Mono ADP-ribose

Inhibitors of mono(ADP-ribose) transferase, lipid peroxidation, and protein synthesis are not effective in reducing SM-induced cell death, while PARP inhibitors are found to be efifective in reducing the cytotoxic effects of SM in human lymphocytes. The compound s ability to inhibit PARP has a direct correlation to its ability to reduce SM-induced cell death. PARP plays an important role in SM-induced cell death (Clayson et al., 1993). The effectiveness of various therapeutic agents, namely niacin,... 

PolyfADP-ribose) is synthesized by PARP-1 and hydrolyzed by enzymes known as polyfADP-ribose) glycohydrolase (PARC), phosphodiesterases (PDases) and ADP-ribosyl protein lyase. Among these, PARC serves as an enzyme that hydrolyzes poly(ADP-ribose) chains quite efficiendy, including the branched pordon, and finally leaves the protein-proximal mono-ADP-ribose molecule, which might be removed by ADP-ribosyl protein lyase or released spontaneously at neutral pH. Such de-modificadon would enable PARP-1 to use the same acceptor protein in a new cycle of poly(ADP-ribos )adon (F 1). 

ADP-ribosyltransferase catalyzes the transfer of the ADP-ribose moiety of NAD to acceptors, as arginine and other guanidino compounds and proteins, and forms mono-(ADP-ribose)-acceptor adducts. In eukaryotes, this enzyme was first detected in turkey erythrocyte by Moss and associates who went on to purify and characterize the enzyme [1]. 

ADP-ribose) synthetase and the latter fraction involves ADP-ribosyltransferase. To clarify that the radioactive compound formed by the latter fraction was indeed the mono(ADP-ribose) molecule, the acid-insoluble reaction product was treated with alkali at 37°C for 2 h. The radioactive material solubilized was adjusted to pH 7.0 and subjected to high performance liquid chromatography with reverse phase column. The eluate was monitored by UV and fractionated and radioactivity of the fraction was measured. The retention time of the radioactive product coincided with that of authentic mono(ADP-ribose). Furthermore, by treatment with snake venom phosphodiesterase this radioactive peak, tentatively considered to be ADP-ribose, migrated to the position corresponding to the 5 -AMP. These results indicate that hen liver nuclei contain ADP-ribosyltransferase. We purified this enzyme to a homogeneous state through salt extraction, gel filtration, hydroxyapatite, phenyl-Sepharose, Cm-cellulose, and DNA-Sepharose [3]. 

We have attempted to dissect the incorporation of NAD into these two reactions by snake venom phosphodiesterase (SVPDE) digestion. The rate of incorporation of label into 5 -AMP gives an estimate of the rate of initiation and the rate of incorporation into phosphoribosyl-AMP(PR-AMP) estimates the rate of elongation. AE-cellulose chromatography can be used to monitor the proportion of 5 -AMP deriving from mono- and poly(ADP-ribose) residues respectively. This provides an important check since, in permeabilised cells, mono(ADP-ribose) residues can be produced other than in the nucleus and also by non-enzymic reactions [9],... 

Bredehorst R, Goebel M, Renzi F, Kittler M, Klapproth K, Hilz H (1979) Intrinsic ADP-ribose transferase activity versus levels of mono(ADP-ribose) protein conjugates in proliferating Ehrlich ascites tumor cells. Hoppe-Seyler s Z Physiol Chem 360 1737-1743... 

Adamietz P, Wielckens K, Bredehorst R, Lengyel H, Hilz H (1981) Subcellular distribution of mono(ADP-ribose) protein conjugates in rat liver. Biochem Biophys Res Commun 101 96-103... 

When labeled SMP were analyzed on SDS-PAGE, radioactivity was found almost exclusively in the region of proteins of mol.wt. of about 32,000 [13]. Treatment of the labeled protein with snake venom phosphodiesterase Uberated predominantly 5 -AMP, indicating modification of the protein by mono(ADP-ribose). 

