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 116,000 and 90,000, were prepared from D. discoideum by using poly(A) hybridized to oligo(dT)-cellulose as affinity material. The Km is 20 microM NAD+ for the 90,000-M(r) protein and 77 microM NAD+ for the 116,000-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.
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July 1993
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Research Article|
July 01 1993
Purification and characterization of NAD+:ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum
B Kofler;
B Kofler
*Institut für Biochemie (Nat. Fak.), Universitat Innsbruck, A-6020 Innsbruch, Austria
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E Wallraff;
E Wallraff
†Max-Planck-Institut für Biochemie, D-8033 Martinsried, Germany
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H Herzog;
H Herzog
*Institut für Biochemie (Nat. Fak.), Universitat Innsbruck, A-6020 Innsbruch, Austria
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R Schneider;
R Schneider
*Institut für Biochemie (Nat. Fak.), Universitat Innsbruck, A-6020 Innsbruch, Austria
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B Auer;
B Auer
*Institut für Biochemie (Nat. Fak.), Universitat Innsbruck, A-6020 Innsbruch, Austria
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M Schweiger
M Schweiger
*Institut für Biochemie (Nat. Fak.), Universitat Innsbruck, A-6020 Innsbruch, Austria
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1993 The Biochemical Society, London
1993
Biochem J (1993) 293 (1): 275–281.
Citation
B Kofler, E Wallraff, H Herzog, R Schneider, B Auer, M Schweiger; Purification and characterization of NAD+:ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum. Biochem J 1 July 1993; 293 (1): 275–281. doi: https://doi.org/10.1042/bj2930275
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