Two Different Types of Bonds Linking Single ADP‐Ribose Residues Covalently to Proteins

Abstract

Single ADP‐ribose residues covalently bound to protein of rat liver and Ehrlich ascites tumor cells were rendered acid soluble by treatment with neutral hydroxylamine. Incubation of the acid‐soluble extract with 1 M N a OH at 56°C converted the released ADP‐ribose selectively to 5′‐AMP, which could then be quantified by a highly specific radioimmunoassay for 5′‐AMP. Direct treatment of the acid‐insoluble fractions with alkali yielded a higher amount of ADP‐ribose equivalents than NH 2 OH treatment, indicating the release of additional ADP‐ribose residues linked to the acceptors by an NH 2 OH‐resistant, alkali‐labile linkage, as already observed in vitro . In adult rat liver 5300 pmol mono(ADP‐ribose) residues/mg DNA linked by NH 2 OH‐labile bonds, and 12600 pmol mono(ADP‐ribose) residues released by NaOH were found. The corresponding values for Ehrlich ascites tumor cells (stationary growth phase) were 480 pmol and 1660 pmol respectively. Opposite to the ratios found in vitro , and in spite of the nearly tenfold difference in total releasable mono(ADP‐ribose) residues, both tissues exhibited higher levels of ADP‐ribose residues bound to the acceptors by the NH 2 OH‐resistant linkage than residues linked by NH 2 OH‐susceptible bonds. These data also show that the bulk of the ADP‐ribosylated proteins in eukaryotic cells is modified by single ADP‐ribose rather than by poly(ADP‐ribose) chains. Extraction of histone H1 with perchloric acid prior to the determination of ADP‐ribose residues indicated that histone H1 in vivo carried only a very small fraction of the total protein‐bound mono(ADP‐ribose) residues.