RESUMEN
The presented study was designed to estimate the ability of hypericin to interact with the anticancer drug doxorubicin. The hetero-association of hypericin and doxorubicin was investigated with absorption and fluorescence spectroscopy methods in aqueous solution of DMSO in two-component mixtures: doxorubicin-hypericin and three component mixtures: DNA-doxorubicin-hypericin. The data indicate that hypericin forms complexes with doxorubicin and that the association constants are on the order of 300,000 M-1 in a buffer with 30% DMSO content. The absorption spectra of the hypericin - doxorubicin complexes were examined as well. Owing to its ability to interact with flat aromatic compounds, hypericin may potentially be used as an interceptor molecule to detoxification of patients after chemotherapy.
Asunto(s)
Dimetilsulfóxido , Perileno , Antracenos , Doxorrubicina/química , Doxorrubicina/farmacología , Humanos , Perileno/análogos & derivados , Perileno/química , Espectrometría de FluorescenciaRESUMEN
1H Nuclear Magnetic Resonance relaxometry has been applied to reveal dynamical properties of water molecules embedded into egg yolk and white of three species: turkey, chicken and quail. Two fractions of water molecules, referred to as confined-water and free-water fractions, have been revealed. It has been demonstrated that translation diffusion of the confined-water fraction is three-dimensional. The dynamics of the confined-water has been quantitatively described in terms of diffusion coefficients and rotational correlation times. The parameters have been compared for egg yolk and white for all the species. In addition to these quantities, the number of the confined-water molecules per unit volume has been provided for all cases. The obtained parameters provide insight into the dynamics of water in eggs of different origin and allow to identify similarities and differences between them in connection to the structure of the network formed by the macromolecular fraction of egg yolk and white.
Asunto(s)
Imagen por Resonancia Magnética , Agua , Difusión , Huevos , Espectroscopía de Resonancia MagnéticaRESUMEN
Chlorophyll was shown to spontaneously form a complex with cadmium, which is incorporated at the central position of the chlorophyll molecule porphyrin ring, where it replaces magnesium. The rate of complex formation depended on the ratio of Cd2+ ions to chlorophyll concentration in the solution. In solutions with chlorophyll concentration of C = 1 × 10-5 M and Cd2+ concentrations of C = 1 × 10-5 M, C = 1 × 10-3 M and C = 9 × 10-3 M, Cd-Chl complex formation was completed after 200 h, 50 h and 33 h, respectively. The formation of Cd-Chl complex followed the second order over all substrates reaction order, first order over Cd2+ concentration and first over Chl concentration. The pseudo second order reaction rate constant k, when Cd2+ concentration was equal Chl concentration have been obtained as k = 1.510 ± 0.023 × 10-4 M-1min-1. Quantum chemistry computations showed that Cd-chlorophyll complex existed in two conformations in the methanol solution with cadmium ion placed either below or above the coordination plane. Two times smaller overlap integral of the Chl fluorescence spectrum with the Cd-Chl absorption spectrum IChl,Cd-Chl= 2.4223 × 10-13 cm3/M in comparison with the overlap integral of the Chl fluorescence spectrum with the Chl absorption spectrum IChl,Chl= 4.6210 × 10-13 cm3/M (twice lower probability of energy transfer Chl∗ â Cd-Chl than Chl∗ â Chl) and lower Förster critical distance for resonance energy transfer: RoChlâCd-Chl= 46.773 Å, RoChlâChl= 52.086 Å, indicated that in plants intoxicated with cadmium, taken up from the contaminated soil, the energy transfer between Chl and Cd-Chl in antennas will be disturbed, which may be one of the reasons for the inhibition of photosynthesis.
Asunto(s)
Cadmio/química , Clorofila/química , Fotosíntesis/efectos de los fármacos , Clorofila/metabolismo , Transferencia de Energía , Fluorescencia , Iones , Plantas/metabolismo , Espectrometría de FluorescenciaRESUMEN
This paper contains the results of a study on the interactions between mitoxantrone and some bioactive polyphenols. It has been demonstrated that polyphenols can intercept mitoxantrone. Quercetin shows the highest affinity for complexing with mitoxantrone, in contrast to resveratrol, which shows the lowest affinity. The main process underlying the association between cytostatic and polyphenols occurs in the ground state. The values of the constants of the association reactions between the analysed compounds and mitoxantrone are large enough to generate an evident intercepting effect in the three-component system (mitoxantrone-DNA-polyphenol). The affinity of the analysed plant-origin compounds is approximately 1000-fold weaker than the interaction of mitoxantrone with the DNA, which implies that the presence of these compounds in food should not adversely affect oncological therapy but rather could successfully aid oncological treatment by regulating the quantities of the drug in its active form.
