Poly (ADP‐ribose) Polymerase from Ehrlich Ascites Tumor Cells

Abstract

Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells, partially purified by chromatography on DNA-agarose, was obtained as a more than 80 ' %, homogeneous preparation by isoelectric focusing in a sucrose gradient. The polymerase activity was shown to be associated with the major protein in the preparation. Results obtained by electrophoresis in the presence of sodium dodecylsulfate indicated that poly(ADP-ribose) polymerase consists of a polypeptide chain with a molecular weight of 130 000. Ultracentrifugation at non-denaturating conditions indicated that the active enzyme may be an oligomeric form of this polypeptide chain. The isoelectric point of the polymerase was 9.40. The effects of various additions to the assay mixture on the synthesis of poly(ADPribose) as well as some kinetic data, are given. It is shown that poly(ADP-ribose) is a highly efficient inhibitor of its own synthesis, and results are presented which suggest that the well-known stimulatory effect of DNA on the synthesis is due to reduction of this inhibitory effect of the product.Eukaryotic cell nuclei contain an enzyme which incorporates the ADP-ribose moiety of NAD into poly(ADP-ribose) [l -31. Poly(ADP-ribose) of various sizes, with chain length varying from 1-40, is bound to nuclear proteins, both histones and non-histones [4,5]. Although the biological role of the polymer is not yet understood, its involvement in the control of DNA synthesis [6][7][8], DNA repair [9-121, cell proliferation [13-151 and cell differentiation has been reported.Recently, several procedures for the isolation of highly purified poly(ADP4bose) polymerase from different sources have been reported [I9 -221. The availability of purified polymerase may be of great importance, both in elucidating the biological role of poly(ADP-ribose) and in other kinds of studies. It is, for instance, not known how the cell regulates the degree of ADP-ribosylation of the different acceptor proteins and whether there exist several polymerases with different specificities towards the acceptor proteins. Also, polymerase purification will make it possible to determine whether the higher rate of synthesis of poly(ADP-ribose) reported in transformed cells [23] is due to higher polymerase concentrations or to the stances, has the major protein present after purification been shown to be responsible for the polymer synthesis. In the present work we will describe a relatively fast and simple method for obtaining an almost homogeneous preparation of poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells. The major protein present is shown to be able to synthesize poly-(ADP-ribose). Some properties of the polymerase are given, as well as a possible explanation of the wellknown DNA requirement of the polymer synthesis [24,25].

Poly(ADP-Ribose) Biosynthesis

“…A two-to threefold change in the apparent Km of the polymerase for its substrate was observed when histones were added to the reaction mixture (Okayama et al 1977;Kristensen and Holtlund 1978) and an allosteric mechanism was implied on the basis of this and other evidence (Okayama et al 1977;Yoshihara et al 1978;Jump and Smulson 1980). A number of different mechanisms have been proposed to account for this effect.…”

Eukaryotic nuclear ADP-ribosylation reactions

“…Enzyme activity is lacking, however, in granulocytes and mammalian erythrocytes and is greatly diminished in, if not absent from, terminally differentiated epidermal cells (Ikai et al, 1981). The ADPRT from many sources has been purified and characterized (Man- Kristensen & Holtlund, 1978;Tsopanakis et al, 1978;Ito et al, 1979;Niedergang et al, 1979;Jump & Smulson, 1980;Agemori et al, 1982;Carter & Berger, 1982, and others) and in all cases is a basic protein.…”

A Comparison of Purified Poly (ADP‐ribose) Polymerases from Ehrlich Ascites Tumor Cells, Pig Thymus, and HeLa S3 Cells

“…In all cases, the isolated enzyme is a fairly basic protein. The molecular masses reported, however, vary from 50000 D a for the rat liver enzyme [I] to 130000 Da for polymerase from bovine thymus [4] and from Ehrlich ascites tumor cells [8]. These variations may indicate the presence of more than one poly(ADP-ribose) polymerase in an organism.…”