537th MEETING, CANTERBURY
695
PoPy(Adenosine Diphosphate Ribose) in Physarum polycephalum
WILLIAM J. D. WHISH, PETER R. STONE, M. K. O’FARRELL and
SYDNEY SHALL
Biochemistry Laboratory, University of Sussex, Falmer, Brighton BN1 9 QG, U.K.
The presence of poly(ADP-ribose) polymerase, a chromatin-bound enzyme, in
eukaryote nuclei is well documented (Chambon et al., 1966; Nishizuka et al., 1967:
Reeder et al., 1967; Fujimura et al., 1967). Poly(ADP-ribose) polymerase polymerizes
NAD+, with the loss of the nicotinamide moiety to form a polymer. The product is
covalently bound to a variety of nuclear proteins (Nishizuka et a[., 1969; Otake et al.,
1969). In Physarum polycephalurn approximately 50 % of the protein-bound polymer
is soluble in incubating buffer, which normally does not extract histone; 15 ”/, is extracted
with IM-CaCI,, which in this organism extracts histone (Shall et al., 1972).
We have developed two methods for the rapid determination of average polymer
chain-length in systems in vitro. Nuclei from P. polycephalum or mouse fibroblast (LS)
cells were incubated with various concentrations of r3H]NAD+ (1 ~ M - I mM) for
between 2 and 60min to give poly(ADP-ribose). The average chain-length of the
polymer was estimated and from these results the average rate of initiation and chain
extension could be calculated. The nuclei, after incubation with [3H]NAD+,were precipitated and washed with a 66% ethanol-0.1 M-sodium acetate mixture (pH5.4) until
free of ethanol-soluble radioactivity. The nuclear pellet was solubilized for 15min in
0.05 M-NaOH, neutralized with 0.05M-HCI,and digested with snake-venom phosphodiesterase. The digest was applied to a small column of Dowex 1 (formate form) and
sequentially eluted with 20ml batches of 0 . 0 3 ~ - 0, . 3 ~ and
- 3.0~-formicacid to elute
[3H]adenosine, [3H]AMP and 2’-(5”-[3H]phosphoribosyl)-5’-AMPrespectively.
The 0.03~-formicacid wash was a control for phosphatase activity that may have
been present in the snake-venom phosphodiesterase preparation, because phosphatase
activity would yield dephosphorylated AMP and phosphoribosyl-AMP and the products
would appear in this fraction. Rarely did any radioactivity appear in this fraction. The
batches were evaporated to dryness at 60°C, treated with Hyamine and counted in a
liquid-scintillation counter.
The [3H]AMP and 3H-labelled phosphoribosyl-AMP were also separated and
estimated by using polyethyleneimine-cellulose thin-layer chromatography. A LiCIacetic acid solvent was used to separate [3H]AMPand 3H-labelledphosphoribosyl-AMP.
Both methods were rapid and gave radioisotope recoveries of greater than 80%. The
chain length was calculated from the total counts recovered from the column or thin
layer (AMP+phosphoribosyl-AMP) divided by the AMP counts.
The concentration of NAD+ had little effect on the chain-length. The total amount
of polymer was proportional to the NAD+concentrationbetween 1 WM and 0.1 mM. These
results indicate that the initiationof new polymer was dependent onNAD+ concentration.
The chain length in Physarum was independent of incubation time [n = 3 in (ADPribose).]. In mouse L cells the chain-length increased from I I = 2 at 2min to n = 4
at 60min.
A relationship between nuclear NAD+metabolism, poly(ADP-ribose) polymerase and
DNA synthesis has been postulated. It would seem possible from the results presented
that control might be exerted through the number of poly(ADP-ribose) chains rather
than through their length. The number of chains might be related to the nuclear concentration of NAD+.