Asunto(s)
Mitoxantrona/química , Extractos Vegetales/química , Plantas/química , Polifenoles/química , ADN/químicaRESUMEN
7-Methylguanosine (m(7)G) nucleotides labelled with acetylpyrene (AcPy) were synthesized as fluorescent mRNA 5' end (cap) analogues. The unique fluorescent properties of m(7)G-AcPy conjugates, different from G-AcPy, can be applied to studying various mRNA cap-related processes including the evaluation of putative inhibitors of DcpS enzyme-a therapeutic target in neuromuscular diseases.
Asunto(s)
Guanina/análogos & derivados , Nucleótidos/química , Pirenos/química , Fluorescencia , Guanina/química , Espectrometría de FluorescenciaRESUMEN
The above-ground parts of celery plants were exposed to two polycyclic aromatic hydrocarbons (PAHs): 3-ring anthracene (ANT) and 5-ring benzo[k]fluoranthene (BkF), and the combination of ANT and BkF. After 43 days of exposure (overall dose of 1325µg/plant), celery plants retained only 1.4% of the total dose of ANT and 17.5% of the total dose of BkF. After exposure to a combination of ANT and BkF (1325µg of each compound per plant), the average ANT concentrations were more than twofold higher in/on leaf blades, whereas BkF levels were insignificantly higher. Under natural photoperiod conditions equivalent to a normal day, the combined application of ANT and BkF to the above-ground parts of celery plants slowed down physicochemical transformations of ANT. A similar effect was observed when PAHs were applied to glass surfaces. The combination of both PAHs probably led to stacking interactions, which decreased volatilization, in particular of ANT. Phytotoxicity of ANT and BkF could not be unambiguously established based on the results of this study. In all analyzed treatments, the chlorophyll content of leaf blades remained unchanged. Foliar application of ANT reduced ascorbic acid levels in all analyzed plant parts and increased the total acidity of celery leaves. In all experimental treatments, the total phenolic content of leaves increased up to 15%. Interestingly, ANT and BkF did not produce cumulative effects when applied in combination (when total PAH concentrations per plant were twofold higher).
Asunto(s)
Antracenos/toxicidad , Apium/efectos de los fármacos , Fluorenos/toxicidad , Antracenos/farmacocinética , Apium/metabolismo , Fluorenos/farmacocinética , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismoRESUMEN
We describe the synthesis and properties of five dinucleotide fluorescent cap analogues labelled at the ribose of the 7-methylguanosine moiety with either anthraniloyl (Ant) or N-methylanthraniloyl (Mant), which have been designed for the preparation of fluorescent mRNAs via transcription in vitro. Two of the analogues bear a methylene modification in the triphosphate bridge, providing resistance against either the Dcp2 or DcpS decapping enzymes. All these compounds were prepared by ZnCl2-mediated coupling of a nucleotide P-imidazolide with a fluorescently labelled mononucleotide. To evaluate the utility of these compounds for studying interactions with cap-binding proteins and cap-related cellular processes, both biological and spectroscopic features of those compounds were determined. The results indicate acceptable quantum yields of fluorescence, pH independence, environmental sensitivity, and photostability. The cap analogues are incorporated by RNA polymerase into mRNA transcripts that are efficiently translated in vitro. Transcripts containing fluorescent caps but unmodified in the triphosphate chain are hydrolysed by Dcp2 whereas those containing a α-ß methylene modification are resistant. Model studies exploiting sensitivity of Mant to changes of local environment demonstrated utility of the synthesized compounds for studying cap-related proteins.
