Synthesis and Degradation of Poly(ADP-ribose) in Zebrafish Brain Exposed to Aluminum.

Poly(ADPribosyl)ation is a post-translational protein modification, catalyzed by poly(ADP-ribose) polymerase (PARPs) enzymes, responsible for ADP-ribose polymer synthesis (PAR) from NAD+. PAR turnover is assured by poly(ADPR) glycohydrolase (PARGs) enzymes. In our previous study, the altered histology of zebrafish brain tissue, resulting in demyelination and neurodegeneration also with poly(ADPribosyl)ation hyperactivation, was demonstrated after aluminum (Al) exposure for 10 and 15 days. On the basis of this evidence, the aim of the present research was to study the synthesis and degradation of poly(ADP-ribose) in the brain of adult zebrafish exposed to 11 mg/L of Al for 10, 15, and 20 days. For this reason, PARP and PARG expression analyses were carried out, and ADPR polymers were synthesized and digested. The data showed the presence of different PARP isoforms, among which a human PARP1 counterpart was also expressed. Moreover, the highest PARP and PARG activity levels, responsible for the PAR production and its degradation, respectively, were measured after 10 and 15 days of exposure. We suppose that PARP activation is related to DNA damage induced by Al, while PARG activation is needed to avoid PAR accumulation, which is known to inhibit PARP and promote parthanatos. On the contrary, PARP activity decrease at longer exposure times suggests that neuronal cells could adopt the stratagem of reducing polymer synthesis to avoid energy expenditure and allow cell survival.

Antioxidant Characterization of Six Tomato Cultivars and Derived Products Destined for Human Consumption.

The consumption of fresh tomatoes and processed tomato products is widespread in the Mediterranean diet. This fruit is a valuable source of antioxidants and plays an important role in preventing oxidative stress. This study aimed to investigate the content of antioxidants and measure the total antioxidant capacity (ABTS and DPPH assays) in the peel, pulp, and seed fractions of six tomato cultivars. Finally, some bioactive compounds and total antioxidant activity were also determined in homemade tomato purees, since such homemade production is commonplace in Southern Italy. The level of antioxidants and total antioxidant capacity in each fraction were also calculated based on their actual fresh weight in the whole tomato. The overall results indicated that the peel and seeds of all analysed tomato cultivars contribute significantly to the antioxidant charge of the fruits. Consequently, consuming tomatoes without peel and seeds results in a substantial loss of compounds beneficial for human health. Our results also showed that phenolic and lycopene content, as well as antioxidant activities in all purees are higher than in fresh tomatoes. Based on this evidence, producing homemade tomato puree is a good practice, and its consumption helps prevent oxidative stress damage.

A Molecular Mechanism to Explain the Nickel-Induced Changes in Protamine-like Proteins and Their DNA Binding Affecting Sperm Chromatin in Mytilus galloprovincialis: An In Vitro Study.

Nickel is associated with reproductive toxicity, but little is known about the molecular mechanisms of nickel-induced effects on sperm chromatin and protamine-like proteins (PLs). In the present work, we analyzed PLs from Mytilus galloprovincialis by urea-acetic acid polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and assessed their binding to DNA by Electrophoretic Mobility Shift Assay (EMSA) after exposing mussels to 5, 15, and 35 µM NiCl2 for 24 h. In addition, a time course of digestion with MNase and release of PLs from sperm nuclei by the NaCl gradient was performed. For all exposure doses, in AU-PAGE, there was an additional migrating band between PL-III and PL-IV, corresponding to a fraction of PLs in the form of peptides detected by SDS-PAGE. Alterations in DNA binding of PLs were observed by EMSA after exposure to 5 and 15 µM NiCl2, while, at all NiCl2 doses, increased accessibility of MNase to sperm chromatin was found. The latter was particularly relevant at 15 µM NiCl2, a dose at which increased release of PLII and PLIII from sperm nuclei and the highest value of nickel accumulated in the gonads were also found. Finally, at all exposure doses, there was also an increase in PARP expression, but especially at 5 µM NiCl2. A possible molecular mechanism for the toxic reproductive effects of nickel in Mytilus galloprovincialis is discussed.

Aluminium exposure leads to neurodegeneration and alters the expression of marker genes involved to parkinsonism in zebrafish brain.