The mono(ADP-ribose) residue modifying the protein turns over rapidly. This was indicated by two lines of evidence [13]. First, when SMP were incubated with [ H]-NAD and, after attaining a steady state level of modification as judged by incorporation of tritium, were then supplied with [ C]-NAD, there was loss of tritium and simultaneously incorporation of [ C] into the protein. Secondly, when ATP was added to SMP, mono(ADP-ribose) was lost from the protein within seconds. Whether the removal of mono(ADP-ribose) from the protein is enzyme-catalyzed remains to be seen. The bond between mono(ADP-ribose) and the 32,000 mol.wt. protein in SMP is acid stable, alkali sensitive, and labile in neutral hydroxyl amine. Formation of it is inhibited by arginine blocking reagents [13]. 

When inner mitochondrial membranes hydrolyze NAD a protein with mol.wt. of about 32,000 is modified by mono(ADP-ribose). The modification turns over rapidly. A NAD glycohydrolase can be isolated from SMP. Its mol.wt. as judged by SDS-PAGE is about 32,000. In the presence of NAD the enzyme undergoes auto(ADP-ribosylation). 

To confirm that the incorporated radioactive moiety was mono(ADP-ribose), the acid-insoluble fractions of SR vesicles incubated with labeled NAD in the presence and absence of poly L-lysine were treated with alkali and the respective supernatant was analyzed by reverse phase HPLC. In both cases, radioactive peaks co-eluted with authentic ADP-ribose and 5 AMP (data not shown). 

Fig. 1. Release of cysteine-linked ADP-ribose by mercuric ion. The acid insoluble fraction of rat liver (15) was dissolved in 98% ice cold formic acid and radiolabeled mono-ADP-ribosylated protein was added. The solution was diluted with five volumes of ice cold H2O and precipitated by addition of 100% (w/v) ice cold trichloroacetic acid to a final concentration of 20% (w/v). The sample was held on ice for 10 min and subjected to centrifugation. The precipitate was resuspended in ice cold, 98% formic acid and stored at -20° C for subsequent use. To release cysteine-linked ADP-ribose, the sample in ice cold 98% formic acid was diluted with an equal volume of ice cold H2O or a freshly prepared solution of 20 mM mercuric acetate and the resulting solution was incubat at 37°C 10 min. The samples were then placed on ice, 5 volumes of ice cold H2O were added followed by 100% (w/v) trichloroacetic acid to a final concentration of 20%. After 10 min on ice, the samples were collected by centrifugation and the supernatant was removed. A sample was taken to determine released radiolabeled mono-ADP-ribose. The pellet containing the remaining protein-bound mono-ADP-ribose was dissolved in 250 mM ammonium acetate, 10 EDTA and 6 M guanidine before sampling for radioactivity. ( ) presence, (O) absence of mercuric ion. Panel A shows a time course at 10 mM mercuric ion and Panel B shows a 10 min incubation at the indicated concentrations of mercuric ion.

Recent reports question the assumption that mitochondrial ADP-ribosylation is a true ADP-ribosyl transferase catalyzed enzymic reaction. This is mainly because mono-ADP-ribose produced by NAD-glycohydrolase can be bound to mitochondrial protein acceptors nonenzymatically (12). 

DNA strand breaks, induced by exposure to chemicals or ionizing radiation stimulate poly(ADP-ribose) polymerase activity (1, 2). The resultant protein modifications have been postulated to comprise an important step in the DNA repair process (3). Inhibitors of the polymerase have been shown to sensitize human fibroblasts (4) and certain tumor cells (5) to ionizing radiation and to inhibit the repair of potentially lethal radiation injury (6, 7). That the response of the tumor cell lines vary, with some showing sensitivity to inhibitors of poly(ADP-ribosyl)ation and irradiation while others do not, suggested a need for detailed investigation of the ADP-ribosylation process in these tumor cell lines. In the present study we report the quantitative variations in protein-bound mono(ADP-ribose) levels as well as poly(ADP-ribose) pol)unerase activities and cellular NAD levels of various tumor cells. To this end, we also describe die development and characterization of polyclonal antisera to mono(ADP-ribose) and its potential use as a probe for studies of ADP-ribosylation. 

