The activity and properties of poly(adenosine diphosphate ribose) polymerase in vitro during the embryonic development of the South African clawed toad Xenopus laevis

DEVELOPMENTAL

BIOLOGY

i’2,254-265

(1979)

The Activity and Properties of Poly(Adenosine Diphosphate Ribose) Polymerase in Vitro during the Embryonic Development of the South African Clawed Toad Xenopus laevis FARZIN FARZANEH' AND COLIN K. PEARSON Department

of Biochemistry,

Received

University

October

of Aberdeen, Marischal United Kingdom

6, 1978; accepted

in revised

College,

form

Aberdeen

February

AB9

IAS,

Scotland,

26, 1979

Characterization of poly(ADPribose) polymerase in isolated nuclei of Xenopus laevis embryos shows that maximum activity in vitro occurs at 25°C in 10 mM Tris-HCl buffer, pH 8.0, containing 20 mM MgC12, 1 mM dithiothreitol, and 3 mM NaF. Under these conditions the apparent K, for NAD’ was 0.125 m&f. The activity of the polymerase during embryogenesis was measured using both a whole embryo extract and isolated embryonic nuclei. Both of these sources of enzyme demonstrated an increase of approximately eightfold in the activity per cell, between early cleavage (stages 2-4; Nieuwkoop and Faber, 1956, “Normal Table of Xenopus Zaevis (Daudin),” NorthHolland, Amsterdam) and late neurula (stages 23-24). From late neurula to early tadpole (stages 38-39) the activity of the extracted enzyme, calculated per cell, decreased by 64%. During the same period the activity of the enzyme in isolated nuclei increased by 40% to reach its maximum activity in early tail bud (stages 27-28), and thereafter decreased by 23%. These results indicate a possible involvement of poly(ADPribose) polymerase in these embryos in the cell differentiation processes, rather than in cell proliferation.

processes are sequentially well separated (Gurdon, 1974; Deuchar, 1972; Benbow et al., 1975). Therefore, in an attempt to correlate the activity of this enzyme with any one or more of such processes the in vitro activity of the polymerase during embryogenesis was studied.

INTRODUCTION

The eukaryotic enzyme poly(ADPribose) polymerase catalyzes the formation of a homopolymer of ADPribose linked together by l’-2’ glycosidic bonds. NAD’ is the specific substrate for this reaction and is cleaved into nicotinamide and ADPribose, units of which are then added successively onto an initial ADPribose unit, reported to be covalently bound to nuclear proteins including both histones and nonhistones. The biological function of the polymerase and of the polymer it synthesizes in unknown, although a number of proposals suggest a role in the regulation of eukaryotic DNA replication, DNA repair, cell division, and differentiation (see Hilz and Stone (1976) and Hayaishi and Ueda (1977) for reviews). During the embryonic development of Xenopus laevis many of these ’ Present address: Biochemistry Laboratory (CRPC), School of Biological Sciences, University Sussex, Brighton, Sussex, United Kingdom.

MATERIALS

METHODS

Materials All reagents used were AnalaR grade or the most pure available. (NH&SO+ cysteine hydrochloride, MgC12, NAD’, NaH2P04, trichloroacetic acid, and toluene (scintillation grade) were obtained from BDH Chemicals Ltd., Poole, Dorset. DLdithiothreitol, DNA (highly polymerized calf thymus, type I), deoxyribonuclease I, histones (calf thymus, type II AS), 2-mercaptoethanol (type I), micrococcal nuclease (grade VI), Pronase (type V), ribonuclease (type I-A), snake venom phosphodiesterase (type VII), spleen phosphodiesterase (type

of 254

0012-1606/79/100254-12$02.00/O Copyright 0 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.

