RESUMO
Hypotonic extraction of bovine retinal rod outer segments after bleaching in isotonic buffer yielded an extract exhibiting activated cGMP phosphodiesterase properties. Since this extract was virtually devoid of other proteins involved in the rod outer segment cGMP enzymatic cascade, it was used to study phosphodiesterase catalytic activity. The hypotonic extract required Mg2+ in the range 0.1-1.0 mM for optimal cGMP hydrolysis. At these Mg2+ concentrations hydrolysis could be effectively inhibited by Ca2+ at concentrations which might be attainable in rod outer segments. Since higher Ca2+ concentrations were required to give a chosen degree of inhibition at higher Mg2+ concentrations, this inhibition was probably due to competition by Ca2+ for Mg2+ binding site(s) on the phosphodiesterase catalytic unit. Other divalent cations were also able to inhibit cGMP hydrolysis, many of them (especially those with ionic radii close to that of magnesium) more effectively than calcium. It is suggested that Ca2+ may play a role in phototransduction by participating in the control of photoreceptor sensitivity, and that this is achieved by modulating rod outer segment cGMP hydrolysis.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Cálcio/farmacologia , Células Fotorreceptoras/enzimologia , Segmento Externo da Célula Bastonete/enzimologia , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Cátions Bivalentes , Bovinos , GMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Luz , Magnésio/farmacologia , Protaminas/farmacologia , Tripsina/farmacologiaRESUMO
Conformational changes in the extradiscal regions of rhodopsin induced by illumination were investigated by modifying the visual pigment by mild treatment with cyanogen bromide prior to and after light exposure. Light induced an increased yield of cleavage of the Met bond 253-254 and a new cleavage at the Met bond 155-156 of the rhodopsin polypeptide chain. These residues, located at the beginnings of the membrane-buried helices 6 and 4, respectively, were concluded to become extradiscally exposed upon illumination.
Assuntos
Pigmentos da Retina/análise , Rodopsina/análise , Animais , Bovinos , Brometo de Cianogênio/farmacologia , Eletroforese em Gel de Poliacrilamida , Proteínas do Olho/análise , Luz , Conformação Proteica , Segmento Externo da Célula Bastonete/análise , Opsinas de Bastonetes , EspectrofotometriaRESUMO
Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The alpha subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [alpha-32P]nicotinamide adenine dinucleotide (NAD+), the alpha and beta subunits of transducin were found to be radiolabeled. The labeling was decreased by snake venom phosphodiesterase I (PDE I). The modification was shown to be mono ADP-ribosylation by analyses on thin layer chromatography of the PDE I-hydrolyzed products which revealed only 5'AMP residues. In addition we report that sodium nitroprusside activates the ADP-ribosylation of transducin.
Assuntos
Difosfato de Adenosina/metabolismo , Ribose/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Transducina/metabolismo , Cromatografia em Camada Fina , Eletroforese em Gel de PoliacrilamidaRESUMO
CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/análogos & derivados , Pigmentos da Retina/análise , Rodopsina/análise , Tionucleotídeos/metabolismo , Animais , Bovinos , Brometo de Cianogênio/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/metabolismo , Luz , Conformação Proteica , Segmento Externo da Célula Bastonete/análiseRESUMO
Amongst the chromogranin B (CGB) derived fragments naturally generated in bovine chromaffin granules and detected in the extracellular space, we recently identified a major peptide corresponding to the 614-626 sequence of CGB. This peptide, named secretolytin, shared an interesting sequence homology with the lytic domain of cecropins and displayed a potent antibacterial activity. The aim of the present study was to determine the structural features of secretolytin necessary for this biological activity. Our results suggest that an alpha-helical amphipathic structure common to secretolytin, cecropins and pig myeloid antibacterial peptide may account for the antibacterial activity.
