PURIFICATION AND CHARACTERIZATION OF NAD+ADP-RIBOSYLTRANSFERASE (POLYMERIZING) FROM DICTYOSTELIUM-DISCOIDEUM

A novel affinity-purification scheme based on the tight binding of NAD+:ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose) polymerase; EC 2.4.2.30] to single-strand nicks in DNA, single-stranded patches and DNA ends has been developed to facilitate the purification of this enzyme from the lower eukaryote Dictyostelium discoideum. Two homogeneous forms of the enzyme, with M(r) values of 116000 and 90000, were prepared from D. discoideum by using poly(A) hybridized to oligo(dT)cellulose as affinity material. The K(m) is 20 muM NAD+ for the 90000-M(r) protein and 77 muM NAD+ for the 116000-M(r) protein. The optimum conditions for the enzyme activity in vitro are 6-10-degrees-C and pH 8. The time course is linear during the first 10 min of the reaction only. As in enzymes of higher eukaryotes, the activity is dependent on DNA and histone H1 and is inhibited by 3-methoxybenzamide, nicotinamide, theophylline, caffeine and thymidine.