AND

FARZANEH

AND PEARSON

Poly(ADPribose)

I), and tris(hydroxymethyl)aminomethane were from Sigma Chemical Company Ltd., Kingston-upon-Thames, Surrey. [14C]NAD, nicotinamide [U-14C]adenine dinucleotide, ammonium salt (300 mCi/ mmole, 25 &i/ml), was purchased from the Radiochemical Centre, Amersham, Buckinghamshire. Scintillation fluors PPO and POPOP were from Koch-Light Laboratories, Buckinghamshire. Glass-fiber filters (GF/C) were obtained from Whatman Ltd., London. Polyethyleneimine-cellulose sheets (PEIcellulose) were from Brinkmann Instruments, New York. Adult Xenopus laevis and chorionic gonadotropin were purchased from Harris’s Biological Supplies Ltd. Buffers Citric acid-NazHP04. Ten-milliliter portions of 50 mM citric acid were titrated with the required amount of 50 mM Na2HP04 to obtain solutions in the pH range 3.0 to 6.5. Glycine-NaOH. Ten-milliliter portions of 50 mM glycine were titrated with 50 mM NaOH to obtain buffer solutions in the pH range 8.5 to 10.0. Universal buffer. Ten-milliliter portions of a solution containing 26.27 mg of citric acid, 17.01 mg KH2P04, 7.73 mg H3B03 and 23.03 mg of diethylbarbituric acid were titrated with 50 mM NaOH to obtain solutions in the pH range 5.0 to 11.0. Tris-HCZ. Trizma base 5 to 50 mM was titrated to the required pH with HCl. Isolation

of Nuclei

Production of embryos and the isolation of nuclei were performed as previously described by the authors (Farzaneh and Pearson, 1978a). Extraction ase from

of Poly(ADPribose) Whole Embryos

Polymer-

Dejellied embryos of stages 2 to 39 (Nieuwkoop and Faber, 1956) were homogenized (20 embryos/ml) in 200 mM sodium

Polymerase

in Xenopus

Embryos

255

phosphate buffer, pH 8.0, containing 2 mM 2-mercaptoethanol and 2 mM MgC12, at 2000 rpm using 10 strokes of a tightly fitting Teflon pestle powered by a T&R-Stir-R motor. After standing in ice for 30 min with occasional stirring, homogenates were centrifuged at lOO,OOOg,, (27,000 rpm) for 1 hr at 2°C in a 6 X 5.5~ml swing-out rotor in an MSE 65 Superspeed ultracentrifuge. The yolk and lipids which collected at the surface were removed by absorbing them to tissue paper; the clear supernatants were removed and the pellets reextracted. Less than 2% of the poly(ADPribose) polymerase activity remained in the final pellet. Solid (NH&S04 was added to the combined supernatants to a final concentration of 20% (w/v). After 30 min on ice the precipitated proteins, including all of the detectable poly(ADPribose) polymerase, were pelleted by centrifuging for 15 min at SOOOg,, in a 12 x 1.5~ml fixed-angle rotor in the Eppendorf5412 centrifuge. Supernatants were discarded and the pellets were washed with 20% (w/v) (NH4)&S04. The pellets were finally dissolved in 1.0 ml of 25 mM Tris-HCl, pH 8.0 at 25’C, containing 20 mM MgClz and 1.0 mM dithiothreitol. The concentrations of sodium phosphate and (NH4)2S04 used are not those which enable the polymerase of the highest specific activity to be obtained, but were chosen because they allow a near quantitative recovery of the enzyme from embryos. Measurement of Poly(ADPribose) erase Activity

Polym-

Reactions were initiated by adding 0.5 to 1.0 X lo5 nuclei or 50 ~1 of the extracted poly(ADPribose) polymerase to a mixture which contained, in a final volume of 200 ~1, 10 mM Tris-HCl, pH 8.0 at 25°C 20 mM MgCh, 3 mM NaF, 1.0 mM dithiothreitol, and 0.83 PM nicotinamide ,[U-‘“Cladenine dinucleotide (300 mCi/mmole), except for the experiment shown in Table 2 where the NAD+ was at 1 mM. They were terminated at required times by adding 400 ~1 of ice-cold 10% (w/v) trichloroacetic acid

