RESUMEN
Although the pseudohalide thiocyanate (SCN(-)) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN(-)/H(2)O(2) system and activated eosinophils as roughly equimolar amounts of OSCN(-) (hypothiocyanite) and OCN(-) (cyanate). Red blood cells exposed to increasing concentrations of OSCN(-)/OCN(-) are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN(-)/OCN(-) inactivates red blood cell membrane ATPases 10-1000 times more potently than do HOCl, HOBr, and H(2)O(2). Exposure of glutathione S-transferase to [(14)C]OSCN(-)/OCN(-) causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN(-)/H(2)O(2) reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.
Asunto(s)
Eosinófilos/enzimología , Eritrocitos/efectos de los fármacos , Oxidantes/metabolismo , Oxidantes/toxicidad , Peroxidasas/metabolismo , Tiocianatos/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Cianatos/metabolismo , Peroxidasa del Eosinófilo , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Eritrocitos/enzimología , Eritrocitos/metabolismo , Glutatión/metabolismo , Glutatión Transferasa/antagonistas & inhibidores , Glutatión Transferasa/metabolismo , Hemólisis/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Neutrófilos/enzimología , Neutrófilos/metabolismo , Oxidantes/química , Peroxidasa/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Temperatura , Acetato de Tetradecanoilforbol/farmacología , Tiocianatos/química , Tiocianatos/toxicidadRESUMEN
Omapatrilat, a potent vasopeptidase inhibitor, is currently under development for the treatment of hypertension and congestive heart failure. This study describes the plasma profile along with isolation and identification of urinary metabolites of omapatrilat from subjects dosed orally with 50 mg of [(14)C]omapatrilat. Only a portion of the radioactivity in plasma was unextractable (40-43%). Prominent metabolites identified in plasma were S-methyl omapatrilat, acyl glucuronide of S-methyl omapatrilat, and S-methyl (S)-2-thio-3-phenylpropionic acid. Omapatrilat accounted for less than 3% of the radioactivity. However, after dithiothreitol reduction all of the radioactivity was extractable and was characterized to be omapatrilat and its hydrolysis product (S)-2-thio-3-phenylpropionic acid, both apparently bound to proteins via reversible disulfide bonds. Urinary profile of radioactivity showed no parent compound but the presence of several metabolites that can be grouped into three categories. 1) Three metabolites, accounting for 56% of the urinary radioactivity, resulted from the hydrolysis of the exocyclic amide bond of omapatrilat. Two metabolites were diastereomers of S-methyl sulfoxide of (S)-2-thio-3-phenylpropionic acid, and the third was the acyl glucuronide of S-methyl (S)-2-thio-3-phenylpropionic acid. 2) One disulfide, identified as the L-cysteine mixed disulfide of omapatrilat, accounted for 8% of the radioactivity in the urine. 3) Five metabolites, derived from omapatrilat, accounted for 30% of the radioactivity in the urine. Two of these metabolites were mixtures of diastereomers of S-methyl sulfoxide of omapatrilat and the third was the S-methyl omapatrilat ring sulfoxide. The other two metabolites were S-methyl omapatrilat and its acyl glucuronide. These results indicate that omapatrilat undergoes extensive metabolism in humans.
