ADP-ribosylation in inner membrane of rat liver mitochondria.

Abstract

NAD' glycohydrolase activity is found at high levels in submitochondrial particles. It leads to the reaction products ADP-ribose, nicotinamide, and small amounts of 5'-AMP. Furthermore, submitochondrial particles catalyze the exchange reaction: [adenosine-14C]ADP-ribose + NAD+ -[adenosine-'4C]_ NAD+ + ADP-ribose. When submitochondrial particles are incubated with NAD+, mono(ADP-ribosyl)ation of protein molecules migrating with an apparent molecular weight of 30,000 in sodium dodecyl sulfate/polyacrylamide gel electrophoresis is demonstrable. Inhibitor studies suggest attachment of ADP-ribose to arginine residues. ADP-ribose bound to submitochondrial particles is rapidly turning over. The release of ADP-ribose from the protein is probably enzyme catalyzed. The rapid turnover, the specificity of the modification, and the inhibition of ADP-ribosylation by ATP and nicotinamide suggest a regulatory role of mono(ADP-ribosyl)ation of a protein in the inner mitochondrial membrane.ADP-ribosylation is a posttranslational protein modification receiving growing interest. Several reviews on both the biological and methodological aspects have been published (1-3). ADPribose is attached to acceptor proteins as a monomer, oligomer, or polymer. Mono(ADP-ribosyl)ation has so far been found to be catalyzed mainly by prokaryotic enzymes-e.g., diphtheria toxin (4), Pseudomnas exotoxin A (5), choleragen (6), and Escherichia coli heat-labile enterotoxin (7). Catalysis of mono-ADPribosylation by enzymes from rat liver (8) and erythrocytes from turkey and man (9, 10) has also been reported. Poly(ADP-ribosyl)ation, on the other hand, is found mainly in the nuclei of eukaryotic cells. The accumulating evidence indicates a close relationship between poly(ADP-ribosyl)ation and chromatin activities (11,12).During our studies on the mechanism of hydroperoxide-induced release of calcium from rat liver mitochondria we observed the hydrolysis of intramitochondrial pyridine nucleotides at the ,BN-glycosidic bond between nicotinamide and ADPribose in intact mitochondria (13,14). The hydrolysis of NAD+ is accompanied by a covalent protein modification at the inner side of the inner mitochondrial membrane by a NAD+-derived compound. Pyridine nucleotide hydrolysis, protein modification, and release of calcium from rat liver mitochondria are inhibited by ATP (15). On the basis of these findings we put forward the hypothesis that this covalent modification is part of a mechanism controlling calcium release.Kun et a! (16,17) have described ADP-ribosylation of a soluble protein derived from sonicated mitochondria. In recent papers Hilz and co-workers (18,19) analyzed the distribution of mono-and poly(ADP-ribose) in liver cells. We describe here a covalent modification by mono(ADP-ribose) of a protein in the inner mitochondrial membrane. The modification is found almost exclusively in protein molecules with a molecular weight of 30,000 in NaDodSO4/polyacrylamide gel electrophoresis. The modifying group has a high turnover. The fast removal of ADP...

Evidence for ADP‐ribosylation in the mechanism of rapid thyroid hormone control of mitochondria

“…Two groups [26,27] have recently shown that rat liver mitochondria possess an active ADPribosyltransferase or perhaps an NAD glycohydrolase followed by a surprisingly specific nonenzymatic ADP-ribosylation [28]. This activity mono ADP-ribosylates an inner membrane protein whose molecular mass is around 31 kDa on SDS-PAGE [27] and which shows rapid turnover of the covalent modification in vivo.…”

Unexpected stimulation of mitochondrial ADP‐ribosylation by cyanide

“…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

“…However, the oxidation of pyridine nucleotides is not sufficient to induce the release of Ca 2* from mitochondria. In addition, hydrolysis of NAD ÷ to nicotinamide and ADP-ribose by NAD+glycohydrolase is required [6]. It has been reported that ADPR generated by this enzyme might lead to ADP-ribosyla-*Corresponding author.…”

“…The respective NAD+glycohydrolase was assigned to the inner mitochondrial membrane and a 60-to 70-fold enriched preparation from rat liver showed a main protein band with a molar mass of 62,000 [8]. The putative ADPR acceptor protein was proposed to be a protein of the inner mitochondrial membrane which has a molar mass of 30,000 [6]. A recent report suggested that the mitochondrial NAD+-glycohydrolase is located in the outer mitochondrial membrane [9].…”