Glucocorticoid-induced reduction of poly (ADP-ribose) synthetase in nuclei from chick embryo liver.

The effect of glucocorticoid hormone administration on the nuclear poly(ADP-ribose) synthetase activity of chick embryoliver was investigated. Compared with the values obtained with control nuclei, the enzyme activity was markedly reduced in the nuclei of liver prepared from chick embryo treated with 0.1 mg hydrocortisone for 12 hours or longer. The possible relationship between the reduction of poly(ADP-ribose) synthetase activity and decrease in DNA synthesis is discussed.     

Poly (ADP-ribose) synthetase activity in rat testis mitochondria

A quite active poly (ADP-ribose) synthetase was found in isolated rat testis mitochondria. Similar levels of activity were found in mitochondria isolated from bull and hamster testis. In contrast, mitochondria isolated from rat brain or liver, and demembraned sperm, showed negligible activity. Centrifugation of testis mitochondria through a linear sucrose gradient, showed that, poly (ADP-ribose) synthetase cosediment together with succinatecytochrome c reductase and mitochondrial proteins. Furthermore, treatment with digitonin indicated that, the enzyme is localized in the inner membrane-matrix complex. Finally, kinetic studies demonstrated that, the apparent Km for NAD⁺ of the mitochondrial enzyme, was 22 μM compared with 210 μM for the nuclear enzyme.     

Developmental pattern of poly (ADP-ribose) synthetase and NAD glycohydrolase in the brain of the fetal and neonatal rat

Poly (ADP-ribose) synthetase and NAD glycohydrolase were examined in nuclear fractions from rat brain at sequential times during late fetal and the first two weeks of neonatal life. In whole brain, both enzymes were demonstrable at all stages of development, but followed separate patterns. Activity of the synthetase which was greatest in fetal life, fell steadily with fetal maturation from 3.90±0.06 nmol/mg DNA at 16 days, to reach a nadir of 1.36±0.09 nmol/mg DNA on the 4th postnatal day. Subsequently it underwent a non sustained neonatal rise reaching a peak of 2.46±0.07 nmol/mg DNA on the 8th day. By contrast, NAD glycohydrolase activity increased steadily throughout late fetal and during the first two weeks of neonatal life, from 12.77±0.40 nmol/mg DNA on day 16 of gestation to 25.80±.95 nmol/mg DNA on neonatal day 12. In neonatal cerebellum the activity of poly (ADP-ribose) synthetase was greater at 8 than at 4 days, could be stimulated with graded concentrations of sonicated DNA up to 100 g, but was inhibited by higher concentrations of DNA and by all concentrations of exogenous histone. In an in vitro culture system of fetal rat brain cells, the activity of poly (ADP-ribose) synthetase increased steadily over six days. Cycloheximide 10^–3 M completely inhibited the activity of this enzyme. NAD glycohydrolase activity increased progressively in vitro, and after 6 days in cycloheximide (10^–3 M), the cultures contained significantly greater levels of enzyme activity. It is suggested that changing activities of poly (ADP-ribose) synthetase and NAD glycohydrolase could both provide potential markers for brain cell differentiation in this system.     

Theophylline reduces poly (ADP-ribose) synthetase from chick embryo liver nuclei

The effect of theophylline on poly(ADP-ribosyl)ation was investigated. The poly(ADP-ribose) synthetase activity in vitro was markedly reduced in the liver nuclei prepared from theophylline-treated chick embryo. This reduction was not due to the enzyme inhibition by theophylline contamination in the nuclear fraction. The hydroxyapatite column chromatographic analysis of [3H]adenosine-labelled poly(ADP-ribose)molecules formed in vivo revealed that the in vivo formation of poly(ADP-ribose)molecules was also decreased by theophylline administration. The theophylline-induced reduction of poly(ADP-ribose) synthesis was not due to either low NAD levels or to a decrease in the chain length of the poly(ADP-ribose) molecule, rather this reduction was derived from a decrease in the number of poly(ADP-ribose) molecule. Possible mechanisms related to reduction of poly(ADP-ribose) synthesis in vivo are discussed.     

