Localization of an endogenous ADP-ribose acceptor, p33, in polymorphonuclear cell granules in chicken liver interlobular connective tissue

We investigated immunohistochemically the localization of p33, an endogenous substrate protein for an arginine-specific ADP-ribosyltransferase in chicken liver. Polymorphonuclear-pseudo-eosinophilic granulocytes (heterophils) in interlobular connective tissues of the liver were exclusively and strongly stained with the antibody against p33. Strong reactivity was associated with granules in cytoplasm of the heterophils. When the chicken liver nuclear fraction was washed, the transferase activity was released into the 600 x g supernatant fraction while a nuclear enzyme poly(ADP-ribose) synthetase was retained in the pellet fraction. These results indicate that p33 and probably also ADP-ribosyltransferase, found in the liver nuclear fraction [Tanigawa et al. (1984) J. Biol. Chem. 259, 2022-2029, Mishima et al. (1988) Eur. J. Biochem. 179, 267-273], originate from interlobular heterophils of the chicken liver.     

Reduction of mono(ADP-ribosyl)ation of 20 kDa protein with maturation in rat testis: involvement of guanine nucleotides

When the homogenate prepared from immature rat testes was incubated with [32P]NAD, several proteins (90, 39 and 20 kDa) were ADP-ribosylated in the absence of bacterial toxins. This observation suggested the existence of an endogenous ADP-ribosyltransferase and substrates. The data that the digested product by phosphodiesterase of ADP-ribosylated 20 kDa protein was 5'-AMP suggested that 20 kDa protein was mono(ADP-ribosyl)ated. In addition, the mono(ADP-ribosyl)ation of 20 kDa protein was enhanced by guanine nucleotides such as GTP, GDP and GTP[gamma S], and decreased by the concentrations of 10 mM Mg2+. In contrast, the incorporation of ADP-ribose moiety from NAD to both 90 and 39 kDa proteins was not changed by guanine nucleotides. On the other hand, mono(ADP-ribosyl)ation of 20 kDa protein was not observed in the homogenate prepared from other tissues of the same rats. Furthermore, we found that mono(ADP-ribosyl)ation of 20 kDa protein was decreased with the maturation of the rats and that an endogenous mono(ADP-ribosyl)transferase and 20 kDa protein were located in the nuclei.     

Mono(ADP-ribosyl)ation of Gi by eukaryotic cysteine-specific mono(ADP-ribosyl) transferase attenuates inhibition of adenylate cyclase by epinephrine

Eukaryotic cysteine-specific mono(ADP-ribosyl)transferase, named ADP-ribosyltransferase C (Tanuma, S., Kawashima, K. and Endo, H. (1988) J. Biol. Chem. 263, 5485-5489), attenuates inhibition of adenylate cyclase in human platelet membranes by epinephrine. This attenuation appeared to result from mono(ADP-ribosyl)ation by ADP-ribosyltransferase C of the inhibitory guanine nucleotide-binding protein (Gi) of adenylate cyclase. These results indicate a role of ADP-ribosyltransferase C in regulation of hormonal control of the adenylate cyclase system.     

ADP-ribosylation of actins by arginine-specific ADP-ribosyltransferase purified from chicken heterophils.

We reported the purification and characterization of an arginine-specific ADP-ribosyltransferase and acceptor protein p33 in granules of chicken peripheral polymorphonuclear leukocytes (heterophils) [Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K. & Shimoyama, M. (1991) J. Biochem. (Tokyo) 110, 388-394]. In the present study, we obtained evidence that chicken non-muscle beta/gamma-actin, skeletal muscle alpha-actin and smooth-muscle gamma-actin were ADP ribosylated by the heterophil ADP-ribosyltransferase. The stoichiometry of ADP-ribose incorporation into these actins was 1.2 mol, 1.0 mol and 2.0 mol ADP-ribose/mol of beta/gamma-actin, alpha-actin and gamma-actin, respectively. The optimal pH for the ADP ribosylation was at pH 8.5, with the respective actin. Km values for NAD were calculated to be 30 microM with beta/gamma-actin, 35 microM with alpha-actin and 20 microM with gamma-actin. The Km values for the actin isoforms were 15 microM for beta/gamma-actin, 2.5 microM for alpha-actin and 10 microM for gamma-actin. ADP ribosylation of actin inhibited its capacity to polymerize, as determined by the increase in fluorescence intensity with N-(1-pyrenyl)iodoacetamide-labelled actin. Filamentous actin (F-actin) polymerized with the respective actin isoform was also ADP ribosylated, although the extent of the modification of F-actin was lower than that of globular actin (G-actin). In situ ADP ribosylation of beta/gamma-actin was evidenced with chicken peripheral heterophils permeabilized with saponin. Thus, the endogenous ADP ribosylation of actin in the heterophils may be involved in the cellular processes such as phagocytosis, secretion and migration.     

