Purification and Characterization of Poly(ADP-ribose) Synthetase from Calf Thymus*

Poly(ADP-ribose) synthetase calf thymus has been purified homogeneity a simple and rapid method with a recovery of 10 to 20%. The enzyme activity absolutely the of DNA. further stimulates the reaction.

Poly(ADP-ribose) synthetase has been partially purified from a variety of sources (15)(16)(17)(18)(19).  (21) have reported that the highly purified enzyme from the same source does require DNA absolutely for the reaction, but it migrates as two bands on gel electrophoresis.
In order to resolve the above discrepancy and obtain further insight into the mechanism of the poly(ADP-ribose) synthe- tase reaction, we have purified the enzyme to homogeneity with a rapid and simple method and determined the physicochemical properties as well as enzymological characteristics of the protein.
In this paper, evidence is presented that poly(ADP-ribose) synthetase is a simple globular protein composed of a single polypeptide of M, = 110,000 that absolutely requires the addition of DNA for the reaction.

EXPERIMENTAL PROCEDURES AND RESULTS
Details of the experimental procedures used and of the results are presented in the miniprint supplement which follows.' DISCUSSION We have purified calf thymus poly(ADP-ribose) synthetase to apparent homogeneity and characterized the molecular properties of the enzyme. These are summarized in Table I . The frictional coefficient ratio, f/f0 = 1.39, indicates that it is a globular protein with slight asymmetry. Since neutral sugar and phospholipid are not detected in the final enzyme preparation, the enzyme appears to be a simple protein.
The enzyme is a basic protein having a p1 value of 9.8. Amino acid composition in Table V   (-) and the latter by the curve-fitting (---), as shown in Fig. 3B.
the gel under the same conditions, indicating that the enzyme consists of a single polypeptide.
It is possible, however, that the purified enzyme has different mobilities on the gel under the above conditions when some residues of glutamine and asparagine in the enzyme protein have been converted into glutamic acid and aspartic acid during manipulation.
In this connection, Mandel et al. (20) recently reported that their enzyme preparation from calf thymus has a pI value of 6.5. In this case, however, their enzyme preparation appears to be an enzyme. DNA complex, thus exhibiting an acidic p1 since any procedure to remove DNA from the enzyme fraction is not used by their method and, in fact, their enzyme does not require the addition of DNA for full activity. In our purification procedures, separation of DNA from the enzyme fraction was accomplished by protamine sulfate treatment and thereafter, the enzyme always required DNA for the reaction.
The addition of histone stimulated the synthesis of poly(ADP-ribose) approximately 2-fold and resulted in an increase in the average chain length of the polymer without any increase in the number of sites for ADP-ribosylation. These results indicate that, in the purified enzyme system, histone acts as a kind of allosteric activator rather than acceptor for ADP-ribosylation as described by Okayama et al. (24) and Yoshihara el al. (25). Therefore, the acceptor for ADP-ribosylation in the purified system appears to be the enzyme itself or an endogenous molecule attached to or copurified with the enzyme protein. Then, the initial attachment of poly (ADP-ribose) to the acceptor and subsequent transfer of the polymer to histone may be catalyzed by two different enzymes. These problems, including definite determination of the sites for ADP-ribosylation in the purified system as well as those in the crude system, are now under investigation in our laboratory.