Aluminium, despite being extremely widespread in the world, is a non-essential metal to human metabolism. This metal is known to have toxic effects on a variety of organs including the brain and is considered an etiological factor in neurodegenerative diseases. However, the molecular mechanisms by which aluminium exerts neurotoxic effects are not yet completely understood. Zebrafish is an animal model also used to study neurodegenerative diseases since the overall anatomical organization of the central nervous system is relatively conserved and similar to mammals. Adult zebrafish were exposed to 11 mg/L of Al for 10, 15, and 20 days and the neurotoxic effects of aluminium were analysed by histological, biochemical, and molecular evaluations. Histological stainings allowed to evaluation of the morphology of the brain parenchyma, the alteration of myelin and the activation of neurodegenerative processes. The expression of the Glial Fibrillary Acidic Protein, a marker of glial cells, was evaluated to observe the quantitative alteration of this important protein for the nervous system. In addition, the poly(ADP-ribose) polymerase activity was measured to verify a possible oxidative DNA damage caused by exposure to this metal. Finally, the evaluation of the markers involved in Parkinsonism was assessed by Real-Time PCR to better understand the role of aluminium in the regulation of genes related to Parkinson's neurodegenerative disease. Data showed that aluminium significantly affected the histology of cerebral tissue especially in the first periods of exposure, 10 and 15 days. This trend was also followed by the expression of GFAP. At longer exposure times, there was an improvement/stabilization of the overall neurological conditions and decrease in PARP activity. In addition, aluminium is involved in the deregulation of the expression of genes closely related to Parkinsonism. Overall, the data confirm the neurotoxicity induced by aluminium and shed a light on its involvement in neurodegenerative processes.

Role of Poly(ADP-Ribose) Polymerase (PARP) Enzyme in the Systemic Acquired Acclimation Induced by Light Stress in Phaseolus vulgaris L. Plants.

Plants are able to acclimate to environmental constraints through functional modifications that may also occur in tissues that are not directly exposed to stress. This process is termed "systemic acquired acclimation." The present study aims to evaluate the involvement of PolyADP-ribose) polymerase (PARP) protein in the acclimation process to high light (HL) stress in Phaseolus vulgaris plants. For this purpose, some leaves located at the top of the plant, in the apical position, were directly exposed to HL ("inducing" leaves), while others on the same plant, distal from the top, continued to be exposed to growth light ("receiving" leaves) to verify the hypothesis that an "alert" message may be transferred from injured tissues to distal ones. Biochemical and eco-physiological analyses, namely PARP activity, H2O2 and water- and fat-soluble antioxidants (i.e., ascorbic acid, tocopherol, glutathione (GSH), phenols, carotenoids, etc.) content, and chlorophyll fluorescence measurements were performed on both "inducing" and "receiving" leaves. Even if no change in PARP expression was found, its activity increased in "receiving" unstressed leaves in response to the light stress duration experimented by "inducing" leaves, while antioxidant capacity declined. When the "receiving" leaves were exposed to HL, the PARP activity returned to the control value, while antioxidant capacity photosynthetic electron transport rate (Jf) decreased and increased, respectively, compared to Control. Our results seem to show an acclimation pathway triggered in remote tissues not yet subjected to stress, likely involving a reactive oxygen species wave activating the PARP enzyme in a mechanism still to be clarified. In addition, the increased tolerance of plants directly exposed to HL could implicate a boosted synthesis of soluble antioxidants accompanied by a reduction of PARP activity to reduce excessive consumption of NAD(P).

Chemistry of Tropical Eucheumatoids: Potential for Food and Feed Applications.

The use of seaweeds as additives in animal nutrition may be a valid option to traditional feed as they represent a rich source of minerals, carbohydrates and antioxidants. The aim of this study was to analyze the chemical composition and in vitro antioxidant capacity of two tropical eucheumatoids, Kappaphycus alvarezii and Kappaphycus striatus, in Malaysian wild offshore waters. The chemical analysis was performed via inductively coupled plasma-optical emission spectroscopy for evaluating the concentration of toxic (Cd, Pb, Hg, As) and essential elements (Mn, Fe, Cu, Ni, Zn, Se); NMR spectroscopy was used for carrageenans investigation. Furthermore, the soluble and fat-soluble antioxidant capacities were determined by FRAP, DPPH and ABTS assays. The chemical analysis revealed a higher content of trace elements in K. alvarezii as compared to K. striatus, and both exhibited a high mineral content. No significant differences in metal concentrations were found between the two species. Both samples showed a mixture of prevailing κ- and t-carrageenans. Finally, the levels of soluble and fat-soluble antioxidants in K. alvarezii were significantly higher than in K. striatus. Our findings suggest that K. alvarezii could be used as a potential feed additive because of its favorable chemical and nutritional features.

