Tsopanakis C, Leeson E, Tsopanakis A, Shall S
Eur J Biochem. 1978 Oct;90(2):337-45. doi: 10.1111/j.1432-1033.1978.tb12610.x.
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 mu mol NAD+ incorporated x min-1 x mg-1 of protein at 37 degree 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 Km for NAD+ was 23 micrometer.