Target protein for eucaryotic arginine-specific ADP-ribosyltransferase

Among ADP-ribosyltransferases reported in eucaryotes, arginine-specific transferases from turkey erythrocytes, chicken heterophils and rabbit skeletal muscle have been purified and extensively studied. They were reported to modify a number of proteinsin vitro. ADP-ribosylation of Ha-ras-p21 and transducin by the turkey erythrocyte transferase inhibits their GTPase and GTP-binding activities. Chicken heterophil enzyme modifies several substrate proteins for protein kinases and decreases the phosphate-acceptor activity. Rabbit skeletal muscle Ca²⁺-ATPase is inhibited by ADP-ribosylation catalyzed by the muscle transferase. Three transferases all ADP-ribosylate small molecular weight guanidino compounds such as arginine, arginine methylester and agmatine and poly-L-arginine and nuclear histones. However, the observation that muscle transferase did not ADP-ribosylate casein or actin, both of which can be modified by the heterophil transferase under the same conditions indicates that substrate specificity of these two enzymes are different. Substrate-dependent effects were observed with polyions of nucleotides such that polyanions stimulate the ADP-ribosylation of possible target protein, p33 by chicken heterophil transferase but has no effect on the modification of casein by the same enzyme.     

DNA-regulated arginine-specific mono(ADP-ribosyl)ation and de-ADP-ribosylation of endogenous acceptor proteins in human neutrophils

Arginine-specific mono(ADP-ribosyl)ation and de-ADP-ribosylation reactions of endogenous acceptor proteins were examined using human neutrophils. The cells contained arginine-specific ADP-ribosyltransferase, acceptor proteins and hydrolase catalyzing the release of ADP-ribose from the ADP-ribose/acceptor conjugate. One major acceptor protein with an apparent molecular mass of 27 kDa was detected in the neutrophils. The ADP-ribosylation of this protein was greatly enhanced when double-stranded DNA was added. The release of ADP-ribose from the ADP-ribosyl core-histones was suppressed. These findings provide clues as to the physiological function of neutrophil ADP-ribosyltransferase.     

ADP-Ribosylation of Histones in Nuclei Isolated from Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

Two-dimensional electrophoresis (2D-PAGE) of a histone fraction isolated from nuclei of embryos of the sea urchin Hemicentrotus pulcherrimus exhibited almost all histone species at all stages examined. At the gastrula stage, a spot of H1A became evident and three spots closely associated with one another were found in place of a single spot of H2A.1. In the histone fraction isolated from [adenylate-³²P] NAD⁺-treated nuclei of all stages examined, autoradiograms of 2D-PAGE exhibited spots of mono [ADP-ribosyl] ated H1 and polymodified H2B.2, H3.1, H3.3 and H4 but did not show ADP-ribosylated H2A.1, H2A.2 or H2B.1. Poly [ADP-ribosyl] ated H3.2, found in morulae, was not detectable in blastulae and gastrulae. Treatment with dimethylsulfate, known to activate ADP-ribosylation in other cell types, induced poly [ADP-ribosyl] ation of H2A.2 and H2B.1 in embryos at all stages examined, and also polymodification of H3.2 in gastrulae. ADP-ribosylation of H1, H2B.2, H3.1 and H3.3 was hardly affected by dimethylsulfate treatment, though modification of H4 was blocked by this treatment. Probably, strong regulation of ADP-ribosyltransferase reactions causes failures of modification of H2A.2 and H2B.1 throughout early development and also of H3.2 at the gastrula stage. Regulation of histone ADP-ribosylation is thought to alter chromatin structures and the rate of gene expression, contributing to cell differentiation.     