256

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containing 1% (w/v) nicotinamide, a competitive inhibitor of poly(ADPribose) polymerase, and 2% (w/v) neutralized tetrasodium pyrophosphate to reduce the control values by competing with NAD’ for NAD-binding proteins. Controls were obtained by terminating reactions immediately after adding nuclei or the extracted enzyme. Samples were left on ice for 45-90 min to precipitate the acid-insoluble material which was then collected on glass-fiber disks (GF/C, 2.5 cm diam). These were washed with liberal quantities of the acid solution, followed by 95% (v/v) ethanol. The filters were then dried and counted for radioactivity using a toluene-based scintillation fluid (Pearson et al., 1976). In assays of the polymerase in the presence of exogenous DNA and acceptor proteins, 15 pg of highly polymerized calf thymus DNA (stimulator of the polymerase) and 15 pg of calf thymus hi&ones (poly(ADPribose) acceptor proteins) were added to the incubation medium described above. One unit of poly(ADPribose) polymerase activity is defined as 1 pmole of ADPribose incorporated per minute.

Identification

of Poly(ADPribose)

The acid-insoluble material obtained after incubating nuclei with [14C]NAD’ was collected by centrifuging samples at 0-4°C for 5 min at SOOOg,, in a 12 x 1.5-ml fixedangle rotor in an Eppendorf-5412 centrifuge. The pellets were washed twice with the trichloroacetic acid solution and twice with ethanol (as described above; see polymerase assays) to remove unreacted [14C]NADf. The washed pellets were then suspended (final volume 200 ~1) in 50 mM Tris-HCl, pH 6.8 at 37”C, containing 20 mM MgCL and the appropriate amount of specified enzyme as follows: 50 pg of deoxyribonuclease I, 50 pg of ribonuclease A, 0.1 units of micrococcal nuclease, 50 pg of spleen phosphodiesterase, 50 pg of snake venom phosphodiesterase, and 50 pg of Pronase. Mixtures were incubated at 37°C for

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1 hr, and the reaction was then terminated by adding 400 ~1 of ice-cold 10% (w/v) trichloroacetic acid solution. After 30 min on ice the acid-precipitable material remaining was collected onto filter disks and subsequently counted for radioactivity as described above. In one set of experiments the snake venom phosphodiesterase-treated samples were digested with 50 pg of Pronase for another hour before terminating the reactions (see Table 1). The acid-soluble products of the snake venom phosphodiesterase digestions were analyzed by chromatography on polyethyleneimine cellulose thin layers (Farzaneh and Pearson, 1978b; Stone et al., 1973). RESULTS

Optimum Conditions for Poly(ADPribose) Polymerase Activity Figures 1 and 2 show the effects of incubation temperature, pH, type of buffer, and buffer concentration on the polymerase acTABLE

1

ENZYMIC DIGESTION OF THE ACID-INSOLUBLE MATERIAL PRODUCED AFTER INCUBATING ISOLATED NUCLEI WITH [‘%]NAD+” Enzyme

Control Deoxyribonuclease I Ribonuclease A Micrococcal nuclease Pronase Spleen phosphodiesterase Snake venom phosphodiesterase Snake venom phosphodiesterase followed by Pronase

Acid-insoluble radioactivity remaining after enzyme digestion (cm)

Proportion of acid-insoluble radioactivity digested @J)

7525 7773 7535 7403 2068 7598 2805

Nil Nil Nil 2 72 Nil 63

308

96

“For this experiment nuclei were obtained from stage 27-28 embryos. Similar results were obtained using isolated nuclei or the extracted enzymes at developmental stages 13 to 36. Conditions of incubation are described under Materials and Methods.

