Amino Acid-specific ADP-ribosylation STABILITY OF THE REACTION PRODUCTS OF AN NADzARGININE ADP-RIBOSYLTRANSFERASE TO HYDROXYLAMINE AND HYDROXIDE*

[adenine-U-14C]ADP-ribose-agmatine and [adenineU-’4C]ADP-ribose-histone were synthesized by an NAD:arginine ADP-ribosyltransferase from [’*C]NAD and agmatine and histone, respectively. The pseudofirst order rate constants for breakdown of the two components either in 0.4 N NaOH or in 0.4 M neutral hydroxylamine were identical. Hydroxylamine treatment of [‘4C]ADP-ribose-agmatine or [32P]ADP-ribose-histone yielded a single radioactive product which was separated by high pressure liquid chromatography and identified as ADP-ribose-hydroxamate by the formation of a ferric chloride complex. Hydrolysis of ADP-ribose-hydroxamate with snake venom phosphodiesterase resulted in the formation of 5’AMP, consistent with the presence of a pyrophosphate bond. Incubation of ADP-rib~se-[’~C]agmatine, synthesized by the ADP-ribosyltransferase from NAD and [I4C]agmatine, with 0.4 M neutral hydroxylamine resulted in the release of [I4C]agmatine rather than phosphorib~syl[‘~C]agmatine. In addition, neither NAD nor ADP-ribose reacts with hydroxylamine; i.e. there was no evidence of nucleophilic attack by hydroxylamine at the pyrophosphate bond. The ADP-ribosyl-protein linkage formed by the NAD:arginine ADP-ribosyltransferase is considerably more stable to hydroxylamine than is the ADP-ribose-glutamate bond. The presence of ADP-ribose-arginine and ADP-ribose-glutamate synthesized by the ADP-ribosyltransferase and poly(ADP-ribose) synthetase, respectively, may be the chemical basis for the “hydroxylamine-stable” and “hydroxylamine-labile” bonds described by Hilz (Hilz, H. (1981) Hoppe-Seyler’s 2. Physiol. Chem. 362, 1415-1425).