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture.

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15-60 microM) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher Km than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [32P]NAD+ and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.     

Differences in the poly(ADP-ribosyl)ation patterns of ICP4, the herpes simplex virus major regulatory protein, in infected cells and in isolated nuclei.

Infected-cell protein 4 (ICP4), the major regulatory protein in herpes simplex viruses 1 and 2, was previously reported to accept 32P from [32P]NAD in isolated nuclei. This modification was attributed to poly(ADP-ribosyl)ation (C. M. Preston and E. L. Notarianni, Virology 131:492-501, 1983). We determined that an antibody specific for poly(ADP-ribose) reacts with ICP4 extracted from infected cells, electrophoretically separated in denaturing gels, and electrically transferred to nitrocellulose. Our results indicate that all forms of ICP4 observed in one-dimensional gel electrophoresis are poly(ADP-ribosyl)ated. Poly(ADP-ribose) on ICP4 extracted from infected cells was resistant to cleavage by purified poly(ADP-ribose) glycohydrolase unless ICP4 was in a denatured state. Poly(ADP-ribose) added to ICP4 in isolated nuclei was sensitive to this enzyme. This result indicates that the two processes are distinct and may involve different sites on the ICP4 molecule.     

Retinyl phosphate mannose synthesis in rat liver membranes. Phospholipase sensitivity and phospholipid requirement.

A remarkable and immediate decrease in GDP-mannose:retinyl phosphate mannosyltransferase activity was found on pre-incubation of rat liver postnuclear membranes with phospholipase A2 or phospholipase C. Under the same conditions of pre-incubation (1 min at 37 degrees C) trypsin did not affect the enzyme activity, whereas pre-incubation for 30 min with trypsin and Pronase abolished enzyme activity. The lipid extract of untreated rat liver membranes partially restored enzyme activity after phospholipase treatment. Sphingomyelin was as active as the endogenous lipids. Other phospholipids were less active in the following order: phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol = phosphatidylserine. Dolichyl phosphate mannose synthesis was inhibited less (33%) by phospholipase C than was Ret-P-Man synthesis (98.5%) under identical conditions of incubation, which included 0.025% Triton. However, retinyl phosphate mannose synthesis by purified endoplasmic reticulum was found to be resistant to phospholipase C. Mixing experiments failed to demonstrate an inhibitory effect of the phospholipase-treated postnuclear membrane fraction on the synthetic activity of the endoplasmic reticulum, thus excluding the release of an inhibitory factor from the postnuclear membranes.     

Isolation of ADP-ribosyltransferase by affinity chromatography

An affinity adsorbent for ADP-ribosyltransferase (EC 2.4.2.30) has been synthesized by coupling 3-aminobenzamide to Sepharose 4B. Using this material, ADP-ribosyltransferase from human placenta has been purified from crude extract to homogeneity within a few hours. The enzyme has an apparent Km for NAD+ of 52 microM. Its molecular mass is 115,000 as determined by gel electrophoresis. The enzyme is DNA dependent and stimulated by histone, its temperature optimum is at 25 degrees C, and its pH optimum is around pH 9. alpha-NAD+, thymidine, caffeine, theophylline, theobromine, 3-methoxybenzamide, and nicotinamide inhibit the enzyme. Purification of ADP-ribosyltransferases from horse, rat, and chicken liver was also achieved with the method described.     

Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes

An NAD- and guanidine-dependent ADP-ribosyltransferase has been purified more than 500,000-fold from turkey erythrocytes with an 18% yield. The enzyme in the 100,000 X g supernatant fraction was bound to phenyl-Sepharose, eluted with 50% propylene glycol, and further purified by sequential chromatographic steps on carboxymethylcellulose, NAD-agarose and concanavalin A-agarose. The transferase was specifically eluted from concanavalin A-agarose with alpha-methylmannoside. The enzymatic activity was extremely labile following the first purification step. Both propylene glycol and NaCl stabilized the transferase; significant increases in enzyme recovery were obtained by conducting the NAD- and concanavalin A-agarose chromatography in buffer containing propylene glycol. The purified protein exhibits one predominant protein band on SDS-polyacrylamide gels with an estimated molecular weight of 28,300. On Ultrogel AcA54 chromatography, single coincident peaks of ADP-ribosyltransferase activity and protein were observed. Enzyme activity was independent of DNA; the highly purified transferase was inhibited by thymidine, nicotinamide, and theophylline. The specific activity of the purified enzyme (350 mumol of ADP-ribose transferred from NAD to arginine methyl estermin-1mg-1) is comparable to that reported for purified NAD glycohydrolases and poly(ADP-ribosyl)transferases.     

Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase

Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.     

ADP-ribosyltransferase from hen liver nuclei. Purification and characterization

Chromatin-bound ADP-ribosyltransferase from adult hen liver nuclei was purified to a homogeneous state through salt extraction, gel filtration, hydroxyapatite, phenyl-Sepharose, Cm-cellulose, and DNA-Sepharose. The ADP-ribosyltransferase has a pH optimum at 9.0 and does not require DNA for reaction. The purified enzyme has a molecular weight of 27,500 +/- 500. Agmatine sulfate, arginine methyl ester, histones, and casein proved to be effective acceptors for the ADP-ribose molecule. Among histones, H3 was most active, followed by H2a, H4, and H2b, in that order, the lowest activity seen with H1. With all the acceptors tested, the rate of nicotinamide release was in excess of the ADP-ribosylation. However, changes in the ratio of nicotinamide release to ADP-ribosylation seemed to depend on concentrations of the acceptor used. ADP-ribose-whole histones X adducts formed by ADP-ribosyltransferase served as initiators for poly(ADP-ribose) synthesis when these adducts were incubated in the presence of NAD, DNA, Mg2+, and the purified poly(ADP-ribose) synthetase, in which poly(ADP-ribose) formation can occur.     

ADP-ribosylation of proteins in nuclei and mitochondria from tissues rats of various age exposed gamma-radiation

Constitutive and gamma-induced ADP-ribosylation of nuclei and mitochondrial proteins in 2- and 29-month-old rats was studied. ADP-ribosylation was determined by binding of [3H]-adenin with the proteins after incubation of cellular organells in reaction mixture supplemented with [adenin-2,8-3H]-NAD. It was detected that the level of total protein ADP-ribosylation in the nuclei is 4.5-6.2 times higher than in the mitochondria. By inhibition of poly(ADP-ribose) polymerase (PARP) with 3-aminobenzamidine and treatment of ADP-ribosylated proteins with phosphodiesterase I, it was demonstrated that about 90% of [3H]-adenin bound by proteins in the nuclei and 70% in the mitochondria was the result of PARP activity. The level of total ADP-ribosylation of nuclear and mitochondrial proteins in the tissues of old rats was reliably lower than in young animals. This reduction of ADP-ribosylation in old animals is the result of the lower activity of PARP, not of mono(ADP-ribosyl) transferase (MART). The level of ADP-ribosylation of proteins in the nuclei of brain and spleen cells of 2-month-old rats irradiated with of 5 and 10 Gy was by 49-109% higher than in the control. At the same doses of radiation, the level of ADP-ribosylation of nuclear proteins in brain and spleen of old rats increased only by 29-65% compared to the control. Unlike cell nuclei, the radiation-induced activation of ADP-ribosylation in mitochondria was less expressed: the level of ADP-ribosylation increased by 34-37% in young rats and by 11-27% in old animals. This increased binding of ADP-ribose residues by the proteins of nuclei and mitochondria from tissues of gamma-irradiated rats is exceptionally conditioned by activation of poly(ADP-ribosyl)ation because the level of mono(ADP-ribosyl)ation remains constant. The results of this study enable the suggestion that poly(ADP-ribosyl)ation also occurs in the mitochondria of brain and spleen cells of the gamma-irradiated rats, though less pronounced than in cell the cell nuclei of these tissues. Thus, one of the probable causes of the less efficient repair of radiation-induced DNA damage in old organisms is a decline of both constitutive and induced poly(ADP-ribosyl)ation of proteins in cell nucleus and mitochondria.     

