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Protein bound mono ADP-ribose

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. 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.
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. [Pg.94]

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). [Pg.99]

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. [Pg.433]

Table 1. Protein-bound mono( ADP-ribose) in rat liver mitochondria ... 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. [Pg.434]

When freshly isolated rat liver miotochondria were assessed for endogenous protein-bound mono(ADP-ribose) using the method of Jacobson and co-workers (7, 8), 23.8 and 6.0 pmol of ADP-ribose/mg of protein were detected following incubation of the mitochondrial proteins for 12 hr in the presence and absence, respectively, of 1 M hydroxylamine (Table 1). Mcubation with 3 M neutral hydroxylamine during 12 hr liberated 54.3 pmol of ADP-ribose/mg of mitochondrial protein. Treatment of the mitochondrial proteins with 1 M sodium hydroxide for 2 hr released... [Pg.434]

Cell Type Radiation Survival Parameters Radiation Sensitization by3AB orBZ Poly(ADP-ribose) Polymerase Activity Permeabilized Sonicated Protein-Bound Mono(ADP- Cellular Ribose NAD ... [Pg.99]

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. [Pg.330]

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). [Pg.32]

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. [Pg.99]

The transfer of the ADP-ribose moiety from NAD onto a biological macromolecule (in almost all cases an amino acid side chain of a protein) is referred to as ADP-ribosylation. Subsequently, another ADP-ribose unit can be attached to the protein-bound ADP-ribose. Further elongation of the ADP-ribose chain will result in poly-ADP-ribosylation. This process will not be further addressed in this chapter. However, mono-ADP-ribosylation, i.e., attachment of a single ADP-ribose unit to a protein, has also been established as a specific protein modification with important r ulatory functions. [Pg.133]

The conversion of polymer to monomer formation is shown in Table 1. No. 1 indicates the mono- and poly(ADP-ribose) formation without added DNA and No. 2 the effect of DNA, both with the native enzyme. No. 3 The blocking of lysine end groups results in drastic decrease in polymer, but little change in monomer formation. In No. 4, matrix bound polymerase was incubated with lysine modified enzyme, and after the enzymatic reaction the modified free protein was reisolated and products analyzed. Similar to Nr. 3, monomer formation was close to the rate catalyzed by the native enzyme without DNA (No. 1), but oligomer formation was low. The matrix bound enzyme with added DNA synthesized relatively small quantities of polymers (No. 5), demonstrating that the physical state of the enzyme itself can modify the nature of products. [Pg.72]


See other pages where Protein bound mono ADP-ribose is mentioned: [Pg.528]    [Pg.528]    [Pg.435]    [Pg.526]    [Pg.570]    [Pg.51]    [Pg.94]    [Pg.29]    [Pg.528]    [Pg.529]    [Pg.85]    [Pg.445]   
See also in sourсe #XX -- [ Pg.528 ]




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