Theophylline reduces poly(ADP-ribose) synthetase from chick embryo liver nuclei

The effect of theophylline on poly(ADP-ribosyl)ation was investigated. The poly(ADP-ribose) synthetase activity in vitro was markedly reduced in the liver nuclei prepared from theophylline-treated chick embryo. This reduction was not due to the enzyme inhibition by theophylline contamination in the nuclear fraction. The hydroxyapatite column chromatographic analysis of [³H]adenosine-labelled poly(ADP-ribose) molecules formed in vivo revealed that the in vivo formation of poly(ADP-ribose) molecules was also decreased by theophylline administration. The theophylline-induced reduction of poly(ADP-ribose) synthesis was not due to either low NAD levels or to a decrease in the chain length of the poly(ADP-ribose) molecule, rather this reduction was derived from a decrease in the number of poly(ADP-ribose) molecules. Possible mechanisms related to reduction of poly(ADP-ribose) synthesis in vivo are discussed.     

3T3-L1 preadipocyte differentiation and poly(ADP-ribose) synthetase

Differentiation of 3T3-L1 preadipocytes, induced by methyl-isobutylxanthine (MIX), dexamethasone (DEX), and insulin, results in cells with the morphological and biochemical characteristics of adipocytes. Following incubation of 3T3-L1 cells with MIX, DEX, and insulin, poly(ADP-ribose) synthetase activity decreased abruptly, remained low for several hours and then increased; this rise was delayed by readdition of MIX, DEX, and insulin. The transient reduction in poly(ADP-ribose) synthetase activity in 3T3-L1 cells occurred prior to the appearance of the adipocyte phenotype induced by the above agents. It was not observed when preparations were assayed in the presence of DNase I, indicating that poly(ADP-ribose) synthetase activity was masked following treatment with MIX, DEX, and insulin. The change in synthetase activity represents the earliest alteration of a specific enzyme yet detected during the differentiation of 3T3-L1 cells. It appears to be differentiation specific since nondifferentiating 3T3-C2 control cells did not exhibit changes in poly(ADP-ribose) synthetase activity when treated with MIX, DEX, and insulin. The transient reduction in activity may be an early event in differentiation which reflects changes in chromatin structure.     

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.     

Increased ionic strength: effects on DNA fragmentation and poly(ADP-ribose) polymerase activity in HeLa nuclei

Poly(ADP-ribose) polymerase activity was measured in a crude nuclear fraction isolated from HeLa cells. It was found that the addition of ammonium sulfate or other salts to the standard incubation medium inhibited the formation of poly(ADP-ribose). Through the use of alkaline sucrose density gradients it was also noted that this same increase in ionic strength inhibited the in vitro breakdown of the HeLa DNA. Additional experiments with alkaline sucrose density gradients and deoxyribonuclease I showed that the in vitro activity of poly(ADP-ribose) polymerase is largely dependent upon DNA fragmentation but that DNA fragmentation at least in vitro is not dependent upon the formation of poly(ADP-ribose). These observations imply that this nuclear enzyme is not extremely sensitive to changes in the ionic strength of the reaction media but is affected indirectly, supposedly through changes in the endonuclease activity of the HeLa nuclei. If this proves to be true, then the addition of salt to the incubation medium for poly(ADP-ribose) polymerase could prove to be a valuable tool for the study of ADP-ribosylation reactions.     

Measurement of poly(ADP-ribose) glycohydrolase activity by high resolution polyacrylamide gel electrophoresis: Specific inhibition by histones and nuclear matrix proteins

We have developed a novel enzyme assay that allows the simultaneous determination of noncovalent interactions of poly(ADP-ribose) with nuclear proteins as well as poly(ADP-ribose) glycohydrolase (PARG) activity by high resolution polyacrylamide gel electrophoresis. ADP-ribose chains between 2 and 70 residues in size were enzymatically synthesized with pure poly(ADP-ribose) polymerase (PARP) and were purified by affinity chromatography on a boronate resin following alkaline release from protein. This preparation of polymers of ADP-ribose was used as the enzyme substrate for purified PARG. We also obtained the nuclear matrix fraction from rat liver nuclei and measured the enzyme activity of purified PARG in the presence or absence of either histone proteins or nuclear matrix proteins. Both resulted in a marked inhibition of PARG activity as determined by the decrease in the formation of monomeric ADP-ribose. The inhibition of PARG was presumably due to the non-covalent interactions of these proteins with free ADP-ribose polymers. Thus, the presence of histone and nuclear matrix proteins should be taken into consideration when measuring PARG activity.     

