Purification of ADP-ribosylated nuclear proteins by covalent chromatography on dihydroxyboryl polyacrylamide beads and their characterization.
Nuclear proteins modified by mono or poly ADP-ribosylation were selectively isolated and purified by covalent chromatography on a dihydroxyboryl polyacrylamide bead column that specifically interacts with cis-diol-containing compounds. From rat liver nuclei that had been incubated with NAD+, histones and some nonhistone proteins were extracted with 0.25 M HCl. Approximately 60% of the ADP-ribose incorporated into 20% trichloroacetic acid-precipitable material was recovered in this extract. The ADP-ribosylated material was then isolated from the extract by covalent chromatography on a borate gel column and further purified by carboxymethylcellulose column chromatography. As judged by electrophoretic mobilities in various gel systems and by amino acid compositions, approximately 50% of the ADP-ribose recovered in the carboxymethylcellulose fractions was associated with several nonhistone proteins with molecular weights of 2-6 x 10(4), while 35% aand 15% were associated with histones H2B and H1, respectively. Since the average chain length of the polymer bound to any of these proteins was less than two ADP-ribos-l units, the percentage distribution reflects the number of ADP-ribosylated sites rather than the chain length.
D000818:Animals; D002467:Cell Nucleus / Q000032:analysis; D002850:Chromatography, Gel / Q000379:methods; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification* / Q000378:metabolism; D006657:Histones / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000032:analysis; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D051381:Rats
D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D009702:Nucleoside Diphosphate Sugars; D011064:Poly Adenosine Diphosphate Ribose
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