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...

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 acidsoluble extract with 1 M NaOH 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 ADPribose equivalents than N H 2 0 H treatment, indicating the release of additional ADP-ribose residues linked to the acceptors by an NH20H-resistant, alkali-labile linkage, as already observed in vitro. In adult rat liver 5300 pmol mono(ADP-ribose) residues/mg DNA linked by NHrOH-labile bonds, and 12 600 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 NH2OH-resistant linkage than residues linked by NH2OH-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.Nuclei of mammalian cells contain an enzyme system which transfers ADP-ribose residues from NAD to form an ADP-ribose homopolymer [I -31. Most if not all of the (ADP-ribose), residues formed were shown to be covalently linked to nuclear proteins [4,5]. ADP-ribosylation of proteins by oligomeric and polymeric (ADP-ribose), chains as well as by single ADP-ribose units has been described, and these reactions were implicated in the regulation of various functions of nucleic acids and chromatin (for a review see [6]). It was postulated that the bond linking the (ADP-ribose), residues to the acceptor proteins resembled an ester-type linkage because of its lability towards neutral hydroxylamine and towards alkaline conditions [4]. Supporting evidence was derived from the Abbreviations. ADP-ribose, adenosine diphosphate ribose; (ADP-ribose)., oligomeric or polymeric ADP-ribose; EAT, Ehrlich ascites tumor.Enzyme, Alkaline phosphatase (EC 3.1.3.1).amino acid composition of the ADP-ribose linkage region in histone H1 ADP-ribosylated in vitro [7]. Other reports postulated a linkage of ADP-ribose to histone H1 through a phosphoserine residue [8]. Studies in this laboratory have shown that total ADPribose residues transferred in vitro from NAD were bound to nuclear proteins by at least two different types of bonds, one exhibiting the properties of an ester-type linkage (susceptibility towards neutral NH20H and alkali) the other being NH2O...

Kinetic analyses of mRNA and 28-S RNA labeling by [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP. [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleoside attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA.The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 pM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.Incorporation of labeled uridine into RNA has often been used as a measure of RNA synthesis. It also forms the basis of new half-life determinations of mRNA [I -41. In all these determinations it was assumed that one UTP pool served all the different RNA species. When we applied newly developed methods for the direct determination of specific radioactivities of individual RNA species [5, 61 and of UTP [7, 81 indications of rather different steady-state specific radioactivities for 28-S RNA zierms mRNA and (total) UTP were obtained [9, 101. These data pointed to the existence of different precursor pools for the various RNA species. This means that the turnover determinations mentioned above are inaccurate, as well as the conclusions drawn from labeling experiments with pyrimidine nucleotide precursors. Therefore, a study on steady-state specific radioDedicated to Feodor Lynen on the occasion of his 65th birthday. activities of uridine and cytidine in several RNA species under carefully controlled conditions of logarithmic growth and constant exogenous [3H]uridine concentrations over several generation times was started. The data obtained provide strong evidence that ribosomal and messenger RNA formation depend on separate pyrimidine nucleotide precursor pools.

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