Preferential Perinuclear Localization of Poly(ADP-ribose) Glycohydrolase

The transient nature of poly(ADP-ribosyl)ation, a posttranslational modification of nuclear proteins, is achieved by the enzyme poly(ADP-ribose) glycohydrolase (PARG) which hydrolyzes the poly(ADP-ribose) polymer into free ADP-ribose residues. To investigate the molecular size and localization of PARG, we developed a specific polyclonal antibody directed against the bovine PARG carboxy-terminal region. We found that PARG purified from bovine thymus was recognized as a 59-kDa protein, while Western blot analysis of total cell extracts revealed the presence of a unique 110-kDa protein. This 110-kDa PARG was mostly found in postnuclear extracts, whereas it was barely detectable in the nuclear fractions of COS7 cells. Further analysis by immunofluorescence revealed a cytoplasmic perinuclear distribution of PARG in COS7 cells overexpressing the bovine PARG cDNA. These results provide direct evidence that PARG is primarily a cytoplasmic enzyme and suggest that a very low amount of intranuclear PARG is required for poly(ADP-ribose) turnover.     

Cellular regulation of ADP-ribosylation of proteins. IV. Conversion of poly(ADP-ribose) polymerase activity to NAD-glycohydrolase during retinoic acid-induced differentiation of HL60 cells.

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.     

Poly(ADP-ribose) degradation by post-nuclear extracts from human cells

The nuclear metabolism of poly(ADP-ribose) is mainly regulated by poly(ADP-ribose) polymerase-1 (PARP-1) and by poly(ADP-ribose) glycohydrolase (PARG). A PARP-like enzyme, V-PARP, and a PARG isoform are present in the extra-nuclear compartment of mammalian cells, even if poly(ADP-ribose) has never been detected therein. In this work, we demonstrate the ability of post-nuclear extracts from HeLa and HL60 cells to degrade synthetic 32P-polymers of ADP-ribose to ADP-ribose and, further, to AMP. This reaction implies the combined action of PARG and of an ADP-ribose-degrading activity, possibly corresponding to a phosphodiesterase and/or to an ADP-ribose pyrophosphatase. The inhibition of PARG or ADP-ribose-degrading enzymes allowed the demonstration that in vitro synthesized 32P-poly(ADP-ribose) is first digested to ADP-ribose monomers by a typical PARG reaction, and that ADP-ribose is further rapidly converted into AMP by an Mg(2+)-dependent activity. Collectively, our results demonstrate the ability of the human cell post-nuclear fraction to convert synthetic poly(ADP-ribose) into utilizable AMP units by the concerted action of PARG and ADP-ribose-degrading activities.     

The relationship between adenosine diphosphate-ribosylation and mammary gland differentiation

Poly(adenosine diphosphate [ADP]-ribosyl)ation, although associated with differentiation in many systems, exhibited a reciprocal relationship with mammary gland differentiation, and both the synthetic and degradatory pathways complemented each other in this regard. Poly(ADP-ribosyl)synthetase activity declined during pregnancy and lactation, while poly(ADP-ribose) degradatory activity rose late in pregnancy and peaked during lactation. In explant cultures, similar changes occurred and appeared to be under separate hormonal control; prolactin suppressed the synthetase activity, whereas insulin stimulated the poly(ADP-ribosyl)glycohydrolase activity. This latter effect may be mediated by a decline in cAMP levels for the following reasons: the glycohydrolase is known to be inhibited by cAMp, insulin decreased cAMP concentrations in mammary explants by 70%, and cholera toxin blocked the effects of insulin on poly(ADP-ribose) degradation. This reciprocal relationship between poly(ADP-ribosyl)ation and mammary gland differentiation is further supported by pharmacological studies: in the presence of insulin, cortisol, and prolactin, an inhibitor of the synthetase stimulated alpha-lactalbumin three-fold over hormone stimulation alone. However, this inhibitor was unable to induce differentiation in the absence of prolactin. Therefore, although there is a close association between a decline in enzyme activity and mammary differentiation, the data are insufficient to support a causal relationship.     

