Poly synthetase calf thymus

NAD glycohydrolases from rat liver nuclei, 66, 151 poly(ADP-ribose) synthetase from rat liver nuclei, 66, 154 poly(ADP-ribose) synthetase from calf thymus, 66, 159 extraction and quantitative determination of larger than tetrameric endogenous polyadenosine diphosphoribose from animal tissues, 66, 165 covalent modification of proteins by metabolites of NAD, 66, 168 coenzyme activity of NAD bound to polymer supports through the adenine moiety, 66, 176 use of differently immobilized nucleotides for binding NAD -dependent dehydrogenases, 66, 192. 

DNA topoisomerases have been found to be subject to covalent modifications that modulate their DNA-relaxing activities. An example of this is the ADP-ribosylation of calf thymus topoisomerase I by poly(ADP-ribose) synthetase (Ferro et al., 1983 Jongstra-Bilen et al., 1983 Ferro and Olivera, 1984). The modified enzyme is less active in DNA relaxation. The possibility that topoisomerase I and poly(ADP-ribose) synthetase are associated in vivo was suggested by the observation that they copurify (Ferro et al., 1983 Jongstra-Bilen et al., 1983). 

Histones have been reported to be required for stimulation of synthetase activity in the presence of DNA (43, 60, 93,100,105,117,125, 148,158,159,220,221,233). As noted, however, near maximal rates of poly(ADP-ribose) formation could be obtained utilizing poly(dA) poly(dT), or "active DNA in the absence of added histones (83). Stimulation of poly ADP-ribosylation was observed when histones were added to intact (100%) or partially denatured calf thymus DNA (400%). Yoshihara suggested (83) that added histone binds to denatured DNA and masks its inhibitory action. Histones did not activate by serving as ADP-ribose acceptor in the enzyme reaction catalyzed by the purified bovine thymus poly(ADP-ribose) synthetase. It was known, however. 

Immobilization was carried out using a homogeneous preparation of poly(ADP-ribose) synthetase obtained from calf thymus [6] and commercially available BrCN-activated Sepharose 4B. For successful immobilization of active enzyme, it was important to block a majority (% two-thirds) of the active groups of the gel by pretreatment with dithiothreitol. The treated gel was mixed with the enzyme solution in the presence of 0.35 M KCl, 0.2 mAf dithiothreitol, 20% glycerol, and 0.1 A/K phosphate buffer (pH 8.0). Figure 1 shows the time course of immobilization of the enzyme activity. The decrease in the soluble enzyme activity reflects mainly immobilization of the enzyme, whereas the decrease in the immobilized enzyme activity is indicative of inactivation of the enzyme. In order to avoid the increase of inactive... 

Ito S, Shizuta Y, Hayaishi O (1979) Purification and characterization of poly(ADP-ribose) synthetase from calf thymus. J Biol Chem 254 3647-3651... 

Poly(ADP-ribose) synthetases were purified from calf thymus [4], mouse testis [5], and human placenta [6]. Proteolytic digestion was performed as described previously [7, 8]. 3-(Bromoacetyl)pyridine was prepared according to the method of Woenckhaus... 

Poly(ADP-ribose) synthetases were purified to apparent homogeneity from calf thymus, mouse testis, and human placenta. The major characteristics of these enzymes are presented in Table 1. In addition to molecular mass, sedimentation constant, isoelectric point, and partial specific volume, the apparent for NAD and DNA as well as V ,ax of the reaction are all common to these three enzymes. Amino acid compositions of the enzymes are shown in Table 2. Here again, the numbers of each amino acid residue are very similar to each other, although some differences as denoted by star symbols are noted. 

Fig. 1. Interspecies cross-reactivity of poly(ADP-ribose) synthetases as detected by immunoblotting. DNA binding proteins prepared from calf thymus (lane 7), HeLa cells (lane 2), mouse testis (lane 3), and chicken liver (lane 4) were immunostained with antisera against calf thymus poly(ADP-ribose) synthetase after SDS gel electrophoresis and trans-blotting...

Fig. 2. Effect of ethanol on calf thymus poly(ADP-ribose) synthetase. Initial velocities were obtained by incubating partially purified enzyme with 100 juM [ PJNAD in 0.2 ml reactions containing 50 vnM Tris-HCl, pH 8.0, 5 mAf EDTA, and 0.06 Mg Hae III fragments with 5 -hydro-xyls (A), 0.3 Mg ml Hae III fragments with 5 -phosphates ( ), or 3 Mg ml Hpa II fragments (°). The results of one representative experiment are shown and the data for each DNA are expressed as activity relative to the activity in the absence of ethanol (165, 183, 137 pmol ADPR/2 min, respectively)...

Poly(ADP-ribose) synthetase is a chromatin-bound enzyme which produces a protein bound homopolymer of ADP-ribose using NAD as a substrate (1). The enzyme from calf thymus has been purified to homogeneity and extensively characterized in several laboratories (2-4). Using limited proteolysis, we recently demonstrated that the enzyme (Mr =... 

Fig. 1. (left) Effects of the antibodies on poly(ADP-ribose) synthetase activity. Antibodies were produced by immunizing BALB/c mice and IgG fractions were purifi from antisera. The purified enzyme (0.25 unit) from calf thymus was preincubated in 50 mM Tris-HCl buffer (pH 8.0) containing 10% glycerol and 200 mM NaCl at 0°C for 30 min with varying amounts of al20K (O), a54K ( ), or control (A) in a total volume of 20 pi, and the mixture assayed for the enzyme activity. The enzyme activity in the absence of IgG was taken as 100%. 

To investigate whetiher these antibodies could be used for analyzing the metabolic turnover of poly(ADP-ribose) synthetase in vivo, we first examined the ability of these IgG firactions to immunoprecipitate the enzyme in a crude extract Nuclei were isolated from calf thymus and incubated... 

Fig. 4. (right) Possible cleavage sites of poly(ADP-ribose) synthetase in calf thymus nuclei. 

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