Search

Acceptor proteins for poly(ADP-ribose) have been purified from mouse testis nuclei. Nuclear proteins were labelled in vitro with [14C]ribose and [3H]adenine, extracted with 5% (v/v) HClO4 and 0.25 M-HCl and separated by ion-exchange chromatography. Non-histone proteins were found to be the major acceptors in both the 5% (w/v)-HClO4-soluble and 5%-HClO4-insoluble HCl-extractable fractions. Of the two groups of non-histone proteins associated with chromatin, the LMG (low-mobility-group) proteins were preferentially ADP-ribosylated. HMG (high-mobility group) proteins were labelled to lower specific radioactivity. Six LMG proteins were purified to approx. 90% homogeneity and were identified from their mobility on polyacrylamide gels at pH 2.9 and from their amino acid composition. The average length of the poly(ADP-ribose) chain was estimated to be four to six repeating ADP-ribose units. It is suggested that ADP-ribosylation of LMG proteins, a long-neglected group of chromatin-associated proteins, is important during spermatogenesis for the production of spermatozoa with intact and competent DNA.

The complete amino-acid sequence of BS-RNAse, a dimeric ribonuclease isolated from bovine seminal plasma, was determined. The reduced and S-carboxymethylated subunit chain of the enzyme was cleaved by trypsin and chymotrypsin. The resulting peptides, purified by cation-exchange chromatography were sequenced by dansyl-Edman, subtractive Edman degradation and carboxypeptidase A and B digestion. Chymotryptic peptides were used for the alignment. Automated Edman degradation of the native protein, through the N-terminal 41 amino-acid residues, completed the sequence information. The subunit chain of BS-RNAse, composed of 124 amino-acid residues, with a molecular mass of 13,610 Da, is highly homologous (81%) to pancreatic ribonuclease A. A good degree of homology (31%) was also found with human angiogenin. No N-linked carbohydrate-attachment sites, such as Asn-X-Ser/Thr, were found in the protein.

Poly(ADP-ribosylation) of nuclear proteins has been investigated in rat testis under different experimental conditions to determine whether it is associated with somatic or germinal cells. Isolated, intact nuclei were incubated with [14C]NAD and extracted sequentially with 5% HClO4 and 0.25 M HCl, and labelled soluble proteins were analysed by reverse-phase high-performance liquid chromatography and acetic acid-urea polyacrylamide gel electrophoresis (pH 2.9). Results show that in normal adult testis a major acceptor protein for poly(ADP-ribose) in HClO4 extracts is the tissue-specific histone, H1t. Core histones and three proteins (alpha, beta and gamma) with low mobility on acetic acid-urea gels were the major acceptors identified in HCl extracts. Poly(ADP-ribosylation) of all the aforementioned proteins is very low in isolated intact nuclei of testis from 8-day-old animals (only spermatogonia present in seminiferous tubules), increases significantly by 16-day (pachytene spermatocytes appear) and reaches adult proportions by 32 days (condensing spermatids present). In the nuclei from cryptorchid testes, poly(ADP-ribosylation) of nuclear proteins resembles 8-day-old testis. It is concluded that (a) poly(ADP-ribosylation) of nuclear proteins in rat testis is closely correlated with spermatogenesis and can be inferred that is particularly active in the early stages of meiosis; (b) testis-specific proteins (histone H1t and low mobility proteins, alpha, beta and gamma) are poly(ADP-ribosylated) to higher specific radioactivity than somatic histones.

Poly(ADP-ribosylation) of nuclear proteins is one of a number of post-translational events which has been demonstrated in eukaryotic cells. The reaction is catalysed by poly(ADP-ribose) synthetase which transfers ADP-ribose from NAD to a suitable acceptor protein. Histone H1 and HMG proteins 1, 2, 14 and 17 have been reported as acceptors in a variety of tissues ranging from trout testis [1] to mammary carcinoma cells [2]. Poly(ADP-ribosylation) of HMG proteins is of particular interest because of the reported association of these proteins with actively transcribed genes [3]. Functionally, poly(ADP-ribosylation) has been implicated in a variety of regulatory events such as DNA synthesis [4], DNA excision repair [5, 1], gene expression [6] and cell differentiation [7].