In vivo ischemia and storage injury cause alterations in the activity of poly(adenosine diphosphate ribose) synthetase

The objective of this study was to determine if DNA damage caused by ischemic insult (blood depletion) causes an alteration in the activity of endogenous mouse kidney poly(ADP-ribose) synthetase. The results show that kidneys made nonviable by warm (37 degrees C) in vitro ischemia (organ storage to study the effects of blood loss at normal body temperature) and in vivo ischemia (surgical depletion of the blood supply by arterial clamping) exhibit decreased levels of enzyme activity. Kidneys made nonviable by cold (0 degrees C) storage injury (organ storage as utilized for transplantation), however, possess elevated levels of enzyme activity. The DNA isolated from ischemic kidneys was shown to have a stimulatory effect upon exogenous calf thymus poly(ADP-ribose) synthetase. Also, electron microscopy analysis of DNA from ischemic kidneys showed that cold storage injury leads to the formation of large (average size = 500 bases) single-stranded regions. The results suggest that the activities of both endogenous and exogenous poly(ADP-ribose) synthetase are related to the nature of DNA damage resulting from ischemic insult.     

Purification and properties of poly(ADP-ribose) synthetase from mouse testicle

Poly(ADP-ribose) synthetase has been purified to apparent homogeneity from mouse testicle by a rapid and simple procedure using column chromatography on DNA-agarose and on Cibacron blue F₃G-A-Sephadex G-150. The purified enzyme absolutely requires DNA for activity, and half-maximal activation occurs at a DNA concentration of 25 μg/ml. The K_m for NAD and V at pH 8.0 and 25 °C are 47 μm and 1400 nmol/min/ mg, respectively. The molecular weight is 116,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicates that the mouse testicle enzyme is very similar to calf thymus enzyme, but there is a difference in the contents of several amino acid residues between the two enzymes. This difference appears to reflect species or tissue specificity of poly(ADP-ribose) synthetase.     

Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.     

Immunohistochemical demonstration of poly(adenosine diphosphate-ribose) synthetase in bovine tissues

The inter- and intracellular localization of poly(adenosine diphosphate-ribose)(poly(ADP-ribose] synthetase was investigated using an indirect immunofluorescence technique and a specific antibody against the enzyme purified from calf thymus. In various bovine tissues, including liver, heart, pancrease, thyroid, spleen, adrenal, and skeletal muscle, the specific immunofluorescence of poly(ADP-ribose) synthetase was localized exclusively in the nucleus. Immunostaining was inhibited by preabsorption of the antibody with purified calf thymus poly(ADP-ribose) synthetase. Nuclear immunofluorescence appeared to be more prominent in the marginal area than in the central region in most nuclei. This staining pattern is similar to that of naturally occurring poly(ADP-ribose). In bovine peripheral blood the immunofluorescence of poly(ADP-ribose) synthetase was detected in nuclei of lymphocytes, but not in granulocytes, in agreement with the finding that the enzymatic activity of poly(ADP-ribose) synthetase was barely detectable in nuclei isolated from granulocytes.     

Growth hormone and rat liver mitochondria effects on urea cycle enzymes

Effects of hypophysectomy and subsequent growth hormone administration on mitochondrial enzymes of the urea cycle were investigated in rat liver. Hypophysectomy increased the activities of the two mitochondrial enzymes, carbamyl phosphate synthetase and ornithine transcarbamylase but not of the cytosolic enzyme, argininosuccinate synthetase. The activity of mitochondrial phosphate dependent glutaminase was not affected. Administration of bovine growth hormone (100 μg/100 g body weight) for two weeks decreased the activities of carbamyl phosphate synthetase and ornithine transcarbamylase almost to the normal level. These results suggest a specific effect of growth hormone on mitochondrial enzymes of the urea cycle and serve to explain the increased urea formation in hypopituitarism.