RESUMEN
The kinetics of the hydrolysis of P(1)-(7-methylguanosinyl-5') P(3)-(guanosinyl-5') triphosphate (m(7)GpppG), P(1)-(7-methylguanosinyl-5') P(4)-(guanosinyl-5') tetraphosphate (m(7)GppppG), and diadenosine 5', 5'( ')-P(1),P(3) -triphosphate (ApppA) in the presence of several Cu(2+) or Zn(2+) ions complexed with bi- or terpyridine has been studied at pH 8.0 and 60 °C. Time-dependent product distributions at various metal complex concentrations have been determined by capillary zone electrophoresis and reversed-phase high performance liquid chromatography. The results show that the predominant hydrolytic reaction is the cleavage of 5',5'-oligophosphate bridge, with Cu(2+) complexes being approximately 15-fold more efficient catalysts than Zn(2+) chelates. In addition, the effect of metal ions complexes at pH 7.0 and 8.0 on the imidazole ring opening in m(7)Gua mononucleotides has been studied. The influence of Cu(2+) complexes on imidazole ring cleavage of mononucleotides is modest, whereas Zn(2+) complexes are almost inactive.
Asunto(s)
Complejos de Coordinación/síntesis química , Cobre/química , Nucleótidos de Guanina/síntesis química , Piridinas/síntesis química , Zinc/química , Catálisis , Cromatografía de Fase Inversa , Complejos de Coordinación/química , Electroforesis Capilar , Nucleótidos de Guanina/química , Calor , Concentración de Iones de Hidrógeno , Hidrólisis , Imidazoles/química , Iones/química , Cinética , Imitación Molecular , Estructura Molecular , Piridinas/química , Ribonucleasas/químicaRESUMEN
Kinetics of the hydrolysis of a P(1)-(7-methylguanosinyl-5') P(3)-(guanosinyl-5') triphosphate (m(7)GpppG), P(1)-(7-methylguanosinyl-5') P(4)- (guanosinyl-5') tetraphosphate (m(7)GppppG), diadenosine-5',5'''-P(1),P(3)-triphosphate (ApppA), and diadenosine-5',5'''-P(1),P(4)-tetraphosphate (AppppA) promoted by Cu(2+) or Zn(2+) has been investigated. Time-dependent products distributions at various metal ion concentrations have been determined by CZE and HPLC-RP. The results show that in acidic conditions, in the presence of metal ion, the predominant hydrolytic reaction is the cleavage of 5',5'-oligophosphate bridge. The 5',5'-oligophosphate bridge of the dinucleotides studied is hydrolyzed by Cu(2+) more efficiently than by Zn(2+). At the catalyst concentration of 2 mM the cleavage of the 5',5'-triphosphate bridge of m(7)GpppG was â¼3.6 times faster, and that of the tetraphosphate bridge of m(7)GppppG â¼2.3-fold faster in the presence of Cu(2+) compared to the Zn(2+) ion, applied as catalysts. Dependence of the rates of hydrolysis on the catalyst concentration was in some instances not linear, interpreted as evidence for participation of more than one metal ion in the transition complex.
Asunto(s)
Cobre/química , Análogos de Caperuza de ARN/química , Zinc/química , Hidrólisis , Iones/química , Cinética , Estructura MolecularRESUMEN
Human diet may contain many mutagenic or carcinogenic aromatic compounds as well as some beneficial physiologically active dietary components, especially plant food phytochemicals, which act as mutagenesis or carcinogenesis inhibitors. This study compared the binding properties of natural compounds in the human diet (caffeine, theophylline, theobromine, and resveratrol) with a water-soluble derivative of chlorophyll to bind to acridine orange, a known mutagen. An analysis was conducted to determine which substances were effective binding agents and may thus be useful in prevention of chemical-induced mutagenesis and carcinogenesis. Data indicated that in order to bind 50% of the mutagen in a complex, less than twice the concentration of chlorophyllin was needed, the resveratrol concentration was 20-fold higher, while a 1000-fold or even 10,000-fold excess of xanthines were required to bind acridine orange.