FARZANEH

AND PEARSON

Poly(ADPribose)

Polymerase

in Xenopus

257

Embryos

1975; Stone et al., 1973), we examined the effect of these on our enzyme activity (Fig. 4). Although there was a 16% increase in the polymerase activity with 3 mM NaF, the presence of (NH&SO, clearly inhibited activity at all concentrations examined and therefore was excluded from subsequent assays. Poly(ADPribose) polymerase assays were thus carried out at 25°C in 10 mM Tris-HCl buffer, pH 8.0, containing 20 mM MgC12, 1 mM dithiothreitol, and 3 mM NaF. Kinetics

0

20 Time

40 (mid

60

FIG. 1. The effect of temperature on the activity of poly(ADPribose) polymerase in isolated nuclei of stage 19 embryos. Details of the incubation are given under Materials and Methods. Throughout we have defined a unit as 1 pmole of ADPribose incorporated per minute.

tivity. The enzyme also required divalent cations, MgClz at 20 mM for maximum activity. This activity was reduced by 95% in the presence of 100 mM MgClz (Fig. 3). The inclusion in the incubation medium of the thiol-group protecting agent dithiothreitol at 1 mM promoted an increase in polymerase activity of about 18%; at higher concentrations, up to 10 mM, the stimulation was less pronounced. Dithiothreitol has been shown to have a greater stimulatory effect on poly(ADPribose) polymerase activity in nuclei from rat liver (Chambon et al., 1963), mouse fibroblasts, and Physarum polycephalum (Shall et al., 1972). The relatively mild response of the Xenopus nuclei was possibly due to 2-mercaptoethanol carried over from the nuclear isolation solutions. Since NaF and (NH&SO4 are reported to inhibit phosphodiesterase and glycohydrolase activity, respectively (Miwa et al.,

of NAD’

Incorporation

The initial rate of poly(ADPribose) synthesis in isolated nuclei was measured at different concentrations of added substrate NAD’. The saturating NAD’ concentration was 0.6-0.8 mM and the apparent K,,, determined from a double-reciprocal plot was 0.125 mM (Fig. 5). Identification

of the Reaction

Product

The acid-insoluble material obtained by incubating the isolated nuclei or extracted enzyme with [14C]NAD+ was treated with different degradative enzymes (Table 1). Only snake venom phosphodiesterase and Pronase rendered the incorporated radioactivity acid soluble. The acid-soluble products of the phosophodiesterase digestion were analyzed by polyethyleneimine-cellulose thin-layer chromatography and only the two characteristic products 5’-AMP and 2’-(5”-phosphoribosyl)-5’-AMP were detected. We interpret these results as showing that the isotopically labeled acidinsoluble material was exclusively (ADPrihose),. The incomplete release of acid-insoluble radioactivity by snake venom phosphodiesterase (see Table 1) could be due to the inaccessibility of the bonds attaching the ADPribose units to acceptor proteins (Sugimura, 1973). The radioactivity rendered acid soluble by Pronase was probably due to the released poly(ADPribose) chains too short to be acid insoluble when freed

258

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I 20 C TRIS-HCI

PH

1 30

I 40

I 50

ICmM)

2. Effect of pH, buffer, and buffer concentration on poly(ADPribose) polymerase activity in isolated nuclei. All buffers in (A) were at 50 mM and included Tris-HCl (O), citric acid-Na2HP04 (0), universal buffer (A), and glycine-NaOH (a). Incubations were for 10 min at 25°C and other conditions were as described for Fig. 1. In experiment (B) the pH was 8.0 throughout. FIG.

from the acceptor proteins (Fujimura and Sugimura, 1971). The radioactive acid-insoluble materials produced by incubating nuclei isolated from embryos at stages 13, 16, 18, 20, 22, 28, and 36 were examined as described above and in all cases were exclusively (greater than 98%, within the limits of the techniques used) composed of ADPribose units (Farzaneh and Pearson, 1977).

Poly(ADPribose) ing Embryonic

.F 2

Magnesium

chloride

CmM)

FIG. 3. Effect of MgCl, concentration on the activity of poly(ADPribose) polymerase in isolated nuclei of stage 19 embryos. Incubations were for 10 min at 25°C.

