to 120 min in spite

synthesis.

As for itself

from

polymerase

to a possibility

from

Ehrlich

of an acceptor-free

ascites

tumor poly-

by the enzyme preparation(40). ACKNOWLEDGEMENT

The authors express their gratitude to Dr. T. Kamiya for his continued interest and encouragement through this study. This work was supported in part by a research grant No 101032 from the Ministry of Japan. REFERENCES 1. Chambon,P., Weill,J.D., and Mandel,P.(1963) Biochem. Biophys. Res. Commun., 11, 39-43. 2. Nishizuka,Y., Ueda,K., Nakazawa,K., and Hayaishi,O.(1967) J. Biol. Chem., 242, 3164-3171. 3. Fujimura,S., Hasegawa,S., Shimizu,Y., and Sugimura,T.(1967) Biochim. Biophys. Acta, 145, 247-259. 4. Chambon,P., Weill,J.D., Strosser,M.T., and Mandel,P.(1966) Biochem.Biophys. Res. Commun., 25, 638-643. 5. Reeder,R.H., Ueda,K., Honjo,T., Nishizuka,Y., and Hayaishi,U.(T967) J. Biol. Chem., 242, 3172-3179.

1287

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6. Hasegawa,S., Fujimura,S., Shimizu,Y., and Sugimura,T.(1968) Biochim. Biophys. Acta, 149, 369-376. 7. Nishizuka,Y., Ueda,K., Honjo,T., and Hayaishi,0.(1968) J. Biol. Chem., 243, 3765-3767. 8. Otake,H., Miwa,M., Fujimura,S., and Sugimura,T.(1969) J. Biochem.(Tokyo), 65, 145-146. 9. Ord,M.G., and Stocken,L.A.(1977) Biochem.J., 161,583-592. 10. Tanuma,S., Emoto,T., and Yamada,Y.(1977) Biochem. Biophys. Res. Commun., 74, 599-605. 11. Roberts,J.H., Stark,P., Giri,C.P., and Smulson,M.(1975) Arch. Biochem. Biophys., 171, 305-315. 12. Ueda,K., Omachi,A., Kawaichi,M., and Hayaishi,0.(1975) Proc. Nat. Acad. Sci. USA, 72, 205-209. 13. Smulson,M., and Roberts,J.H.(1973) Poly(ADP-ribose), An Internat. Symp., Fogerty Internat. Center Proc., No 26, Harris,M., edt., ~~141-160. 14. Adamietz,P., Phillips,C., and Hilz,H.(1976) Proceedings of the 4th Internat. Symp. on "Poly(ADP-ribose) and ADP-ribosylation of Proteins", pp.3, Walter de Gruyter, Berlin. 15. Yoshihara,K., Tanigawa,Y., Burzio,L., and Koide,S.S.(1975) Proc. Nat. Acad. Sci. USA, 72, 289-293. 16. MUller,W.E.G., and Zahn,R.K.(1976) Mol. Cell.Biochem., 12, 147-158. 17. Burzio,L., and Koide,S.S.(1970) Biochem. Biophys. Res. Commun., 40, lOl31020. 18. Burzio,L., and Koide,S.S.(1971) Biochem. Biophys. Res. Commun., 42, 11851190. 19. Yoshihara,K., and Koide,S.S.(1973) FEBS Letters, 35, 262-264. 20. Nagao,M., Yamada,Y., Miwa,M., and Sugimura,T.(1972) Biochem.Biophys. Res. Commun., 48, 219-225. and Smulson,M.(1973) Biochem. Biophys. Res. Commun., 21. Roberts,J.H., Stark,P., 52, 43-50. 22. Smulson,M., Henriksen,O., and R deau,C.(1971) Biochem. Biophys. Res. Commun., 43, 1266-1273. 23. Kidwell,W.R., and Burdette,K.E. 1974) Biochem. Biophys. Res. Commun., 61, 766-773. 24. Lehmann,A.R., Kirk-Bell,S., Sha l,S., and Whish,W.J.0.(1974) Exp. Cell Res., 83, 63-72. 25. Caplan,A.I., and Rosenberg,M.J. 1975) Proc. Nat. Acad. Sci. USA, 72, 18521857. 26. Yamada,M., Miwa,M., and Sugimura,T.(1971) Arch. Biochem. Biophys., 146, 579586. 27. Yoshihara,K.(1972) Biochem. Biophys. Res. Commun., 47, 119-125. 28. Yoshihara,K., and Koide,S.S.(1973) FEBS Letters, 30, 261-264. 29. Nishizuka,Y., Ueda,K., Yoshihara,K., Yamamura,H., Takeda,M., and Hayaishi,O. (1969) Cold Spr. Harb. Symp. on Quant. Biol., 34, pp.781-786. and Hayaishi,0.(1976) Proceedings of 30. Okayama,H., Edson,C.M., Fukushima,M., the 4th Internat. Symp. on "Poly(ADP-ribose) and ADP-ribosylation of Proteins", pp.1, Walter de Gruyter, Berlin. 31. Yoshihara,K., Hashida,T., Yoshihara,H., and Kamiya,T.(1976), ibid., pp.1. 32. Yoshihara,K., Hashida,T., Tanaka,Y., Ogushi,H., Yoshihara,H., and Kamiya, manuscrypt in preparation. T 33. L&y,O.H., Rosebrough,N.J., Farr,A.L., and Randall,R.J.(1951) J. Biol. Chem., 193, 265-275. 34. Burton,K.(l956} Biochem. J., 62, 315-323. 35. Yoshihara,K., and Koide,S.S.(1974) Biochem Biophys. Res. Commun.,59, 658665. 36. Hayashi,K., and Ohba,Y.(1972) Protein, Nucleic Acid and Enzyme, 17, 304-311. 37. Weber,K., and Osborn,M.(1969) J. Biol. Chem., 244, 4406-4412. Biochemistry, 12, 3303-3308. 38. Yamada,M., and Sugimura,T.(1973) 39. Fujimura,S., and Sugimura,T.(1971) Methods in Enzymology, 18 B, pp.223-224, Academic Press, New York. 40. Kristensen,T., and Holtlund,J.(1976) Eur. J. Biochem., 70, 441-446. 1288