In Sulfolobus solfataricus, the Poly(ADP-Ribose) Polymerase-Like Thermoprotein Is a Multifunctional Enzyme.

In Sulfolobus solfataricus, Sso, the ADP-ribosylating thermozyme is known to carry both auto- and heteromodification of target proteins via short chains of ADP-ribose. Here, we provide evidence that this thermoprotein is a multifunctional enzyme, also showing ATPase activity. Electrophoretic and kinetic analyses were performed using NAD+ and ATP as substrates. The results showed that ATP is acting as a negative effector on the NAD+-dependent reaction, and is also responsible for inducing the dimerization of the thermozyme. These findings enabled us to further investigate the kinetic of ADP-ribosylation activity in the presence of ATP, and to also assay its ability to work as a substrate. Moreover, since the heteroacceptor of ADP-ribose is the sulfolobal Sso7 protein, known as an ATPase, some reconstitution experiments were set up to study the reciprocal influence of the ADP-ribosylating thermozyme and the Sso7 protein on their activities, considering also the possibility of direct enzyme/Sso7 protein interactions. This study provides new insights into the ATP-ase activity of the ADP-ribosylating thermozyme, which is able to establish stable complexes with Sso7 protein.

Photo-Protective Mechanisms and the Role of Poly (ADP-Ribose) Polymerase Activity in a Facultative CAM Plant Exposed to Long-Term Water Deprivation.

The Crassulacean acid metabolism (CAM) pathway helps plants to alleviate the oxidative stress under drought, but the shift to CAM-idling may expose plants to the overproduction of reactive oxygen species causing cell damages. The facultative CAM species Portulacaria afra L., was subjected to long-term water deprivation to assess the photo-protective strategies and the poly (ADP-ribose) polymerase (PARP) activity during water stress and plant capability to recover from the stress. Measurements of titratable acidity, chlorophyll fluorescence emission, and antioxidant activity were performed during the stress and rewatering. Under water deprivation, plants shifted from C3 to CAM metabolism, reaching the CAM-idling status at the end of the stress period. The daily variation of the titratable acidity and PARP activity increased at the beginning of stress and declined with stress progression, reaching the lowest value at the end of stress treatment. H2O2 content, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities increased with the severity of water stress. The photochemical processes remained high during the entire stress period indicating the presence of alternative sinks to CO2 fixation. The elevated activity of catalase under severe water stress suggests the occurrence of photorespiration in sustaining the photosynthetic electron transport under CAM-idling condition. The overall data indicate that scavenger enzymes, photorespiration and PARP activity modulation contribute to the strong resistance of P. afra to severe water stress, preserving the functioning of photosynthetic apparatus and ensuring plant recovery with rewatering.

Functional and Structural Leaf Plasticity Determine Photosynthetic Performances during Drought Stress and Recovery in Two Platanus orientalis Populations from Contrasting Habitats.

In the context of climatic change, more severe and long-lasting droughts will modify the fitness of plants, with potentially worse consequences on the relict trees. We have investigated the leaf phenotypic (anatomical, physiological and biochemical) plasticity in well-watered, drought-stressed and re-watered plants of two populations of Platanus orientalis, an endangered species in the west of the Mediterranean area. The two populations originated in contrasting climate (drier and warmer, Italy (IT) population; more humid and colder, Bulgaria (BG) population). The IT control plants had thicker leaves, enabling them to maintain higher leaf water content in the dry environment, and more spongy parenchyma, which could improve water conductivity of these plants and may result in easier CO2 diffusion than in BG plants. Control BG plants were also characterized by higher photorespiration and leaf antioxidants compared to IT plants. BG plants responded to drought with greater leaf thickness shrinkage. Drought also caused substantial reduction in photosynthetic parameters of both IT and BG plants. After re-watering, photosynthesis did not fully recover in either of the two populations. However, IT leaves became thicker, while photorespiration in BG plants further increased, perhaps indicating sustained activation of defensive mechanisms. Overall, our hypothesis, that plants with a fragmented habitat (i.e., the IT population) lose phenotypic plasticity but acquire traits allowing better resistance to the climate where they became adapted, remains confirmed.