Asunto(s)
Inhibidores Enzimáticos/farmacocinética , Piridinas/farmacocinética , Tiazepinas/farmacocinética , Inhibidores de la Enzima Convertidora de Angiotensina/sangre , Inhibidores de la Enzima Convertidora de Angiotensina/farmacocinética , Inhibidores de la Enzima Convertidora de Angiotensina/orina , Biotransformación , Radioisótopos de Carbono , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/sangre , Inhibidores Enzimáticos/orina , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Piridinas/sangre , Piridinas/orina , Tiazepinas/sangre , Tiazepinas/orinaRESUMEN
Neutrophil-activating peptide 2 (NAP-2), which demonstrates a range of proinflammatory activities, is a 72-residue protein belonging to the alpha-chemokine family. Although NAP-2, like other alpha-chemokines, is known to self-associate into dimers and tetramers, it has been shown that the monomeric form is physiologically active. Here we investigate the solution structure of monomeric NAP-2 by multi-dimensional 1H-NMR and 15N-NMR spectroscopy and computational modelling. The NAP-2 monomer consists of an amphipathic, triple-stranded, anti-parallel beta-sheet on which is folded a C-terminal alpha-helix and an aperiodic N-terminal segment. The backbone fold is essentially the same as that found in other alpha-chemokines. 15N T1, T2 and nuclear Overhauser effects (NOEs) have been measured for backbone NH groups and used in a model free approach to calculate order parameters and conformational exchange terms that map out motions of the backbone. N-terminal residues 1 to 17 and the C-terminus are relatively highly flexible, whereas the beta-sheet domain forms the most motionally restricted part of the fold. Conformational exchange occurring on the millisecond time scale is noted at the top of the C-terminal helix and at proximal residues from beta-strands 1 and 2 and the connecting loop. Dissociation to the monomeric state is apparently responsible for increased internal mobility in NAP-2 compared with dimeric and tetrameric states in other alpha-chemokines.
Asunto(s)
Péptidos/química , Espectroscopía de Resonancia Magnética , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , beta-TromboglobulinaRESUMEN
A de novo designed 33-residue polypeptide folds as a compact beta-sheet sandwich tetramer in aqueous solution. NMR structural analysis shows that although monomer subunits have the same three-stranded antiparallel beta-sheet fold, two equally populated conformational states are identified. Conformational heterogeneity arises from formation of two distinct dimer folds. Each dimer is formed by continuing the monomer beta-sheet into a six-stranded sheet similar to that found in alpha-chemokines. Dimer heterogeneity arises primarily from a two-residue shift in the alignment of interfacial strands. NOE-based conformational modeling has yielded well-defined structures for both dimer types. While the tetramer beta-sheet sandwich most probably results from association of hydrophobic surfaces from two amphipathic dimers, dimers could combine to form either two types of homotetramers and/or one heterotetramer composed of both dimer types. Even though interdimer NOEs could not be unambiguously identified to resolve this point, thermodynamic arguments based on observation of equal populations of both dimer types favor formation of heterotetramers.
Asunto(s)
Diseño de Fármacos , Péptidos/síntesis química , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Previous studies have produced conflicting interpretations regarding the aggregation state of BPTI in solution. Here, pulsed-field gradient NMR self-association measurements have been performed with BPTI under a variety of temperature, pH, salt, urea conditions, and protein concentrations. Relative to the standard proteins, lysozyme, ribonuclease, and ubiquitin, diffusion constants indicate that BPTI dimerizes at concentrations above about 3 mg/mL and below 280 K. At higher temperatures, a marked self-association is observed above 10 mg/mL. The apparent lack of significant effects from variations in pH and NaCl concentration suggests minimal contribution to the aggregation process from charge-charge interactions. In contrast, in nondenaturing concentrations of urea (2 M), BPTI behaves as a monomer, suggesting that hydrophobic and polar residues modulate BPTI association. The BPTI surface shows that while one side is highly charged, the opposite side, composed mostly of hydrophobic and some hydrophilic residues, is feasible as an interface for BPTI self-association.
