Hen liver nuclei

ADP-Ribosyltransferase from Hen Liver Nuclei Purification, Properties, and Evidence for ADP-Ribosylation>Induced Suppression of Cyclic AMP-Dependent Phospborylation... 

In ontogenic studies of the ADP-ribosylation reactions, we observed that ADP-ribosyltransferase activity in the adult hen liver nuclei was much higher than in the embryo nuclei [2]. We purified this enzyme from hen liver nuclei [3] and noted the ADP-ribosylation-dependent suppression of protein phosphorylation [4, 5]. This article is an accoimt of our recent studies on ADP-ribosyltransferase of hen liver nuclei and the novel properties of ADP-ribosylation-induced suppression of cAMP-dependent phosphorylation are described. 

The hen liver nuclei contain both ADP-ribosyltransferase and poly(ADP-ribose) synthetase. To separate the ADP-ribosyltransferase from poly(ADP-ribose) synthetase, the 0.6 M potassium chloride extract from the nuclei was applied to a Sephadex G-200 column and eluted with the 0.1 Af Tris-buffer, pH 8.0. Each fraction was incubated with 1 vaM [adenine- H]NAD and 100 jug of whole histones, in a total volume of 0.2 ml containing 50 mAf Tris-Cl" buffer, pH 9.0, and the radioactivity in the acid-insoluble fraction was determined. The result shows the two fractions containing the enzyme activities which catalyze the incorporation of the ADP-ribose moiety from NAD to the whole histones. From the hydroxyapatite column chromatographic analysis of the products formed by the respective fraction, we found that the former fraction contains poly(ADP-ribose) synthetase and the latter fraction contains poly-... 

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]. 

Table 1. Purification of ADP-ribosyltransferase from hen liver nuclei...

The purified nuclei (600 mg of DNA) prepared from 350 g (wet wt) hen liver nuclei were extracted with medium containing 0.6 M KCl, and the extract was applied to a Sephadex G-200 column to separate the ADP-ribosyltransferase from poly(ADP-ribose) synthetase. Assay condi- ... 

Tanigawa Y, Tsuchiya M, Imai Y, Shimoyama M (1984) ADP-ribosyltransferase from hen liver nuclei Purification and characterization. J Biol Chem 259 2022-2029... 

Guanidino compound-specific ADP-ribosyltransferase activity is present in certain eukaryote tissues and cells, and the enzymes from turkey erythrocytes and hen liver nuclei have been purified and characterized (1, 2). These experiments were done using exogenous acceptors and much less is known of the endogenous acceptor proteins for ADP-ribosylation. 

ADP-Ribosylation of human c-Ha-ras protein by hen liver ADP-ribosyl transferase. Human c-Ha-ras protein, normal or mutated from glycine to valine at the 12th position, was produced in Escherichia coli and purified (6). These proteins were incubated with ADP-ribosyl transferase purified from hen liver nuclei (7) in the presence of [adenylate- P]NAD. Incorporation of the radioactivity derived from p PJNAD was clearly shown at the positions corresponding to the protein bands of normal and mutated c-Ha-m.y proteins (Fig. lA and IB, lanes 3 and 4). However no significant radioactive band was formed in the absence of c-Ha-ra proteins (Fig. IB, lane 5). The incorporation of the radioactivity into c-Ha-ra.y protein was inhibited by more than 50% in the presence of 40 mM arginine methylester or 40 mM nicotinamide. 

Fbrmation of poly( ADP-ribose) was first demonstrated in hen and rat liver nuclei 45, 69). Rillowing incubation of isolated nuclei with nicotinamide mononucleotide and ATP, the 5 -phosphoribosyl moiety of nicotinamide mononucleotide and the 5 -AMP moiety of ATP were recovered in an acid-insoluble product 44,45, 68, 69,150,196,207,208). Fincher investigation demonstrated that NAD was the actual substrate with nicotinamide mononucleotide and ATP acting as precursors when NAD radiolabeled in various positions was used in the reaction, analysis revealed that all parts of the molecule, with the exception of the nicotinamide moiety, were incorporated into the product 44,150,176). Thus, in this posttranslational modification, the modifying group, ADP-ribose, is derived from the coenzyme, NAD. 

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