Long-term response of Dictyota dichotoma var. intricata (C. Agardh) Greville (Phaeophyceae) to ocean acidification: Insights from high pCO2 vents.

The ocean acidification may severely affect macroalgal communities of the shallowest coastal habitats where they play relevant structural and functional roles. In this paper were investigated the physiological traits of two populations of Dictyota dichotoma var. intricata, living at two different pH for several generations to assess the reasons of the algae reduced abundance at current (8.1) compared to low pH (6.7). Besides, through transplant experiments, the two populations were analysed for the stress response and reversibility of physiological performance at different pH. The long-term acclimation to high pCO2/low pH favours an ecotype characterised by low energetic costs, higher photochemical efficiency and more resistance to the oxidative stress, compared to individuals living at current pH. These traits promoted the growth and reproduction of the community living at pH 6.7, favouring a lower macroalgal diversity, but a higher ecological success under ocean acidification. The similar behaviour observed between Dictyota living at pH 6.7 and transplanted thalli from pH 6.7 to 8.1, suggested a high tolerance to pH changes in the short-term. On the contrary, adaptive responses may have favoured molecular adjustments on the long-term, as showed by the significant differences between the wild populations at pH 8.1 and 6.7. The overall data indicate that both plasticity and adaptive mechanisms may be the reasons for the success of the brown seaweeds under future high pCO2/low pH. The plasticity due to photochemistry adjustments is likely involved in the early response to environmental changes. Conversely, modifications in the photosynthetic biochemical machinery suggest that more complex adaptive mechanisms occurred in the current population of Dictyota living at pH 6.7. Further studies on population genetics will reveal if any differentiation is taking place at the population level or a local adaptation has already occurred in Dictyota and other brown algae under chronic low pH.

The Ageing Process Affects the Antioxidant Defences and the Poly (ADPribosyl)ation Activity in Cistus Incanus L. Leaves.

The ageing process in living organisms is characterised by the accumulation of several deleterious changes occurring in cells and tissues. The increase of reactive oxygen species with the advancement of age is responsible for the oxidative damage to proteins, lipids and DNA, enhancing the risk of diseases. The antioxidant response and the activation of the poly(ADP-ribosyl)ation process represent the first defences activated by organisms at all life stages to counteract damage to cell structures and genomic material. The regulation of poly(ADP ribosyl)ation with age is little known in plants, especially in combination with antioxidant defences modulation. In this study, the relationships between poly (ADP-ribose) polymerase (PARP) activity and enzymatic and non-enzymatic antioxidant pool have been studied together with the photosynthetic apparatus efficiency in the Mediterranean species Cistus incanus L., examining leaves at different developmental stages: young, mature and senescent. The photosynthetic performance was evaluated by chlorophyll a fluorescence measurement, the total soluble and fat-soluble antioxidant capacity, as well as the activities of enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione-S-transferase (GST), were determined by spectrophotometer, PARP activity was assessed by radioactive labelling. The highest photochemical activity was observed in young leaves, together with the highest GST activity. With the progress of the ageing process, the non-enzymatic antioxidant pool (namely ascorbic acid, α-tocopherol) declined, reaching the lowest value in senescent leaves, whereas PARP activity rose significantly. The overall results indicate that the decline of photosynthetic apparatus efficiency during senescence is due to the reduction of specific defences against oxidative damages, which increase the damages to DNA, as demonstrated by PARP activity rise.

Electron Spin Resonance (ESR) for the study of Reactive Oxygen Species (ROS) on the isolated frog skin (Pelophylax bergeri): A non-invasive method for environmental monitoring.