Asunto(s)
Aprotinina/química , Animales , Bovinos , Difusión , Dimerización , Concentración de Iones de Hidrógeno , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Muramidasa/química , Concentración Osmolar , Ribonucleasas/química , Termodinámica , Ubiquitinas/química , UreaRESUMEN
PURPOSE: The aqueous solubility and the extent of solubilization and ionization constant in sodium taurodeoxycholate (NaTDC) solutions of a series of benzoic acid and aniline derivatives were measured as a basis to characterize and thereby help predict the nature of the interaction of drugs with bile aggregates. METHODS: The aqueous solubility and the solubilization of two series of compounds, 4-alkyl benzoic acids and 4-alkyl anilines, was measured as a function of NaTDC in 0 and 150 mM NaCl. The ionization constants were determined in water and in 50 mM NaTDC at sodium chloride concentration of 0, 75 and 150 mM by spectrophotometric titration. The diffusion coefficients of NaTDC and the solutes were measured by pulsed-field gradient spin echo NMR spectroscopy. RESULTS: The aqueous solubilities decreased with increasing alkyl chain length in both series, and the aniline derivatives had larger solubilities than the benzoic acid derivatives. The number of moles of solute solubilized per mole of bile salt ranged from 0.17 to 0.31 for the benzoic acid derivatives and from 1.3 to 3.0 for the aniline derivatives. The pKa values of the benzoic acid derivatives in the presence of NaTDC were higher relative to the controls, and the difference in the pKa (delta pKa,obs) increased with increasing chain length. With the aniline derivatives, the pKa values were also shifted to higher values in NaTDC relative to the control but only in the absence of salt. The presence of the solute caused a decrease in the diffusion coefficient of NaTDC, and the diffusion coefficients of the solutes decreased with increasing alkyl chain length. With the hexyl derivative, the diffusion coefficient of the solute was smaller than the diffusion coefficient of the bile salt. The chemical shift of the protons attached to carbon 18 and 19 of the salt were decreased to a greater extent in the presence of the solutes than the protons attached to carbon 26. CONCLUSION: Both the solubilization and ionization behavior of solutes were affected by the presence of bile salt aggregates. The surface potential and effective polarity of NaTDC aggregates were found to be dependent on the alkyl chain length for these two homologous series of solutes. The solubilization ratio was largely independent of alkyl chain length, but the unitary partition coefficient was dependent on both alkyl chain length as well as ionization state. The derivatives reduced the diffusivity of the micelles suggesting the formation of larger size aggregates and the solutes (hexyl derivatives) appear to favor association with the larger sized aggregates. The phenyl ring of the solutes appears to be oriented parallel to the plane of the steroid frame with preferential positioning near the hydrophobic rings.
Asunto(s)
Compuestos de Anilina/metabolismo , Benzoatos/metabolismo , Ácido Taurodesoxicólico/metabolismo , Ácido Benzoico , Ácidos y Sales Biliares , Difusión , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Micelas , Concentración Osmolar , Solubilidad , SolucionesRESUMEN
Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin. PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal alpha-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process.
Asunto(s)
Arginina , Heparina/metabolismo , Factor Plaquetario 4/química , Factor Plaquetario 4/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Genes Sintéticos , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factor Plaquetario 4/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Native human platelet factor 4 (PF4) is a homotetrameric protein (70 residues/subunit) known for its anticoagulant heparin binding activity. 2D 15N--1H HSQC NMR experiments of native PF4 in solution show the presence of conformational heterogeneity consistent with the formation of asymmetric homo-tetramers as observed in the X-ray crystal structure of both human and bovine PF4. A chimeric mutant of PF4 (called PF4-M2) which substitutes the first 11 N-terminal residues for the first eight residues from homologous interleukin-8 forms symmetric homo-tetramers with essentially the same heparin binding activity as native PF4. The solution structure of PF4-M2 has been investigated by using two- and three-dimensional 1H- and 15N-NMR spectroscopy and NOE-restrained simulated annealing molecular dynamics. As with other members of the CXC chemokine family whose structures are known, the PF4-M2 subunit monomer consists of a mostly hydrophobic, triple-stranded antiparallel beta-sheet onto which is folded an amphipathic C-terminal helix and a less periodic N-terminal domain. Although N-terminal substitution with the less acidic interleukin-8 sequence most affects the quarternary structure relative to native PF4 at the AC and AD dimer interfaces, AB dimer stability is weakened as reflected in reduced equilibrium association binding constants.