The methods were also applied to the qualitative detection of phosphoribosyl-AMP
derived from polymer synthesized in vivo. P. polycephaluwz was grown in submerged
culture with 20mCi of [32P]Pi.Nuclei were isolated and labelled with [3H]NADf under
standard conditions. All buffer-soluble radioactivity was removed from the nuclei, which
were then washed with acidic ethanol to remove any further soluble radioactivity. The
nuclear residue was then solubilized briefly with 0.01 M-NaOH, neutralized, incubated
with Pronase and reprecipitated with acidic ethanol at low temperature. This treatment
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BIOCHEMICAL SOCIETY TRANSACTIONS
released oligomeric phosphoribosyl-AMP into ethanol-soluble form. The ethanol was
removed by evaporation under reduced pressure. The resulting aqueous solution was
buffered at pH7.4 with Tris, and treated with snake-venom phosphodiesterase to give
32P- and 3H-labelled AMP and phosphoribosyl-AMP, which were both shown to be
present by the methods described above. Experiments with [3H]adenosine instead of
[32P]Pishowed similar results. In this case the isolated nuclei were not incubated with
[3H]NAD+.
The chain-lengths of protein-bound polymer from whole nuclei, buffer-soluble and
CaC12-solublefractions in vitro were estimated by using Dowex 1 (Cl- form) chromatography (Fujimura & Sugimura, 1971). The average chain lengths were about 5-7 units
in all three cases (O’Farrell, 1973). We believe that the chain length of the poly(ADPribose) synthesized in vivo in P. polycephalum is between 2 and 4 units.
It is possible that the ADP-ribosylation of nuclear protein in vitro may not be a true
reflection of the situation in vivo; perhaps in intact cells fewer, or only one, protein
serves as an acceptor.
Chambon, P., Weill, J. D., Doly, J., Strosser, M. T. & Mandel, P. (1966) Biochem. Biophys. Res.
Commun. 25, 638-643
Fujimura, S. & Sugimura, T. (1971) Methods Enzymol. 18B, 223-230
Fujimura, S., Hasegawa, S., Shimizu, Y. & Sugimura, T. (1967) Biochim. Biophys. Acta 145,
247-259
Nishizuka, Y., Ueda, K., Nakazawa, K. & Hayaishi, 0. (1967) J. Biol. Chem. 242,3164-3171
Nishizuka, Y., Ueda, K., Yoshihara, K., Yamamura, H., Takeda, M. & Hayaishi, 0. (1969)
Cold Spring Harbor Symp. Quant. Biol. 34, 781-786
O’Farrell, M. K. (1973) Ph.D. Thesis, University of Sussex
Otake, H., Muira, W., Fujimura, S. & Sugimura, T. (1969) J. Biochem. (Tokyo)65,145-146
Reeder, R. H., Ueda, K., Honjo, T., Nishizuka, Y. & Hayaishi, 0. (1967) J. Biol. Chem. 242,
3 172-3 179
Shall, S., Brightwell, M., O’Farrell, M. K., Stone, P. &Whish, W. J. D. (1972) Hoppe-Seyler’s
Z . Physiol. Cheni. 353, 843
Characterization of a Deoxyribonucleic Acid Polymerase from a Replicating
Nuclear System of the Synchronously Growing Physarumpolycephalum
W. SCHIEBEL and U. BAMBERG
Max-Plunck-Institut fur Biochemie, Abteilung fur Experimentelle Medizin,
8033 Martinsried, German Federal Republic
Jsolated nuclei of Physurum polycephalum, grown during part of the S phase on a
bromodeoxyuridine-containing medium, incorporate deoxyribonucleotides in vitro into
density-labelled DNA. Extraction of the nuclei in the presence of high salt concentrations yields a soluble fraction, which stimulates DNA synthesis. The enzyme activity
seems to be constant within the period comprising S and mid-G2 phase. Sucrosegradient centrifugation indicates a sedimentation coefficient of between 7 and 10s.
The Wibonucleic Acid- and Deoxyribonucleic Acid-Dependent Activities
of Rat Liver Nuclear Deoxyribonucleic Acid Polymerase
R. GITENDRA WICKREMASINGHE, ANDREW M. HOLMES and
IRVING R. JOHNSTON
Department of Biochemistry, University College London, Gower Street, London
W C l E 6BT, U.K.
Low-molecular-weight DNA-dependent DNA polymerases have been purified recently
from the cytoplasm and nuclei of several mammalian species. Besides being DNAdependent most of these enzymes can copy the polyribonucleotide strand of synthetic
RNA-DNA hybrids. Although Ross et al. (1971) showed the low-molecular-weight
DNA-dependent DNA polymerase of normal cells was able to use poly(A). poly(dT)
1973