Assuntos
Antibacterianos/química , Cromograninas/química , Fragmentos de Peptídeos/química , Animais , Antibacterianos/farmacologia , Bacillus/efeitos dos fármacos , Sequência de Bases , Bovinos , Cromogranina B , Cromograninas/farmacologia , Simulação por Computador , Resistência Microbiana a Medicamentos , Hormônios de Inseto/química , Micrococcus/efeitos dos fármacos , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Alinhamento de Sequência , Homologia de Sequência , Relação Estrutura-Atividade , SuínosRESUMO
Bovine rhodopsin from retinal rod photoreceptors, a protein of 39,000 molecular weight, was cleaved by BNPS-Skatole at the level of tryptophanyl bonds. This hydrolysis yields five fragments S1, S2, S3, S4 and S5 (molecular weights: 35,000, 28,000, 19,500, and 15,500 and 12,000, respectively) and four peptides. Large fragments were purified by polyacrylamide gel electrophoresis in SDS. S2, S3 and S4 contain the glycanes of native rhodopsin and their N-termini are blocked. S5 has the same C-terminal extremity as rhodopsin and contains the phosphorylated site. Phosphate groups are incorporated in this fragment on serines and threonines.
Assuntos
Pigmentos da Retina , Rodopsina , Animais , Bovinos , Fenômenos Químicos , Química , Eletroforese em Gel de Poliacrilamida , Hidrólise , Peso Molecular , Fragmentos de Peptídeos/análise , Fosforilação , TriptofanoRESUMO
The antifungal peptide named chromofungin is the most active vasostatin-I-derived peptide, corresponding to the sequence 47-66 of chromogranin A. (1)H-NMR analysis revealed that it adopts a helical structure. The mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied by penetration of monolayers and confocal laser microscopy. Chromofungin is able to interact with the cell wall, to cross the plasma membrane, to accumulate in the microorganism, and to inhibit calcineurin activity.
Assuntos
Cromograninas/química , Cromograninas/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos/química , Alternaria/metabolismo , Antifúngicos/química , Aspergillus fumigatus/metabolismo , Inibidores de Calcineurina , Proteínas de Ligação ao Cálcio/metabolismo , Calreticulina , Candida albicans/metabolismo , Membrana Celular/metabolismo , Cromogranina A , Cromograninas/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Ribonucleoproteínas/metabolismoRESUMO
Time resolved fluorescence of the phenylalanine residue (Phe) alone and included in the transmembrane domain (TMD) sequences of the epidermal growth factor receptor (EGFR) and ErbB-2 was studied using the synchrotron radiation source of light, and compared to molecular dynamics (MD) simulations. The fluorescence intensity decay is strongly sensitive to the environment. A mono-exponential decay was obtained for Phe amino acid alone in two different solvents and for Phe included in EGFR transmembrane sequence, with fluorescence lifetime values varying from 1.7 ns (EGFR) to 7.4 ns (Phe dissolved in water). In ErbB-2 transmembrane sequence three lifetimes were detected. The relative amplitude of the shortest one (0.14 ns) is smaller than 10%, whereas the others (0.6 and 2.2 ns) are almost equally represented. They have been attributed to different rotamers exchanging slowly. This interpretation is supported by MD simulations which evidence transitions in time series of the chi 1 dihedral angle of Phe observed in the case of ErbB-2. The anisotropy decays are similar for both peptides and indicate the presence of a correlation time in the nanosecond range (1-4 ns) and the probable existence of a very fast one (< 0.05 ns). Autocorrelation functions computed from MD simulations corroborate these results.
Assuntos
Receptores ErbB/química , Fenilalanina/química , Receptor ErbB-2/química , Sequência de Aminoácidos , Simulação por Computador , Polarização de Fluorescência , Dados de Sequência Molecular , Espectrometria de Fluorescência , TermodinâmicaRESUMO
The effects of fluoride on ROS phosphodiesterase and G-protein have been studied using membrane-free extracts. When G-protein was present NaF, at millimolar concentrations, stimulated PDE activity however, in a G-protein free extract, cGMP hydrolysis was inhibited by high fluoride concentrations. Fluoride was also found to profoundly inhibit the ability of G-protein to bind a GTP analogue, GTP gamma S, both in the presence and absence of rhodopsin. Aluminium greatly modified these effects of fluoride on PDE and G-protein. The possibility that fluoride activates PDE through its effect on G-protein is discussed.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Proteínas do Olho/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP , Células Fotorreceptoras/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Fluoreto de Sódio/farmacologia , Alumínio/farmacologia , Animais , Bovinos , GMP Cíclico/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Rodopsina/farmacologia , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Tionucleotídeos/metabolismo , TransducinaRESUMO
Light-induced conformational changes occurring at the cytosolic surface of rhodopsin were investigated by performing limited digestions of native and illuminated visual pigment with thermolysin, Arg-C endoproteinase, papain and proteinase K. A higher susceptibility of the extradiscal regions of the bleached pigment to the proteases were observed together with altered capacities of the digested bleached rhodopsins to activate the cGMP phosphodiesterase. The overall results strongly suggest that light induces conformational changes not only in the C-terminal end but also in the second and the third extradiscal loop of rhodopsin.
