Nonenzymic ADP-ribosylation of specific mitochondrial polypeptides.

Abstract

The apparent NAD:protein ADP-ribosyl transferase activity of mitochondria and submitochondrial particles from beef heart and rat liver is simulated by a reaction sequence that consists of an enzymic hydrolysis of NAD to ADP-ribose (ADP-Rib) by NAD glycohydrolase(s) and a nonenzymic ADP-ribosylation of acceptor proteins by the free ADP-Rib formed. The nonenzymic ADP-ribosylation of mitochondrial proteins showed two pH optima and exhibited the same remarkable selectivity as the reaction with NAD. The predominant acceptor in beef heart mitochondria was a 30-kDa protein, whereas in mitochondrial extracts of rat liver a 50-55 kDa polypeptide served as an acceptor. No authentic ADP-Rib transferase activity could be detected even when free ADP-Rib was trapped by NH2OH. Once formed, the mitochondrial ADP-Rib conjugates were resistant to hydroxylamine. NH2OH-resistant mono(ADP-Rib)-protein conjugates as found in most cells may also be products of nonenzymic ADP-ribosylation. In mouse tissues, their amounts relate to protein and NAD contents, and they increase specifically and reversibly in the hypothyroid status. Furthermore, intact rat liver mitochondria contain a mono(ADP-Rib)-polypeptide (50-55 kDa) that appeared to be identical with the polypeptide reacting with ADP-Rib in vitro.Various phage enzymes and bacterial toxins catalyze the transfer of single ADP-ribose (ADP-Rib) residues from NAD to one or several protein acceptors (for reviews, see refs. 1-4). Eukaryotic cells also contain endogenous proteins that are modified by single ADP-Rib residues (cf. ref. 5). In rat liver, most of these mono(ADP-Rib)-protein conjugates are located in the cytoplasm, whereas poly(ADP-Rib)-protein conjugates appear to be confined to the nucleus (6). Two types of endogenous mono(ADP-Rib)-conjugates can be distinguished by their sensitivity towards neutral hydroxylamine. They show independent changes and they are distributed unevenly in various organs of the mouse (5, 7).Interest in cytoplasmic ADP-ribosylation was greatly stimulated when diphtheria toxin-and cholera toxin-catalyzed ADP-ribosylation was detected (7-12) and when membrane-bound (12, 13), cytosolic (14), and mitochondrial (15, 16) ADP-ribosyl transferase activities were described. It was also reported that free ADP-Rib can form Schiff bases with amino groups of proteins, especially of the histones, when incubated at slightly alkaline conditions (17), while true ADP-Rib transferase activity in mitochondria was said to be operating at pH values <7 (18). However, the use of various inhibitors in mitochondrial preparations indicated to us that nonenzymic ADP-ribosylation was occurring at lower pH values as well.This paper describes a reaction sequence found in mitochondria (and plasma membranes) that involves the hydrolysis of NAD by NAD glycohydrolases and the nonezymic ADP-ribosylation of specific acceptors to form acid-stable conjugates. This reaction sequence simulated ADP-Rib transferase activity, and the similarity of acceptors in vivo and in vitro provides sugg...

Eukaryotic nuclear ADP-ribosylation reactions

“…Non-enzymic mono-(ADP-ribosyl)ation has been reported by Hilz et al (1984) turnover of poly(ADP-ribose) (reaction q, Fig. 2a).…”

Unexpected stimulation of mitochondrial ADP‐ribosylation by cyanide

“…and 2, respectively) and in lanes 7 and 8 (as in lanes 3 and 4, respectively). It has been reported that ADP-ribosylation can occur in mitochondria by non-enzymatic transfer of ADP-ribose to protein acceptors [8]. When mitochondria were incubated with [32P]ADPribose in the presence of cyanide, there was no cyanide stimulation of ADP-ribosylation (not shown).…”

“…In an attempt to elucidate the controversy concerning ADP-ribosylation in mitochondria [7,8] data are presented here demonstrating that cyanide stimulates ADP-ribosylation in mitochondria or mitoplasts (inner mitochondrial membrane preparation stripped of outer membrane). Cyanide is a potent irreversible inhibitor of cytochrome c oxidase, i.e.…”

“…Thus, one of the pathways of signal transduction is believed to be operated via ADP-ribosylation, although its precise molecular mechanism remains unclear [a]. There is general agreement regarding nuclear ADP-ribosylation while the nature of ADP-ribosylation in mitochondria has been questioned [7,8]. In view of the fact that mitochondria have their own distinct genetic machinery [9], their bacterial origin continues to be Correspondence address: A. Masmoudi, Centre de Neurochimie du CNRS, 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France debated [lo] and the presence of an ADPribosyltransferase system in mitochondria appears a logical corrollary.…”

Identification and purification of a bovine liver mitochondrial NAD+‐glycohydrolase

“…phenylmethylsulfonyl fluoride: SDS, sodium dodecyl sulfate. tion of acceptor proteins in the inner mitochondrial membrane in a nonenzymatic reaction [7]. Specific ADP-ribosylation of an ADPR acceptor protein may then trigger the release of Ca 2+ from mitochondria through regulation of a Ca 2* channel.…”