RESUMO
The interaction of protoporphyrin compounds of human origin with the major bee venom component melittin (26 a.a., Z +6) and its hybrid derivative (CM15, 15 a.a., Z +6) were studied by a combination of various spectroscopic methods. Throughout a two-state, concentration-dependent process, hemin and its metabolites (biliverdin, bilirubin, bilirubin ditaurate) increase the parallel ß-sheet content of the natively unfolded melittin, suggesting the oligomerization of the peptide chains. In contrast, α-helix promoting effect was observed with the also disordered but more cationic CM15. According to fluorescence quenching experiments, the sole Trp residue of melittin is the key player during the binding, in the vicinity of which the first pigment molecule is accommodated presumably making indole-porphyrin π-π stacking interaction. As circular dichroism titration data suggest, cooperative association of additional ligands subsequently occurs, resulting in multimeric complexes with an apparent dissociation constant ranged from 20 to 65 µM. Spectroscopic measurements conducted with the bilirubin catabolite urobilin and stercobilin refer to the requirement of intact dipyrrinone moieties for inducing secondary structure transformations. The binding topography of porphyrin rings on a model parallel ß-sheet motif was evaluated by absorption spectroscopy and computational modeling showing a slipped-cofacial binding mode responsible for the red shift and hypochromism of the Soret band. Our results may aid to recognize porphyrin-responsive binding motifs of biologically relevant, intrinsically disordered peptides and proteins, where transient conformations play a vital role in their functions.
Assuntos
Anti-Infecciosos/química , Pigmentos Biliares/farmacologia , Hemina/farmacologia , Proteínas Intrinsicamente Desordenadas/química , Peptídeos Catiônicos Antimicrobianos/química , Meliteno/química , Estrutura Secundária de Proteína/efeitos dos fármacosRESUMO
The bilin-binding proteins (BBP) from lepidopteran insects are members of the lipocalin family of proteins and play a special role in pigmentation through the binding of biliverdin IXγ. Lopap, a BBP-like protein from the venom of the toxic caterpillar Lonomia obliqua has been reported to act as a serine protease that activates the coagulation proenzyme prothrombin. Here we show that BBPLo, a variant of lopap from the same organism binds biliverdin IXγ, forming a complex that is spectrally identical with previously described BBP proteins. Although BBPLo is nearly identical in sequence to lopap, no prothrombinase activity was detected in our recombinant preparations using reconstituted systems containing coagulation factors Xa and Va, as well as anionic phospholipids. In addition to biliverdin, BBPLo was found to form a 1:1 complex with heme prompting us to examine whether the unusual biliverdin IXγ ligand of BBPs forms as a result of oxidation of bound heme in situ rather than by a conventional heme oxygenase. Using ascorbate or a NADPH(+)-ferredoxin reductase-ferredoxin system as a source of reducing equivalents, spectral changes are seen that suggest an initial reduction of heme to the Fe(II) state and formation of an oxyferrous complex. The complex then disappears and a product identified as a 5-coordinate carbonyl complex of verdoheme, an intermediate in the biosynthesis of biliverdin, is formed. However, further reaction to form biliverdin was not observed, making it unlikely that biliverdin IXγ is formed by this pathway.
Assuntos
Pigmentos Biliares/química , Endopeptidases/metabolismo , Proteínas de Insetos/metabolismo , Lepidópteros/enzimologia , Sequência de Aminoácidos , Animais , Pigmentos Biliares/farmacologia , Endopeptidases/química , Ferredoxina-NADP Redutase/química , Heme/análogos & derivados , Heme/química , Heme/farmacologia , Proteínas de Insetos/química , Ligantes , Dados de Sequência Molecular , Oxirredução , Ligação Proteica , Especificidade por SubstratoRESUMO
In vitro anti-genotoxic properties of bile pigments have been explored and confirmed recently. Despite these reports mechanisms to explain DNA protection by endogenous bile pigments remain unclear. Surprisingly, the quantification of cellular pigment absorption which could represent a fundamental prerequisite for intracellular (e.g., anti-mutagenic) effects, has not been explored. Therefore, we aimed to measure the amounts of un-/conjugated bilirubin as well as biliverdin absorbed into colonies of Salmonella typhimurium, utilising HPLC analyses, and to observe whether intracellular compound concentrations could predict anti-genotoxic effects. HPLC analyses confirmed that bacterial bile pigment absorption was concentration-dependent. Plate bile pigment concentrations were inversely associated with genotoxicity of all tested mutagens, irrespective of strain and test conditions. However, protection against frame-shift mutation in strain TA98 most strongly depended on the bacterial absorption of bilirubin and biliverdin, which indicates that bile pigments can protect by intercepting mutations extracellularly and specifically inhibit frame-shift mutations intracellularly.
