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

A novel affinity-purification scheme based on the tight binding of NAD +:ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose) poly merase; EC 2.4.2.30] to single-strand nicks in DNA, single-stranded pa tches and DNA ends has been developed to facilitate the purification o f this enzyme from the lower eukaryote Dictyostelium discoideum. Two h omogeneous 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 900 00-M(r) protein and 77 muM NAD+ for the 116000-M(r) protein. The optim um 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 reactio n only. As in enzymes of higher eukaryotes, the activity is dependent on DNA and histone H1 and is inhibited by 3-methoxybenzamide, nicotina mide, theophylline, caffeine and thymidine.