Asunto(s)
ADN/efectos de los fármacos , ADN/metabolismo , Dieta , Naranja de Acridina , Antineoplásicos/farmacología , Fenómenos Biofísicos , Cafeína/farmacología , Carcinógenos , Clorofila/farmacología , Clorofilidas , Humanos , Masculino , Mutágenos , Neoplasias , Teobromina , Teofilina , Xantinas/farmacologíaRESUMEN
The concentrations of the heavy metals lead (Pb) and cadmium (Cd) were determined in berries (blackberry, raspberry, bilberry, wild strawberry), and hazelnuts picked from plants in the wild as well as in fruit (blackberry, raspberry, blueberry) and hazelnuts picked from orchard-farmed plants in northeastern Poland. The levels of seven congeners of polychlorinated biphenyls (PCB(7)), gamma isomer of hexachlorocyclohexane (gamma-HCH), and sum of dichlorodiphenyl trichloroethane and its metabolites (SigmaDDT) were also measured in plants and nuts. In addition, the concentrations of Pb, Cd, PCB(7,) gamma-HCH, and SigmaDDT were determined in the surface samples of soil from the sites of fruit picking. The highest acceptable concentrations based upon Polish standards for Pb and Cd were not exceeded in forest fruit. In wild berries, Pb occurred at a level below the detection limit, whereas the concentration of Cd ranged from 6 to 49 microg/kg fresh weight. The levels were Cd 72 microg/kg fresh weight and Pb 290 microg/kg fresh weight in raspberries from orchard plants and exceeded the maximal acceptable limit of 50 microg/kg for Cd and 200 microg/kg for Pb. The level of Pb at 210 microg/kg fresh weight in hazelnuts from orchard plants also exceeded maximal acceptable limits. Individual samples of fruit, regardless of their origin, were found to contain trace amounts of organic pollutants such as 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p'-DDE) and PCB congeners 101 and 118. All soil samples contained from 3.2 to 14.9 mg/kg dry weight concentrations of Pb and most soil samples also contained Cd. Further, individual soil samples were found to contain high levels of SigmaDDT (145 microg/kg), including p,p'-DDT at a concentration of 67 microg/kg. The concentrations of persistent organic pollutants (POP) in wild and orchard-farm-grown fruit in northeastern Poland were generally below threshold permissible limits, and no correlation was found between levels of contaminants in soils and POP concentrations in fruit.
Asunto(s)
Cadmio/análisis , Cadmio/metabolismo , DDT/metabolismo , Diclorodifenil Dicloroetileno/metabolismo , Agricultura , DDT/análisis , Diclorodifenil Dicloroetileno/análisis , Frutas/metabolismo , Límite de Detección , Metales Pesados/análisis , Polonia , Bifenilos Policlorados/análisis , Suelo/análisisRESUMEN
We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.
Asunto(s)
Aminacrina/análogos & derivados , Clorofilidas/farmacología , Daño del ADN , ADN/química , Mutágenos/toxicidad , Compuestos de Mostaza Nitrogenada/toxicidad , Aminacrina/química , Aminacrina/toxicidad , Antimutagênicos/química , Antimutagênicos/farmacología , Unión Competitiva/efectos de los fármacos , Quimioprevención , Clorofilidas/química , ADN/efectos de los fármacos , Células HL-60 , Humanos , Sustancias Macromoleculares/química , Mutágenos/química , Compuestos de Mostaza Nitrogenada/químicaRESUMEN
In aqueous solutions, in the presence of double-stranded DNA, chlorophyllin (CHL) forms complexes with each of the three DNA intercalators: acridine orange (AO), quinacrine mustard (QM), and doxorubicin (DOX). The evidence for these interactions was obtained by measurement changes in the absorption and fluorescence spectra of the mixtures containing DNA and intercalators during titration with CHL. A model of simple competition between DNA and CHL for the intercalator was used to define the measured interactions. The concentrations of the complexes estimated based on this model were consistent with the concentrations obtained by actual measurement of the absorption spectra. The present data provide further support for the role of chlorophyllin as an "interceptor" that may neutralize biological activity of aromatic compounds including mutagens and antitumor drugs.
Asunto(s)
Clorofilidas/química , ADN/química , Sustancias Intercalantes/química , Espectrometría de FluorescenciaRESUMEN
The alpha-guanidino acids derived of 15 proteinaceous amino acids, omega-guanidino acids with gradually increased hydrocarbon chains, and amidinated dipeptides, were tested as the mimetics of antiadhesive peptides in Mycobacteria phagocytosis inhibition. The crystal structure of omega-guanidino acids used was determined by X-ray structural analysis. It follows from our experiments that the proper distance between guanidine and carboxyl groups of effector molecules is of decisive importance for their inhibitory activity.