Polymerase Activity Development

dur-

When poly(ADPribose) polymerase activity was measured in the nuclei isolated from embryos at different stages of development we found that both the initial rate of the reaction and the total amount of poly(ADPribose) synthesized increased up to developmental stage 28 and decreased thereafter (Fig. 6). The initial enzyme activity was 0.15 units (see Materials and Methods) per lo5 nuclei at developmental stages

FARZANEH

AND

PEARSON

4. Effect of sodium polymerase in isolated nuclei

fluoride of stage

Poly(ADPribose)

Ammonium FIG.

Polymerase

sulphate

Embryos

259

CmM)

(A), and ammonium sulfate 19 embryos. Incubations were

2-4 (7.5 times greater than the activity in isolated nuclei from adult Xenopus liver) and this increased to 1.7 units per lo5 nuclei at stages 27-28 (85 times higher than the activity of adult Xenopus liver nuclei). At developmental stages later than 28 the enzyme activity decreased to 1.28 units per lo5 nuclei (stages 38-39).

in Xenopus

(B), on the activity for 10 min at 25°C.

of poly(ADPribose)

When the activity of poly(ADPribose) polymerase extracted from whole embryos was measured, no similar decrease in activity per embryo occurred during the later stages of development (Fig. 7). However, when the results obtained from extracted embryos were used to calculate the activity per cell (values for the number of cells per

260

DEVELOPMENTALBIOLOGY

-40

FIG. 5. Double-reciprocal activity in isolated nuclei. for 10 min at 25°C.

0

40

80

120

VOLUME 72,1979

160

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240

plot of the effect of substrate NAD’ concentration on poly(ADPribose) polymerase The source of the enzyme was 5 x IO” nuclei from stage 19 embryos. Incubations were

embryo at a given stage of development were taken from the published data of Woodland and Gurdon (1968) and Dawid (1965)) the computed initial rate of enzyme activity did show a similar increase in the activity during early embryogenesis and a comparable decrease in the activity during the stages of development between lateneurula and early-tadpole stages (Fig. 8, broken line). Only at developmental stages 28 and later did we detect a small loss of incorporated radioactivity with increasing incubation time (up to 80 min), which shows that the activity of poly(ADPribose)-degrading enzymes, such as poly(ADPribose) glycohydrolase, is very low in this tissue, at least during the developmental period studied (Farzaneh and Pearson, 1977).

Possible Explanations for the Observed Changes in Poly(ADPribose) Polymerase Activity during Development The observed changes poly(ADPribose) polymerase opment could result from an amount of enzyme protein creased enzyme activity as

in activity of during develincrease in the and/or an ina consequence

of changes in putative activator and/or inhibitor concentrations, including that of substrate NAD+. Others report that damage to DNA can result in a stimulation of this enzyme activity in some isolated nuclei (Hilz and Kittler, 1971; Roberts et al., 1975), but we think this is unlikely to account for our results with Xenopus nuclei since we obtained similar results after damaging the nuclei (and presumably the DNA) by sonication for 1 min (see Table 2). Alternatively, the increased availability of new protein acceptor sites for poly(ADPribose), as embryonic development proceeds, would be expected to lead to an apparent increase in polymerase activity. We observed, however, no further change in the enzymic activity of the nuclei (105/assay, disrupted by sonication), isolated from embryos at stages 8 to 37, when we added 15 ,ug of highly polymerized calf thymus DNA (as a stimulator of the polymerase) and 15 pg of the calf thymus histones (as putative acceptor proteins); see Table 2. Similarly, since the activity of the extracted enzyme was analyzed in the presence of a vast excess of exogenous histones and DNA, an increased availability of acceptor sites presumably

FARZANEH

AND

PEARSON

Poly(ADPribose)

25-

32-33

Polymerase

in Xenopus

261

Embryos

(ADPribose), units. Therefore, the rate of incorporation of 14C into acid-insoluble material was taken as a measure of poly(ADPribose) polymerase activity. The optimum temperature for the polymerase activity was found to be 25°C which is slightly higher than the temperature (22-24°C) required for the most efficient growth and development of the embryos. This contrasts with the optimum temperature reported for the in vitro activity of other poly(ADPribose) polymerases, which are optimally active at 5-15°C below the physiological temperature of the organism (Brightwell et al., 1975; Gill, 1972). However, since the measurable synthesis of poly(ADPribose) is the net result of the

rfY/ 13-14 -

23-25

16-18

’ 7

T ._

10

Time

20 (mid

30

40

FIG. 6. Poly(ADPribose) polymerase activity in nuclei isolated from embryos at different stages of development (numbers adjacent to the lines in the figure). Absolute values varied in four experiments but relative enzyme activities were constant as shown.