BACKGROUND: Reactive oxygen species (ROS) in biological tissues of elected biosentinels represent an optimal biomarker for eco-monitoring of polluted areas. Electron spin resonance (ESR) is the most definitive method for detecting, quantifying and possibly identifying radicals in complex systems. OBJECTIVE: A non-invasive method for monitoring polluted areas by the quantitative determination of ROS in frog skin biopsy is presented. METHODS: We assessed by ESR spectroscopy the ROS level in adult male of Pelophylax bergeri, specie not a risk of extinction, collected from the polluted Sarno River (SA, Italy) basin. The spin-trap ESR method was validated by immunohistochemical analysis of the well-assessed pollution biomarkers cytochrome P450 aromatase 1A (CYP1A) and glutathione S-transferase (GST), and by determining the poly(ADPribose) polymerase (PARP) and GST enzymatic activity. RESULTS: ROS concentration in skin samples from frogs collected in the polluted area is significantly higher than that determined for the unpolluted reference area. Immunohistochemical analysis of CYP1A and GST supported the reliability of our approach, even in the absence of evident morphological and ultrastructural differences. PARP activity assay, connected to possible oxidative DNA damage, and the detoxification index by GST enzymatic assay give statistically significant evidence that higher levels of ROS are associated to alterations of the different biomarkers. CONCLUSIONS: ROS concentration, measured by ESR on isolated frog skin, through the presented non-lethal method, is a reliable biomarker for toxicity screening and represents a useful basic datum for future modelling studies on environmental monitoring and biodiversity loss prevention.

Comparison of the DING protein from the archaeon Sulfolobus solfataricus with human phosphate-binding protein and Pseudomonas fluorescence DING counterparts.

DING proteins represent a new group of 40 kDa-related members, ubiquitous in living organisms. The family also include the DING protein from Sulfolobus solfataricus, functionally related to poly(ADP-ribose) polymerases. Here, the archaeal protein has been compared with the human Phosphate-Binding Protein and the Pseudomonas fluorescence DING enzyme, by enzyme assays and immune cross-reactivity. Surprisingly, as the Sulfolobus enzyme, the Human and Pseudomonas proteins display poly(ADP-ribose) polymerase activity, whereas a phosphatase activity was only present in Sulfolobus and human protein, despite the conserved phosphate-binding site residues in Pseudomonas DING. All proteins were positive to anti-DING antibodies and gave a comparable pattern of anti-poly(ADP-ribose) polymerase immunoreactivity with two bands, at around 40 kDa and roughly at the double of this molecular mass. The latter signal was present in all Sulfolobus enzyme preparations and proved not due to either a contaminant or a precursor protein, but likely being a dimeric form of the 40 kDa polypeptide. The common immunological and partly enzymatic behavior linking human, Pseudomonas and Sulfolobus DING proteins, makes the archaeal protein an important model system to investigate DING protein function and evolution within the cell.

Frog (Pelophylax bergeri, Günther 1986) endocrine disruption assessment: characterization and role of skin poly(ADP-ribose) polymerases.

Model of the our research was the adult male amphibian anura, Pelophylax bergeri, poikilotherm species not considered threatened by the IUCN, sampled in representative sites at different degree. In the first phase, a biochemical characterization of the ADP-ribosylation on the skin of barcoded amphibian anura collected from Matese Lake (clean reference site in CE, Italy) was carried out. Two PARP isoforms were evidence: the first of 66 kDa is localized into nucleus and activated by DNA damage; the second of 150 kDa is in cytoplasm, as demonstrated by biochemical and immunohistochemical analysis. Subsequently, the PARP activity, the quantitative expression of androgen receptor gene, and the levels of arsenic and chromium in skin and testis of frog and soil, water, and sediment collected from sites at different degrees of pollution were measured. A significant variation of PARP activity and androgen receptor expression levels was detected in both tissues of barcoded frogs from Sarno and Scafati, along Sarno River (SA, Italy), suggesting that a PARP activation is correlated to pollution and to steroid-regulated physiology disruption.

Physiological responses of a population of Sargassum vulgare (Phaeophyceae) to high pCO2/low pH: implications for its long-term distribution.