Assuntos
Antimutagênicos/farmacologia , Pigmentos Biliares/farmacologia , Mutagênicos/toxicidade , Salmonella typhimurium/efeitos dos fármacos , Bilirrubina/metabolismo , Biliverdina/metabolismo , Testes de Mutagenicidade , Mutação , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMO
Epidemiological studies report a negative association between circulating bilirubin concentrations and the risk for cancer and cardiovascular disease. Structurally related tetrapyrroles also possess in vitro anti-genotoxic activity and may prevent mutation prior to malignancy. Furthermore, few data suggest that tetrapyrroles exert anti-carcinogenic effects via induction of cell cycle arrest and apoptosis. To further investigate whether tetrapyrroles provoke DNA-damage in human cancer cells, they were tested in the single cell gel electrophoresis assay (SCGE). Eight tetrapyrroles (unconjugated bilirubin, bilirubin ditaurate, biliverdin, biliverdin-/bilirubin dimethyl ester, urobilin, stercobilin and protoporphyrin) were added to cultured Caco2 and HepG2 cells and their effects on comet formation (% tail DNA) were assessed. Flow cytometric assessment (apoptosis/necrosis, cell cycle, intracellular radical species generation) assisted in revealing underlying mechanisms of intracellular action. Cells were incubated with tetrapyrroles at concentrations of 0.5, 5 and 17µM for 24h. Addition of 300µM tertiary-butyl hydroperoxide to cells served as a positive control. Tetrapyrrole incubation mostly resulted in increased DNA-damage (comet formation) in Caco2 and HepG2 cells. Tetrapyrroles that are concentrated within the intestine, including protoporphyrin, urobilin and stercobilin, led to significant comet formation in both cell lines, implicating the compounds in inducing DNA-damage and apoptosis in cancer cells found within organs of the digestive system.
Assuntos
Dano ao DNA/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Neoplasias/patologia , Tetrapirróis/metabolismo , Tetrapirróis/farmacologia , Antioxidantes/farmacologia , Pigmentos Biliares/farmacologia , Bilirrubina/análogos & derivados , Bilirrubina/farmacologia , Células CACO-2 , Ensaio Cometa , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo , Células Hep G2 , Humanos , Neoplasias/genética , Concentração Osmolar , Protoporfirinas/farmacologia , Urobilina/farmacologiaRESUMO
Chemical defenses are used by many organisms to avoid predation, and these defenses may function by stimulating predators' chemosensory systems. Our study examined detection mechanisms for components of defensive ink of sea hares, Aplysia californica, by predatory sea catfish, Ariopsis felis. Behavioral analyses show aplysioviolin and phycoerythrobilin are detected intra-orally and by barbels and are deterrent at concentrations as low as 0.1% full strength. We performed electrophysiological recordings from the facial-trigeminal nerve complex innervating the maxillary barbel and tested aplysioviolin, phycoerythrobilin, amino acids, and bile salts in cross-adaptation experiments. Amino acids and bile salts are known stimulatory compounds for teleost taste systems. Our results show aplysioviolin and phycoerythrobilin are equally stimulatory and completely cross-adapt to each other's responses. Adaptation to aplysioviolin or phycoerythrobilin reduced but did not eliminate responses to amino acids or bile salts. Adaptation to amino acids or bile salts incompletely reduced responses to aplysioviolin or phycoerythrobilin. The fact that cross-adaptations with aplysioviolin and phycoerythrobilin were not completely reciprocal indicates there are amino acid and bile salt sensitive fibers insensitive to aplysioviolin and phycoerythrobilin. These results indicate two gustatory pathways for aplysioviolin and phycoerythrobilin: one independent of amino acids and bile salts and another shared with some amino acids.
Assuntos
Aplysia/fisiologia , Peixes-Gato/fisiologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Ficobilinas/farmacologia , Ficoeritrina/farmacologia , Pigmentos Biológicos/farmacologia , Comportamento Predatório/efeitos dos fármacos , Paladar/efeitos dos fármacos , Adaptação Fisiológica/efeitos dos fármacos , Algoritmos , Aminoácidos/farmacologia , Animais , Ácidos e Sais Biliares/farmacologia , Pigmentos Biliares/farmacologia , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Masculino , Maxila/fisiologia , Boca/fisiologia , Vias Neurais/fisiologia , Órgãos dos Sentidos/fisiologiaRESUMO
Bile pigments, including bilirubin and biliverdin, are endogenous compounds belonging to the porphyrin family of molecules. In the past, bile pigments and bilirubin in particular were thought of as useless by-products of heme catabolism that can be toxic if they accumulate. However, in the past 20 years, research probing the physiological relevance of bile pigments has been mounting, with evidence to suggest bile pigments possess significant antioxidant and anti-mutagenic properties. More specifically, bile pigments are potent peroxyl radical scavengers and inhibit the mutagenic effects of a number of classes of mutagens (polycyclic aromatic hydrocarbons, heterocyclic amines, oxidants). Coincidentally, persons with elevated circulating bilirubin concentrations have a reduced prevalence of cancer and cardio-vascular disease. Despite the encouraging in vitro anti-mutagenic effects of bile pigments, relatively little research has been conducted on their inhibitory capacity in bacterial and cultured cell assays of mutation, which might link the existing in vitro and in vivo observations. This is the first review to summarise the published data and it is our hope it will stimulate further research on these potentially preventative compounds.