Fig. 1. Titration of mono(ADP-ribose) antiserum. Appropriately diluted serum was incubated overnight at 4°C with either [ HJNAD, [ HlADP-ribose, or [ HJS AMP ( 30,000 cpm) in PBS-6SA buffer (final volume of 0.5 ml) either in the presence or absence of 0.05% Tween-20. Bound tracer was separated from free by DCC treatment. The bound fraction was counted in 10 ml of aquasol.

Table 1. Specificity of mono(ADP-ribose) antiserum-cross reactivities of related nucleotides.

Poly(ADP-ribose) polymerase activity was assayed according to Berger al. (10) in peimeabilized cells and Chemey etal. (11) cellular homogenates. Total (alkali-labile) protein-bound mono(ADP-ribose) levels were determined as described in this report while sample preparation for RIA was as reported by Bredehorst et al. (9). NAD assay was done by the cycling assay of Bemofsky et al. (12). 

The differences in the endogenous ADP-ribosylation status of proteins are not as striking as the polymerase activity itself in various cell lines. This observation could be interpreted to reflect a short half-life of the polymer or monomer and also the enzyme assay represents the potential of the cell to s)mthesize the polymer while RIA of protein-bound (endogenous levels) mono(ADP-ribose) evaluates the steady state levels. 

Even contradictory results for one and the same diHerentiation system were reported In the conversion of 3T3-L1 preadipocytes to adipocytes reports of an early, transient decrease of poly(ADP-ribose) polymerase activity as determined in isolated nuclei (9) and a stimulation of differentiation by nicotinamide (10) contrast with a paper reporting the prevention of preadipocyte differentiation by nicotinamide and benzamide (11). In none of these reports, however, was protein-bound poIy(ADP-ribose) determined. Here, we describe an analysis of mono(ADP-ribose) and poly(ADP-ribose) status in 3T3-L1 preadipocytes during differentiation to adipocytes and the effect of various poly(ADP-ribose) polymerase inhibitors. 

In rat liver most of the cellular mono(ADP-ribosylated) proteins are associated with the mitochondrial fraction (1). Two mono(ADP-ribosyl)ating systems have been described in mitochondria, one in die soluble (matrix) fraction (2, 3), the other in submitochondrial particles (SMP, inverted inner membrane vesicles) (3, 4). The ADP-ribosylated matrix protein has a molecular mass of 100 kDa and appears to consist of two major subunits of equal mass. In SMP of both rat liver (4) and beef heart (3), there is one major acceptor protein for mono(ADP-ribose), which migrates with an apparent molecular mass of 30 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Mono(ADP-ribosylation) of the acceptor protein of beef heart SMP was suggested to occur non-enzymicaUy (3). In rat liver SMP, ADP-ribosylation of the 30 kDa protein most probably occurs at an arginine residue, and is readily reversible in the presence of ATP (4). The characteristics of this ADP-ribosylation reaction, i.e. protein specificity and sensitivity to ATP, together widi the observation that intramitochondrial hydrolysis of NAD(P)+ is accompanied by release of Ca + from mitochondria suggests a functional link between mitochondrial protein ADP-ribosylation and Ca2+ release (5,6). 

Using a newly developed fluorescent technique (7,8) we have investigated protein ADP-ribosylation in intact mitochondria. Our findings indicate the existence of at least three classes of ADP-ribosylated proteins in rat liver mitochondria and give evidence for a transient increase of protein-bound mono(ADP-ribose) during the NAD(P)+- linked Ca + release. 

Table 1. Protein-bound mono( ADP-ribose) in rat liver mitochondria ...

The acid insoluble material from rat liver mitochondria was dissolved and subjected to G-25 (superfine) colunm centrifuption. The samples (containing 0.8-2.0 mg of protein) were incubated under the conditions given in the Table and assessed for protein-bound mono(ADP-ribose) fluorimetrically as described by Jacobson et al. and Payne et al. Each value represents the average standard deviation of three mitochondrial preparations. For each mitochondrial preparation and incubation condition at least three samples were examined. 

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