> $ ‘z 5 ;a

3

could not account for the increase in the activity of the enzyme during development. DISCUSSION

4 6 a” z

This study describes some fundamental properties of poly(ADPribose) polymerase in nuclei isolated from developing embryos of Xenopus laevis. Since the acid-insoluble material obtained by incubating isolated nuclei, or extracted enzyme, from embryos at different stages of development, with [14C]NAD’ was graded only by snake venom phosphodiesterase, and the products of digestion were recovered exclusively as 5’-AMP and 2’-(5”phosphoribosyb-5’-AMP, we presume that the incorporated acid-insoluble material at all stages of development was composed of

4

00 $ E

0.

0 0

5

10 Time (mid

5

20

FIG. 7. Poly(ADPribose) polymerase extracted from whole embryos at the developmental stages shown (numbers adjacent to each line) was assayed in the presence of calf thymus histones and DNA (details under Materials and Methods). Although the absolute values varied in six different experiments the relative enzyme activities at different stages of development were always as shown.

DEVELOPMENTAL

BIOLOGY

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2.5-

72 .c : ” > .Y .; M

2.0-

1.5 -

a, m” z

E Z 0 P m

l.O-

i YE L pd

0.5-

4

Developmental

1 0

I lo

stage

I

I

I

I

I

20

30

40

50

60

Embryonic

age

CM

FIG. 8. Initial rate of poly(ADPribose) polymerase activity during embryonic development. The figure shows the initial enzyme activity expressed as units/embryo (A) and units/IO5 nuclei (0). The activities of poly(ADPribose) polymerase extracted from whole embryos are also expressed per lo5 cells (broken line; values for the number of cells/embryo at different development stages were taken from Woodland and Gurdon (1968) and Dawid (1965). The poly(ADPribose) polymerase activity of isolated adult Xenopus liver nuclei was 0.02 pmole per minute per IO5 nuclei.

polymerase activity, which catalyzes polymer synthesis, and glycohydrolase activity, which catalyzes its degradation, the apparently atypical temperature optimum for the Xenopus enzyme may be due to the very low activity of any degradative enzyme (Burzio and Koide, 1970; also see Results). On the other hand, the diminished activity of the enzyme at temperatures above the physiological temperature (e.g., 37”C, Fig. 1) could be due to its instability under such conditions, or to a disproportionate activation of the degradative enzymes. The enzyme exhibited a single optimum at pH 8.0 in Tris-HCl buffer, two optima

(pH 7.5 and 9.5) in universal buffer, and a single optimum at pH 9.5 in glycine-NaOH. Satisfactory explanations of these findings are likely to be complex, although they may be due in part to the presence of more than one polymerase enzyme. However, since most poly(ADPribose) was synthesized using Tris-HCl, pH 8.0, this buffer was used routinely. The optimum Tris-HCl concentration was 10 m&f, and either lower or higher concentrations of the buffer reduced the activity of the enzyme, presumably due to inadequate buffering capacity and excess ionic strength, respectively. The enzyme

FARZANEH

AND PEARSON TABLE

Poly(ADPribose)

2

EFFECT OF SONICATION AND EXOGENOUS DNA AND HISTONES ON ACTIVITY OF PoLY(ADP-RIBOSE) POLYMERASE IN ISOLATED EMBRYONIC NUCLEI” Developmental stage