Assuntos
Antimutagênicos/metabolismo , Pigmentos Biliares/metabolismo , Sequestradores de Radicais Livres/metabolismo , Animais , Antimutagênicos/química , Antimutagênicos/farmacologia , Pigmentos Biliares/química , Pigmentos Biliares/farmacologia , Biliverdina/química , Biliverdina/metabolismo , Biliverdina/farmacologia , Proliferação de Células/efeitos dos fármacos , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Humanos , Estrutura Molecular , Oxidantes/antagonistas & inibidores , Oxidantes/metabolismoRESUMO
The aim of this study was to explore the potential pro- and anti-mutagenic effects of endogenous bile pigments unconjugated bilirubin (BR), biliverdin (BV) and a synthetic, water soluble conjugate, bilirubin ditaurate (BRT) in the Ames Salmonella test. The bile pigments were tested over a wide concentration range (0.01-2 micromol/plate) in the presence of three bacterial strains (TA98, TA100, TA102). A variety of mutagens including benzo[alpha]pyrene (B[alpha]P), 2,4,7 trinitrofluorenone (TNFone), 2-aminofluorene (2-AF), sodium azide (NaN(3)) and tertiary-butyl hydroperoxide (t-BuOOH), were used to promote the formation of mutant revertants. Tests were conducted with (B[alpha]P, 2-AF, t-BuOOH) and without (TNFone, NaN(3), t-BuOOH) metabolic activation incorporating the addition of the microsomal liver preparation, S9. The bile pigments alone did not induce mutagenicity in any of the strains tested (p>0.05). Anti-mutagenic effects of the bile pigments were observed in the presence of all mutagens except for NaN(3) and the anti-mutagenic effects appeared independent of the strain tested. For TNFone induced genotoxicity, the order of effectiveness was BR> or =BRT>BV. However, the order was BV> or =BRT> or =BR for 2-AF. Antioxidant testing in the TA102 strain revealed bile pigments could effectively inhibit the genotoxic effect of t-BuOOH induced oxidative stress. The apparent antioxidant and anti-mutagenic behaviour of bile pigments further suggests their presence in biological systems is of possible physiological importance.
Assuntos
Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Pigmentos Biliares/farmacologia , Animais , Antimutagênicos/química , Antioxidantes/química , Pigmentos Biliares/química , Técnicas In Vitro , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Ratos , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMO
In the present in vitro and in vivo study we investigated the pro-oxidant effects of hemoglobin, as well as the antioxidant effects of its metabolites, in the brain. Incubation of rat brain homogenates with hemoglobin (0-10 microM) but not hemin induced lipid peroxidation up to 24 h (EC50 = 1.2 microM). Hemoglobin's effects were similar to ferrous ion (EC50 = 1.7 microM) and were blocked by the chelating agent deferoxamine (IC50 0.5 microM) and a nitric oxide-releasing compound S-nitrosoglutathione (IC50 = 40 microM). However, metabolites of hemoglobin - biliverdin and bilirubin - inhibited brain lipid peroxidation induced by cell disruption and hemoglobin (biliverdin IC50 = 12-30 and bilirubin IC50 = 75-170 microM). Biliverdin's antioxidative effects in spontaneous and iron-evoked lipid peroxidation were further augmented by manganese (2 microM) since manganese is an antioxidative transition metal and conjugates with bile pigments. Intrastriatal infusion of hemoglobin (0-24 nmol) produced slight, but significant 20-22% decreases in striatal dopamine levels. Whereas, intrastriatal infusion of ferrous citrate (0-24 nmol) dose-dependently induced a greater 66% depletion of striatal dopamine which was preceded by an acute increase of lipid peroxidation. In conclusion, contrary to the in vitro results hemoglobin is far less neurotoxic than ferrous ions in the brain. It is speculated that hemoglobin may be partially detoxified by heme oxygenase and biliverdin reductase to its antioxidative metabolites in the brain. However, in head trauma and stroke, massive bleeding could significantly produce iron-mediated oxidative stress and neurodegeneration which could be minimized by endogenous antioxidants such as biliverdin, bilirubin, manganese and S-nitrosoglutathione.