Asunto(s)
Leucocitos Mononucleares/efectos de los fármacos , Mycobacterium kansasii , Oligopéptidos/farmacología , Fagocitosis/efectos de los fármacos , Amidinas/química , Amidinas/farmacología , Aminoácidos/química , Aminoácidos/farmacología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Glicina/análogos & derivados , Glicina/farmacología , Guanidina/análogos & derivados , Guanidina/química , Guanidina/farmacología , Humanos , Leucocitos Mononucleares/fisiología , Modelos Moleculares , Conformación Molecular , Imitación Molecular , Oligopéptidos/síntesis química , Oligopéptidos/químicaRESUMEN
A peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) (U50-59) possesses a very high immunosuppressory activity, comparable to that of cyclosporine, both in the cellular and humoral immune responses. We found that the pentapeptide DGRTL (U52-56) is the shortest, effective immunosuppressory fragment of ubiquitin, although its potency is weaker than that of U50-59. Replacement of each consecutive residue with alanine in U52-56 allowed identification of essential amino acids involved in the immunosuppression. We also evaluated the roles of its N- and C-terminal groups by their acetylation and/or amidation, respectively. The active sequence is located in the external loop of the molecule and therefore it may serve as an important functional epitope for intermolecular binding. Based on the crystal structure of ubiquitin molecule, we designed and synthesized the cyclic analogue with a restricted conformation, cyclo(Glt-Gln-Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Lys)-NH2 (Glt = glutaryl) by reacting the C-terminal Lys side chain with the glutarylated N-terminus. The peptide was designed to mimic the ubiquitin(48-59) loop, in order to obtain the ligand that may interact with hypothetical receptors of the loop. The cyclization product selectively but strongly suppresses the cellular immune response. The results indicate that the 48-59 loop may serve as an important functional epitope in the ubiquitin molecule for intermolecular binding.
Asunto(s)
Inmunosupresores/química , Inmunosupresores/farmacología , Oligopéptidos/química , Ubiquitina/química , Ubiquitina/farmacología , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Ciclosporina/farmacología , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Conformación Proteica , Ubiquitina/análogos & derivados , Ubiquitina/síntesis químicaRESUMEN
Interleukin-1 receptor antagonist (IL-1Ra) and vaccinia virus protein C10L share a VTXFYF motif, with X being Lys or Arg residue, respectively. Peptides of such sequence compete successfully with IL-1 for the cellular receptor. A pair of complementary peptides, based on the Siemion's hypothesis on the periodicity of the genetic code (QWLNIN and QWANIN), and another pair, in which, following the Root- Bernstein theory, Lys was used as complementary amino acid to Phe (QWLKIK and QWAKIK), were investigated for the peptide-antipeptide interactions using mass spectrometry (ESI-MS) and circular dichroism (CD) methods. The CD measurements indicated some conformational changes, more pronounced in the Siemion's pairs, however, no heterodimer formation was found by MS. In the region of IL-1 receptor situated close to the position of IL-1Ra in the IL-1Ra-receptor complex, a KQKL motif is present, suggesting a possibility of complementary recognition of the Root-Bernstein type in the IL-1 receptor. The biological activity of the complementary peptides is similar to that of the original ones. They efficiently compete with IL-1 and show moderate immunosuppressory activity in humoral and cellular immune response. The inhibition of the IL-1-IL-1 receptor interaction may result from the complementary peptides acting as mini-receptors with affinity for IL-1.
Asunto(s)
Receptores de Interleucina-1/química , Receptores de Interleucina-1/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Dicroismo Circular , Interleucina-1/metabolismo , Espectrometría de Masas , Modelos Moleculares , Conformación Proteica , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores de Interleucina-1/genética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Continuing our research on Mycobacteria kansasii phagocytosis inhibition, we have examined in that context three series of peptides derived from the RGDVY and GRGD sequences. It was found that the levels of the inhibitory activity depend on the amino acid composition as well as on the particular peptide sequence. Distinct inhibitory activity was found in the case of thymopentin (RKDVY), the active fragment of thymopoietin. In this case the Mycobacterium phagocytosis inhibition should be combined with general immunostimulatory activity of RKDVY peptide. Our examination of a series of GRGDV analogs with a successively prolonged oligo-Gly linker inserted into the peptide chain showed that the distance between the Arg and Asp residues required for such an activity should be about 9A.