Relationship of phosphorylation and ADP-ribosylation using a synthetic peptide as a model substrate

Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is a good substrate for cholera toxin in comparison with the angiotensin peptides. Because kemptide contains two potential ADP-ribosylation sites and, is also a good substrate for cAMP-dependent protein kinase, it was possible to gain some insight into factors influencing the specificity of cholera toxin and to study the relationship between phosphorylation and ADP-ribosylation. The ADP-ribosylated products of kemptide were purified by high-performance liquid chromatography and characterized by peptide sequence analysis, trypsin digestion, and fast-atom bombardment mass spectrometry. The major product is mono(ADP-ribosyl)ated preferentially on the first arginyl residue and some mono(ADP-ribosyl)ation was observed to occur on the second arginine. The minor product is di(ADP-ribosyl)ated. The Km and Vmax for mono(ADP-ribosyl)ation of kemptide are approximately 4.3 +/- 1.2 mM and 38.1 +/- 5.5 nmol min-1 mg-1, respectively. Phosphorylated seryl residue of kemptide suppresses ADP-ribosylation of the arginyl residues by cholera toxin. Mono(ADP-ribosyl)ated kemptide is a poor substrate for the cAMP-dependent protein kinase in comparison with kemptide. Di(ADP-ribosyl)ated kemptide is not phosphorylated at all. These results suggest that a mere exposure of an arginyl residue in peptides is not a sufficient condition for effective ADP-ribosylation and that a relationship exists between ADP-ribosylation and phosphorylation.     

ADP-ribosyltransferase is highly conserved: purification and characterization of ADP-ribosyltransferase from a fish and its comparison with the human enzyme

Summary Covalent modification of proteins by ADP-ribosylation is a major mode of protein regulation in eukaryotic cells. ADP-ribosyltransferases have been characterized from mammals but little is known about these enzymes in lower vertebrates. We purified an ADP-ribosyltransferase (E.C. 2.4.2.30) from trout (Salmo trutta faris) by affinity chromatography and characterized it. The 11700-fold purified activity shows a major protein band at a molecular mass of 75000 kDa in a SDS-polyacrylamide gel.In situ reactivation of SDS gels showed the 75000 kDa protein to be enzymatically active, and additional enzymatically active bands at molecular masses of 115000, 90000 and 87000 kDa, respectively. The enzyme is capable of poly-ADP-ribosylation. It crossreacts with affinity purified antibodies raised against human poly(ADP-ribose)synthetase and, except for the temperature optimum, its properties strongly resemble the mammalian enzymes, indicating the conserved character of nuclear ADP-ribosyltransferases. The trout enzyme is DNA- and histone-dependent, has an optimal pH between 8 and 9 and an apparentK _m for NAD⁺ of 24 M. The temperature optimum is 10°C compared with 25°C for the human enzyme. Known ADP-ribosyltransferase inhibitors also inhibit the enzyme from trout. ribosyl-)residues from NAD⁺. It plays a central role in processes affecting DNA function such as DNA repair, recombination, differentiation, tumorigenic cell transformation and cell proliferation (Pekala and Moss 1983; Ueda and Hayaishi 1985; Creissen and Shall 1982; Borek et al. 1984; Lunec 1984; Cleaver et al. 1985; Tseng et al. 1987). It seems to be a central controller of cell physiology (Loetscher et al. 1987; Schweiger et al. 1987). ADP-ribosylation is high in proliferating cells whereas it is low in differentiated cells (Surowy and Berger 1983). Due to the important functions of ADP-ribosyltransferase this enzyme has been studied extensively but almost exclusively in mammals (Ueda and Hayaishi 1985). Only little is known about ADP-ribosyltransferases of lower vertebrates and of species of lower phylogenetic families. Hence it appeared of interest to us to study this enzyme in a fish. Here we report the purification to homogeneity and the characterization of ADP-ribosyltransferase from trout liver. We compared the fish enzyme with the human one and found very similar enzymatic properties and immunological crossreactivity. This indicates that ADP-ribosyltransferases are highly conserved.