Purification and Properties of Poly(ADP‐ribose) Polymerase from Pig‐Thymus Nuclei

Abstract

The nuclear enzyme poly(ADP-ribose) polymerase has been purified about 9200-fold from pig thymus nuclei with a 46% yield. An aqueous organic solvent system was used for the isolation of the polymerase from nuclei and for its purification by chromatography at sub-zero temperatures. Electrophoretic analysis under both denaturing and non-denaturing conditions revealed a single protein band suggesting that the preparation was homogeneous and that the enzyme is composed of one polypeptide chain. The molecular weight estimated from sodium dodecyl sulphate-/polyacrylamide gel electrophoresis was 63 500 and from gel filtration through columns of Sephadex G-100, 58 000. The enzyme preparation was free from poly(ADP-ribose)-degrading enzymes and from DNA. The purified polymerase showed an absolute requirement for both DNA and histones. The maximal specific activity of the homogeneous preparation measured by the standardized assay, was 20.7 pmol NAD* incorporated x min-' x mg-' of protein at 37 "C. Amino-terminal group analysis with dansyl chloride did not reveal a terminal amino acid suggesting that the amino-terminal group may be blocked. In the presence of histones, the K,,, for NAD' was 23 pM.

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.…”

“…Circumstantial evidence for the presence of multiple transferases was initially provided by reported differences in the molecular mass of calf thymus ADPRT purified by different laboratories (Tsopanakis et al, 1978;Jongstra-Bilen et al, 1981), and by the range of acceptors of ADP-ribose indicating an extraordinarily wide enzyme specificity. The metabolic consequences, established so far mostly for DNA repair, of inhibiting the transferase also differ in various tissues.…”

“…Tsopanakis et al, 1978) underestimate the molecular mass of the transferase since it has been shown that it is subject to highly specific endogenous proteolytic processing (Surowy & Berger, 1983a;Holtlund et al, 1983). Jongstra-Bilen et al (1981) concluded that transferases from calf and pig thymus and bovine and rat liver, pancreas, brain and kidney and from chicken brain all have a molecular mass of 1-30kDa.…”

Two Proteolytic Degradation Products of Calf‐Thymus Poly(ADP‐ribose) Polymerase Are Efficient ADP‐ribose Acceptors

“…Since it is known that the N-terminal amino group of poly(ADP-ribose) polymerase is blocked, both when isolated from calf thymus [34], pig thymus [35], and Ehrlich ascites tumor cells [36], and thus not detectable by the usual methods, N-terminal analysis of the two fragments should reveal from which end the polymerase is degraded. We have not yet isolated sufficient amounts of the fragments to perform such an analysis.…”

Inhibitors of nuclear ADP‐ribosyl transferase retard DNA repair after N‐methyl‐N‐nitroso‐urea

“…The enzyme is totally dependent on DNA [5] and is markedly stimulated by fragmentation of the DNA [6][7][8][9]. Evidence has accumulated over the last 5 years that (ADP-ribose)n participates in the cellular recovery from DNA damage [10,11 ].…”