Asunto(s)
Antibacterianos/farmacología , Mycobacterium kansasii/efectos de los fármacos , Oligopéptidos/farmacología , Fagocitosis/efectos de los fármacos , Animales , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Leucocitos/efectos de los fármacos , Ratones , Relación Estructura-ActividadRESUMEN
The present study was designed to estimate the ability of chlorophyllin (CHL) to interact with two acridine mutagens, quinacrine mustard (QM) and acridine orange (AO), and with the antitumor anthracycline doxorubicin (Dox). To this end, aqueous solutions of QM, AO or Dox during titration with CHL were subjected to spectrophotometry and spectrofluorimetry to detect possible interactions between these reagents. The data indicate that CHL forms complexes with AO, QM or Dox in these solutions. The presence of the complexes was manifested by a bathochromic shift of the absorption spectra, as well as by strong quenching of the fluorescence of each of these mutagens in the presence of CHL. CHL, thus, may serve as an interceptor of these mutagenic acridines in different in vivo or in vitro applications. Its ability to interact with Dox may potentially be utilized to detoxify patients overdosed with this or similar drugs.
Asunto(s)
Naranja de Acridina/metabolismo , Antimutagênicos/química , Clorofilidas/química , Doxorrubicina/química , Mutágenos/química , Mostaza de Quinacrina/química , Naranja de Acridina/química , Relación Dosis-Respuesta a Droga , Doxorrubicina/metabolismo , Humanos , Estructura Molecular , Mutágenos/metabolismo , Mostaza de Quinacrina/metabolismo , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
Initial entry of Mycobacteria into the cells depends upon the formation of a molecular complex between Antigen 85 (Ag85), located on the bacterial cell wall, and serum protein-fibronectin (FN) [Nat. Struct. Biol. 7 (2000) 141; Nat. Struct. Biol. 7 (2000) 94]. Therefore, a way of preventing a Mycobacteria invasion could be to inhibit the interaction between fibronectin and leucocyte cellular receptors of the integrin type. We found that some antiadhesive peptides (such as RGDVY and GRGD), derived of fibronectin and human leucocyte antigen DQ (HLA-DQ) sequences, are in fact very potent inhibitors of Mycobacterium kansasii phagocytosis. This observation may open new prospects in the search for tuberculosis therapy.
Asunto(s)
Adhesión Bacteriana , Mycobacterium kansasii/metabolismo , Péptidos/química , Adhesión Celular , Pared Celular/química , Fibronectinas/metabolismo , Integrinas/metabolismo , Leucocitos/metabolismo , Fagocitosis/efectos de los fármacos , TemperaturaRESUMEN
Translation initiation factor eIF4E binds the m(7)G cap of eukaryotic mRNAs and mediates recruitment of mRNA to the ribosome during cap-dependent translation initiation. This event is the rate-limiting step of translation and a major target for translational control. In the nematode Caenorhabditis elegans, about 70% of genes express mRNAs with an unusual cap structure containing m(3)(2,2,7)G, which is poorly recognized by mammalian eIF4E. C. elegans expresses five isoforms of eIF4E (IFE-1, IFE-2, etc.). Three of these (IFE-3, IFE-4 and IFE-5) were investigated by means of spectroscopy and structural modelling based on mouse eIF4E bound to m(7)GDP. Intrinsic fluorescence quenching of Trp residues in the IFEs by iodide ions indicated structural differences between the apo and m(7)G cap bound proteins. Fluorescence quenching by selected cap analogues showed that only IFE-5 forms specific complexes with both m(7)G- and m(3)(2,2,7)G-containing caps (K(as) 2 x 10(6) M(-1) to 7 x 10(6) M(-1)) whereas IFE-3 and IFE-4 discriminated strongly in favor of m(7)G-containing caps. These spectroscopic results quantitatively confirm earlier qualitative data derived from affinity chromatography. The dependence of K(as) on pH indicated optimal cap binding of IFE-3, IFE-4 and IFE-5 at pH 7.2, lower by 0.4 pH units than that of eIF4E from human erythrocytes. These results provide insight into the molecular mechanism of recognition of structurally different caps by the highly homologous IFEs.