The thermophilic microorganism Sulfolobus solfataricus is able to grow at low pH (3.5) and high temperature (87°C) and has been isolated from an acidic hot spring in Agnano (Napoli), Italy (1). This bacterium belongs to the archaebacteria, a phylogenetic group of microorganisms that can be distinguished from other bacteria and eukaryotes (2, 3). The archaebacteria group was initially defined on the basis of a partial 16S ribosomal RNA sequence comparison, which has been well supported by other biochemical features (2-4). More recent studies by Woese and collaborators, based on comparison of complete 16S ribosomal RNA sequences, confirmed and extended earlier archaebacteria phylogeny (3). Although archaebacteria taxonomy is complex and controversial, three main phenotypes have been described, characterized by their peculiar ecological niches, namely high salt environments (halophilic and haloalkaliphilic archaebacteria), high temperature environments (thermophilic and sulfur-dependent archaebacteria) and stringent anaerobic conditions (methanogenic archaebacteria). Several features indicate that the sulfur-dependent branch of archaebacteria, to which Sulfolobus solfataricus belongs, is more closely related to eukaryotes than eubacteria or other archaebacteria (3). One example is the diphtheria toxin reaction, which catalyzes the transfer of ADP-ribose to eukaryotic elongation factor 2. Kessel and Klink (5) demonstrated that the toxin is able to catalyze, in several archaebacteria including Sulfolobus solfataricus, the covalent binding of ADP-ribose to elongation factor, EF-2.

The effects of analogs of diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) were examined on the ADP-ribosylation reaction of histone H1 catalysed by purified bovine thymus poly(ADP-ribose)transferase. Among the compounds tested, epsilon Ap4A and ApCH2pppA were shown to be the most efficient inhibitors of the enzyme. From kinetic studies of their action, it appears that epsilon Ap4A and ApCH2pppA might be 'mixed type' inhibitors.

Many experimental data have been obtained regarding the physiological functions of poly(ADP-ribosyl)ation reactions. DNA replication, cell differentiation, transformation of cells, and DNA repair are among the processes in which poly(ADP-ribosyl)-ation is thought to be involved [1–3]. Inhibitors of ADP-ribosyltransferase (ADPRT) have been very useful in the attempt to ascribe a physiological role to these reactions in various cellular functions. 3-Aminobenzamide and 3-methoxybenzamide have been shown to be powerful competitive inhibitors of ADPRT [4]. Some experimental data obtained with the use of these inhibitors clearly indicate that poly(ADP-ribosyl)ation reactions are indeed crucial for cell differentiation [3] and the DNA strand break rejoining process [5]. On the other hand, the use of inhibitors often meets inconveniences in that their effects are not always limited to the desired metabolic pathway [6]. Tanaka et al. [7] have recently reported that diadenosine tetraphosphate, a ligand of a subunit of DNA polymerase α, could act as a very efficient inhibitor of ADPRT, but further data concerning its involvement in ADP-ribosylation reactions have not been reported so far. Poly amines, such as spermine and spermidine, are also known to have inhibitory effects on ADP-ribosylation of histone H1 [8], although their usefulness as a specific inhibitor of ADPRT remains doubtful. In the light of this circumstance, we have tried to examine the effect of various 2′,5′-oligoadenylates (2′,5′A) on the activity of ADPRT.

The enzyme poly(adenosine diphosphate ribose)polymerase was discovered in chicken liver nuclei in 1966 (Chambon et al., 1) and thoroughly studied in a number of laboratories, where its main properties have been investigated; major contributions in this regard have been given by the groups of Hayaishi (2) and Sugimura (3). The enzyme has many peculiar properties.

ABSTR. 62

Summary When native RNAase BS-1 x is treated with cyanogen bromide, one peptide (CB2) is cleaved from the rest of the protein (CB1) and can be isolated by gel filtration. Peptide CB2 has been sequenced and shown to be the N-terminal peptide of the protein, strictly homologous to the peptide 1–13 of RNAase A. A second fragment (peptide PF2) can be separated by gel filtration after performic acid oxidation of the CB1 fragment. The sequence of this peptide is partially homologous to the 14–29 sequence of RNAase A. From the recovery values determined for peptides CB2 and PF2, it appears that each peptide is present in both chains of RNAase BS-l. These and previous findings are briefly discussed in terms of structure to function relationships.

We have previously demonstrated that a significant percentage of poly(ADPR) polymerase is present, as a tightly-bound form, at the third level of chromatin organisation defined by chromosomal loops and nuclear matrix. The present work is focused on the study of poly(ADP-ribosyl)ation of proteins present in these nuclear subfractions. It has been shown that, due to the action of poly(ADPR) polymerase, the ADP-ribose moiety of [14C]NAD is transferred to both loosely-bound and tightly-bound chromosomal proteins, which in consequence are modified by chain polymers of ADP-ribose of different lengths. Moreover, histone-like proteins seem to be ADP-ribosylated in chromosomal loops and nuclear matrix associated regions of DNA loops (MARS). A hypothesis can be put forward that the ADP-ribosylation system is functionally related to the nuclear processes, actively coordinated by the nuclear matrix.

review article