Picomoles ADPribose incorporated/min/ld nuclei Intac;lnu-

8-9 14-15 24-26 28-30 36-37

26 68 108 165 126

Sonicated nuclei

33 69 95 186 132

Sonicated nuclei plus DNA and histones 33 74 98 178 128

n Assays were for 10 min and contained NAD+ at 1 mM; other conditions were as described under Materials and Methods. Nuclei were sonicated at 0°C by six bursts each of lo-set duration using an Artek sonic dismembrator operating at power setting 60. In incubates containing histones and DNA 15 pg of each were added per assay tube.

also required 20 mM MgClz for maximum activity, but at high concentrations it diminished the activity of the enzyme (Fig. 3)) probably due to the raised ionic strength and/or the precipitation of chromatin at high Mg2’ concentrations. An increase of 16% in the activity of the enzyme in the presence of 3 mM NaF suggests the presence of phosphodiesterase activity in the nuclear preparations. NaF was therefore used routinely in order to avoid any influence of this on the measured activity of poly(ADPribose) polymerase. Using the isolated nuclei to measure kinetic characteristics of the polymerase the apparent K,,, for NAD+ was 0.125 mM. Other reported K, values for this enzyme include 0.04 mM in isolated quail oviduct nuclei (Miiller and Zahn, 1976) and 1.5 mM in LS-cell nuclei (Stone and Shall, 1973). We routinely used 0.83 pM NAD+ in polymerase assays for the sake of economy. During the embryonic development between stages 2-4 (early cleavage) and stages 23-24 (late neurula) there was an increase of approximately eightfold in the activity of the enzyme from 0.15 units/lo5 nuclei at early cleavage to 1.2 units/lo5 nuclei at late

Polymerase

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Embryos

263

neurula (Fig. 8). During this period the number of cells in the embryo increases some 6000-fold from 16 to approximately 100,000 (Woodland and Gurdon, 1968; Dawid, 1965). This represents an increase of 50,000-fold in the activity of the enzyme per embryo. From late neurula to early tadpole (stages 38-39) the activity of the extracted enzyme, calculated per cell, decreased by 64%. During the same period the activity of the enzyme in isolated nuclei increased by 40% to reach its maximum activity in early tail bud (stages 27-28)) and thereafter decreased by 23%. The difference in values between the activity of the enzyme in isolated nuclei and that calculated from the activity of the extracted enzyme (per cell) is probably due to the error inherent in the values used for the number of cells in embryos at different stages of development, although other explanations are possible since the extracted enzyme was obtained using only the most preliminary procedures for purification of proteins (see Materials and Methods). More important, however, is the difference between the activity of the enzyme in early and late embryogenesis. During the proliferation phase of growth, i.e., stages 2 to 10, a period during which the mitotic index can be as high as 50% at stage 5 (Landstr6m et al., 1975) with a cell cycle time of about 20 min (Graham and Morgan, 1966), the activity of poly(ADPribose) polymerase is low. From stage 10 onward when the cell cycle time is greatly increased together with the onset of major differentiation processes the enzyme activity rapidly increases. This suggests the involvement of poly(ADPribose) polymerase in differentiation rather than cell proliferation processes. In order to overcome possible criticism for using highly unsaturated NAD’ concentrations for measuring poly(ADPribose) polymerase activity we carried out one series of experiments with 1 mM NAD+. Table 2 shows that the use of this higher NAD’ concentration in the assay mixtures en-