Assuntos
Pigmentos Biliares/metabolismo , Encéfalo/metabolismo , Glutationa/análogos & derivados , Hemoglobinas/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Compostos Nitrosos/metabolismo , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/metabolismo , Pigmentos Biliares/farmacologia , Bilirrubina/metabolismo , Bilirrubina/farmacologia , Biliverdina/metabolismo , Biliverdina/farmacologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Ácido Cítrico , Desferroxamina/farmacologia , Compostos Ferrosos/farmacologia , Glutationa/metabolismo , Glutationa/farmacologia , Hemoglobinas/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Manganês/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Compostos Nitrosos/farmacologia , Oxidantes/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , S-Nitrosoglutationa , Extratos de Tecidos/metabolismoRESUMO
Using recently developed molecular-shape description algorithms, we searched the Available Chemical Directory for known compounds similar in shape to the potent HIV-1 protease inhibitor Merck L-700,417; 15 compounds most similar in shape to the inhibitor were selected for testing in vitro. Four of these inhibited the protease at 100 microM or less and the most active of the four were the naturally occurring pigments biliverdin and bilirubin. Biliverdin and bilirubin inhibited recombinant HIV-1 protease in vitro at pH 7.8 with K1 values of approx. 1 microM, and also inhibited HIV-2 and simian immunodeficiency virus proteases. The related pyrrolic pigments stercobilin, urobilin, biliverdin dimethyl ester and xanthobilirubic acid showed similar inhibitory activity at low micromolar concentrations. Biliverdin, bilirubin and xanthobilirubic acid did not inhibit viral polyprotein processing in cultured cells, but they reduced viral infectivity significantly. At 100 microM, xanthobilirubic acid affected viral assembly, resulting in a 50% decrease in the generation of infectious particles. In contrast, at the same concentrations biliverdin and bilirubin exerted little or no effect on viral assembly but blocked infection of HeLaT4 cells by 50%. These results suggest that bile pigments might be a new class of potential lead compounds for developing protease inhibitors and they raise the question of whether hyperbilirubinaemia can influence the course of HIV infection.
Assuntos
Pigmentos Biliares/farmacologia , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/fisiologia , Ácido Aspártico Endopeptidases/metabolismo , Bilirrubina/análogos & derivados , Bilirrubina/farmacologia , Biliverdina/farmacologia , Linhagem Celular , Dipeptídeos/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-2/enzimologia , Humanos , Rim , Cinética , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Vírus da Imunodeficiência Símia/enzimologia , Relação Estrutura-AtividadeRESUMO
Bilirubin, biliverdin and their serum albumin complexes were tested as oxyradical scavengers (superoxide generated by the xanthine/xanthine oxidase system and peroxyl radical-trapping antioxidant ability). As superoxide scavengers the free bile pigments showed activities near to that of serum albumin, higher than the water soluble vitamin E analog Trolox and lower than ascorbic acid. The peroxyl radical-trapping antioxidant abilities of the tested bile pigments were much higher than those of the serum albumin and of the same order as their serum albumin complexes. This interaction with peroxyl radicals showed different stoichiometric factors for bilirubin (approximately 2) and biliverdin (approximately 4).
Assuntos
Antioxidantes/farmacologia , Pigmentos Biliares/farmacologia , Animais , Antioxidantes/química , Pigmentos Biliares/química , Bilirrubina/química , Bilirrubina/farmacologia , Biliverdina/química , Biliverdina/farmacologia , Bovinos , Sequestradores de Radicais Livres , Humanos , Técnicas In Vitro , Oxirredução , Albumina Sérica/química , Superóxidos/químicaRESUMO
1. Density-labelling with 99 atoms% of (2)H(2)O distinguished pre-existing from newly synthesized ribonuclease molecules in sections of developing hypocotyl tissue. 2. Activity profiles of enzyme extracted from the fraction pelletable at 100000g showed heterogeneity after isopycnic centrifugation in CsCl gradients. 3. Measurement of density shifts of the entire heterogeneous band shows that ribonuclease protein is synthesized de novo in both continuous far-red light and darkness. 4. A twofold increase in enzyme activity after irradiation was accompanied by band-broadening and a significantly faster rate of labelling than in darkness. 5. The conclusion is drawn from the experimental evidence and theoretical arguments presented that phytochrome regulates the synthesis of new enzyme molecules against a background of continuous (dark-rate) synthesis and degradation. 6. Further information has been deposited as Supplementary Publication SUP 50033 (3 pages) at the British Library Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.