Assuntos
Luz , Pigmentos da Retina , Rodopsina , Rodopsina/efeitos da radiação , Serina Endopeptidases , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Animais , Bovinos , Endopeptidase K , Endopeptidases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas do Olho/metabolismo , Proteínas do Olho/farmacologia , Proteínas do Olho/efeitos da radiação , Papaína/metabolismo , Fragmentos de Peptídeos/metabolismo , Conformação Proteica/efeitos da radiação , Pigmentos da Retina/metabolismo , Pigmentos da Retina/farmacologia , Pigmentos da Retina/efeitos da radiação , Rodopsina/metabolismo , Rodopsina/farmacologia , Segmento Externo da Célula Bastonete/efeitos da radiação , Opsinas de Bastonetes , Termolisina/metabolismoRESUMO
A structural model of the bovine rhodopsin in the discal membrane is proposed, based on the data obtained from the proteolytic and BNPS skatole fragments of the protein. The striking features of the model are the presence of seven transmembrane segments and of a large extradiscal peptidic loop and the localisation of the retinal site.
Assuntos
Pigmentos da Retina , Rodopsina , Animais , Bovinos , Membrana Celular , Modelos Estruturais , Células FotorreceptorasRESUMO
Vasostatin-I, the natural fragment of chromogranin A-(1-76), is a neuropeptide able to kill a large variety of fungi and yeast cells in the micromolar range. We have examined the antifungal properties of synthetic vasostatin-I-related peptides. The most active shortest peptide, named chromofungin, corresponds to the sequence Arg(47)-Leu(66). Extensive (1)H NMR analysis revealed that it adopts a helical structure. The biophysical mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied, showing the penetration of this peptide with different lipid monolayers. In order to examine thoroughly the antifungal activity of chromofungin, confocal laser microscopy was used to demonstrate the ability of the rhodamine-labeled peptide to interact with the fungal cell wall, to cross the plasma membrane, and to accumulate in Aspergillus fumigatus, Alternaria brassicola, and Candida albicans. Our present data reveal that chromofungin inhibits calcineurin activity, extending a previous observation that the N-terminal region of chromogranin A interacts with calmodulin in the presence of calcium. Therefore, the destabilization of fungal wall and plasma membrane, together with the possible intracellular inhibition of calmodulin-dependent enzymes, is likely to represent the mechanism by which vasostatin-I and chromofungin exert antifungal activity.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Cromograninas/química , Cromograninas/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Cromogranina A , Testes de Sensibilidade Microbiana , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
The chromaffin granules have been shown to be an excellent model to study the processing of proenkephalin-A and chromogranins. Recently, we reported a study dealing with the processing of chromogranin B/secretogranin I and the occurrence of the C-terminal chromogranin B-derived peptide 614-626 which was shown to have antibacterial activity [Strub, J.M., Garcia-Sablone, P., Looning, K., Taupenot, L., Hubert, P., Van Dorsselaer, A., Aunis, D. & Metz-Boutigue, M.H. (1995) Eur. J. Biochem. 229, 356-368]. We also observed that this new antibacterial activity present in chromaffin granules was associated with other endogenous protein-derived fragments yet to be characterized. The present study reports the isolation and characterization of a peptide which possesses antibacterial activity and which corresponds to the C-terminal 209-237 sequence of proenkephalin-A. A detailed study using microsequencing and matrix-assisted-laser-desorption time-of-flight mass spectrometry (MALD-TOF MS) allowed us to correlate the antibacterial activity of this peptide named enkelytin (FAEPLPSEEEGESYSKEVPEMEKRYGGFM) with post-translational modifications. Endogenous bisphosphorylated proenkephalin-A-(209-237) was active on Micrococcus luteus and Bacillus megaterium killing bacteria in the 0.2 - 0.4 microM range but was inactive in similar conditions towards Escherichia coli. Enkelytin shares sequence and structural similarities with the antibacterial C-terminal domain of diazepam-binding inhibitor. According to this similarity, a prediction of secondary structure is proposed for enkelytin and discussed in relationship to its biological activity.