264

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hanced the polymerase activity about BOfold, compared with that observed at 0.83 PM NAD+. However, the relative increase in the enzyme activity at each developmental stage was comparable to that obtained using only 0.83 pM NAD’ (shown in Fig. 6). The physiological role of poly(ADPribose) polymerase, at present, is far from clear and the published data suggest its involvement in the regulation of DNA synthesis, transcription, DNA repair, and cell division (see Hilz and Stone (1976) and Hayaishi and Ueda (1977) for reviews). More recently the involvement of this polymer in differentiation has also been implicated. For example, the differentiation of mesodermal cells of embryonic chick limb in vitro into cartilage, rather than muscle cells, is correlated with the increased net rate of poly(ADPribose) synthesis (Caplan and Rosenberg, 1975). Similarly Rastl and Swetly (1978) have reported an increase in the activity of poly(ADPribose) polymerase associated with the induction of differentiation in erythroleukemic mouse cells. Although no defined function can be assigned to the poly(ADPribose) polymerase based on the present studies we note that its activity is low during the proliferative growth phase (stages 2 to lo), and that after this time activity rapidly increases together with the onset of the major differentiation events. Also, if this increase in polymerase activity is a consequence of the change from a proliferative to a differentiative state of development it is then not too surprising that the activity declines after embryonic stage 28 when the differentiation processes are well under way. Finally, although these results apparently preclude an involvement of this enzyme in DNA synthesis and cell division we should point out that its activity at this time, stages 2-4, is nevertheless 7.5 times greater than it is in adult Xenopus liver nuclei. We thank the Medical Kingdom, for support,

Research and our

Councii, colleagues

United Miss

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72,1979

Grainne McDermott and Dr. Henry Furneaux for their advice and assistance. We would also like to thank Mr. Gwyn Williams at the Biochemistry Laboratory, School of Biological Sciences, University of Sussex, for his comments during the preparation of the manuscript. Finally, we acknowledge the interest and encouragement given to us throughout this work by Professor Sidney Shall, Biochemistry Laboratory, School of Biological Sciences, University of Sussex, and Dr. John McKenzie, Department of Developmental Biology, University of Aberdeen. REFERENCES BENBOW, R. M., PESTELL, R. Q. W., and FORD, C. C. (1975). Appearance of DNA polymerase activities during early development of Xenopus Zuevis. Develop. Biol. 43,159-174 BRIGHTWELL, M. O., LEECH, C. E., O’FARRELL, M. K., WHISH, W. J. D., and SHALL, S. (1975). Poly(adenosine diphosphate ribose) polymerase in Physarumpolycephalum. Biochem. J. 147,119-129. BURZIO, L. O., and KOIDE, S. S. (1970). A functional role of poly(ADPR) in DNA synthesis. B&hem. Biophys. Res. Commun. 40, 1013-1020. CAPLAN, A. I., and ROSENBERG, M. J. (1975). Interrelationship between poly(ADP-Rib) synthesis, intracellular NAD levels, and muscle or cartilage differentiation from mesodermal cells of embryonic chick limb. Proc. Nat. Acad. Sci. USA 72, 18521857. CHAMBON, P., WEIL, J. D., and MANDEL, P. (1963). Nicotinamide mononucleotide activation of a new DNA-dependent polyadenylic acid synthesising nuclear enzyme. Biochem. Biophys. Res. Commun. 11, 39-43. DAWID, I. B. (1965). Deoxyribonucleic acid in amphibian eggs. J. Mol. Biol. 12,581-599. DEUCHAR, E. M. (1972). Xenopus luevis and developmental biology. Biol. Reu. 47, 37-112. FARZANEH, F., and PEARSON, C. K. (1977). The activity of poly(adenosine diphosphate ribose)polymerase during the embryonic development of the South African clawed toad Xenopus laevis. Biothem. Sot. Trans. 5,733-734. FARZANEH, F., and PEARSON, C. K. (1978a). A method for isolating uncontaminated nuclei from all stages of developing Xenopus laevis embryos. J. Embryol. Exp. Morphol. 48, 101-108. FARZANEH, F., and PEARSON, C. K. (197813). Poly(adenosine diphosphate ribose) synthesis by isolated nuclei of Xenopus laevis embryos: In vitro elongation of in vivo synthesised chains. Biochem. Biophys. Res. Commun. 84,537-543. FUJIMURA, S., and SUGIMURA, T. (1971). Polymerization of the adenosine 5’-diphosphate-ribose moeity of NAD. In “Methods in Enzymology” (D. B. McCormick and L. D. Weight, eds.), Vol. ISb, pp. 223-230. Academic Press, New York. GRAHAM, C. F., and MORGAN, R. W. (1966). Changes

FARZANEH

AND PEARSON

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