Ocean Acidification (OA) is likely to affect macroalgal diversity in the future with species-specific responses shaping macroalgal communities. In this framework, it is important to focus research on the photosynthetic response of habitat-forming species which have an important structural and functional role in coastal ecosystems. Most of the studies on the impacts of OA involve short-term laboratory or micro/mesocosm experiments. It is more challenging to assess the adaptive responses of macroalgal community to decreasing ocean pH over long-term periods, as they represent the basis of trophic dynamics in marine environments. This work aims to study the physiological traits of a population of Sargassum vulgare that lives naturally in the high pCO2 vents system in Ischia (Italy), in order to predict the species behaviour in a possible OA future scenario. With this purpose, the photosynthetic performance of S. vulgare was studied in a wild, natural population living at low pH (6.7) as well as in a population transplanted from native (6.7) to ambient pH (8.1) for three weeks. The main results show that the photochemical activity and Rubisco expression decreased by 30% after transplanting, whereas the non-photochemical dissipation mechanisms and the photosynthetic pigment content increased by 50% and 40% respectively, in order to compensate for the decrease in photochemical efficiency at low pH. Our data indicated a stress condition for the S. vulgare population induced by pH variation, and therefore a reduced acclimation capability at different pH conditions. The decline of the PSII maximum quantum yield (Fv/Fm) and the increase of PARP enzyme activity in transplanted thalli further supported this hypothesis. The absence of the species at ambient pH conditions close to the vent system, as well as the differences in physiological traits, suggest a local adaptation of S. vulgare at pH6.7, through optimization of photosynthetic performance.

Proliferative events and apoptotic remodelling in retinal development of common toad (Bufo bufo).

Proliferation and apoptosis are fundamental processes in the development of the retina, and a proper balance of the two phenomena is crucial to correct development of the organ. Despite intense investigation in different vertebrates, only a few studies have analyzed the cell death and the cell division quantitatively in the same species during development. Here we studied the time course of apoptosis and proliferation in the retina of common toad, Bufo bufo, and discuss the findings in an evolutionary perspective. We found cells that were dividing first scattered throughout the retina, then, in later stages, proliferation was confined to the ciliary marginal zone. This pattern was confirmed by the expression of the proliferative marker PCNA. Both proliferation and apoptosis occurred in successive waves, and two apoptotic peaks were detected: one at premetamorphosis 1 and the second at prometamorphosis. PARP-1, a known molecular marker of apoptosis, was used to confirm the data obtained by counting pyknotic nuclei. In summary, proliferative and apoptotic waves display an inverse time-relationship through development, with apoptotic peaks coinciding with low proliferation phases. In a comparative perspective, amphibians follow a developmental pattern similar to other vertebrates, although with different timing.

Synthesis and degradation of poly(ADP-ribose) in plants.

Poly(ADP-ribosylation) is a post-translational modification of proteins involved in a wide range of molecular and cellular processes in mammalian system. The main enzymes responsible for this modification are the poly(ADP-ribose) polymerases that catalyze the transfer of ADP-ribose moieties from NAD + to target protein acceptors, producing long and branched ADP-ribose polymers. The poly(ADP-ribosyl)ation is rapidly reverted by poly(ADP-ribose) glycohydrolase enzymes, which hydrolyzes poly(ADP-ribose) polymers, generating free ADP-ribose. So far, nine proteins with a poly(ADP-ribose) polymerase signature and two poly(ADP-ribose) glycohydrolase enzymes encoded by two adiacent genes were identified in Arabidopsis . The present review will describe the structures and functions of plant poly(ADP-ribose) polymerases and poly(ADP-ribose) glycohydrolases.

Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity.

Purification of the poly-ADP-ribose polymerase-like thermozyme from the archaeon Sulfolobus solfataricus.

Several different protocols have been developed to purify the ADP-ribosylating enzyme from Sulfolobus solfataricus. A number of techniques have been applied in regard to the crude homogenate preparation and protein extraction. Either mechanical cell lysis with DNAase digestion or freeze-thawing with sonication allowed to obtain fairly similar amounts of the thermozyme in the homogenate. While similar recovery of thermozyme was obtained by employing both purification protocols, the proteins were solubilized with different methods, and the affinity chromatography on NAD-Agarose of the first protocol was replaced by a gel filtration step in the second protocol. When enzyme activity was compared with electrophoresis and anti-poly-ADP-ribose polymerase 1 antibody immunoblotting results, it was noticed that lysis by sonication induces aggregation of monomeric PARP-like thermozyme at least in a dimeric form. The dimeric aggregate is also evidenced by treatment of cells with sonication followed by different protein extraction (Method III). Finally, we describe the third purification protocol that allows fast recovery of small amounts of purified ADP-ribosylating enzyme.