Asunto(s)
Factor Plaquetario 4/química , Secuencia de Aminoácidos , Animales , Biopolímeros , Bovinos , Simulación por Computador , Cristalografía por Rayos X , Heparina/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutación , Factor Plaquetario 4/genética , Factor Plaquetario 4/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , SolucionesRESUMEN
We have previously suggested that variations in the 31P chemical shifts of individual phosphates in duplex oligonucleotides are attributable to torsional angle changes in the deoxyribose phosphate backbone. This hypothesis is not directly supported by analysis of the 1H/31P two-dimensional J-resolved spectra of a number of mismatch dodecamer oligonucleotide duplexes including the following sequences: d-(CGTGAATTCGCG), d(CGUGAATTCGCG), d(CGGGAATTCGCG), d(CGAGAATTCGCG), and d(CGCGAATTCACG). The 31P NMR signals of the dodecamer mismatch duplexes were assigned by 2D 1H/31P pure absorption phase constant time (PAC) heteronuclear correlation spectra. From the assigned H3' and H4' signals, the 31P signals of the base-pair mismatch dodecamers were identified. JH3'-P coupling constants for each of the phosphates of the dodecamers were obtained from 1H/31P J-resolved selective proton flip 2D spectra. By use of a modified Karplus relationship, the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. JH3'-P coupling constants were measured for many of the oligonucleotides as a function of temperature. There exists a good linear correlation between 31P chemical shifts and the epsilon torsional angle. This correlation can be further extended to the C3'-O3'-P-O5' torsional angle (zeta) by using a linear relationship between epsilon and zeta obtained from crystal structure studies. The 31P chemical shifts follow the general observation that the more internally the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. In addition, 31P chemical shifts show sequence- and site-specific variations. Analysis of the backbone torsional angle variations from the coupling constant analysis has provided additional information regarding the origin of these variations in 31P chemical shifts.
Asunto(s)
ADN , Oligodesoxirribonucleótidos , Secuencia de Bases , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Fósforo , TemperaturaRESUMEN
Assignment of the 1H and 31P NMR spectra of an extrahelical adenosine tridecamer oligodeoxyribonucleotide duplex, d(CGCAGAATTCGCG)2, has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The downfield 31P resonance previously noted by Patel et al. (1982) has been assigned by both 17O labeling of the phosphate as well as a pure absorption phase constant-time heteronuclear 31P-1H correlated spectrum and has been associated with the phosphate on the 3' side of the extrahelical adenosine. JH3'-P coupling constants for each of the phosphates of the tridecamer were obtained from the 1H-31P J-resolved selective proton-flip 2D spectrum. By use of a modified Karplus relationship the C4-C3'-O3-P torsional angles (epsilon) were obtained. There exists a good linear correlation between 31P chemical shifts and the epsilon torsional angle. The 31P chemical shifts and epsilon torsional angles follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. Because the extrahelical adenosine significantly distorts the deoxyribose phosphate backbone conformation even several bases distant from the extrahelical adenosine, 31P chemical shifts show complex site- and sequence-specific variations. Modeling and NOESY distance-restrained energy minimization and restrained molecular dynamics suggest that the extrahelical adenosine stacks into the duplex. However, a minor conformation is also observed in the 1H NMR, which could be associated with a structure in which the extrahelical adenosine loops out into solution.
Asunto(s)
Adenosina , Espectroscopía de Resonancia Magnética/métodos , Oligodesoxirribonucleótidos , Modelos Moleculares , Conformación de Ácido Nucleico , Fósforo , Protones , TermodinámicaRESUMEN
Assignment of the 31P resonances of a series of six sequenced-related tetradecamer DNA duplexes, d(TGTGAGCGCTCACA)2, d(TATGAGCGCTCATA)2, d(TCTGAGCGCTCAGA)2, d(TGTGTGCGCACACA)2, d(TGTGACGCGTCACA)2 and d(CACAGTATACTGTG)2, related to the lac operator DNA sequence was determined either by site-specific 17O labeling of the phosphoryl groups or by two-dimensional 1H-31P pure absorption phase constant time (PAC) heteronuclear correlation spectroscopy. J(H3'-P) coupling constants for each of the phosphates of the tetradecamers were obtained from 1H-31P J-resolved selective proton flip 2D spectra. By use of a modified Karplus relationship the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. Comparison of the 31P chemical shifts and J(H3'-P) coupling constants of these sequences has allowed greater insight into those various factors responsible for 31P chemical shift variations in oligonucleotides and provided an important probe of the sequence-dependent structural variation of the deoxyribose phosphate backbone of DNA in solution. These sequence-specific variations in the conformation of the DNA sugar phosphate backbone of various lac operator DNA sequences can possibly explain the sequence-specific recognition of DNA by DNA binding proteins, as mediated through direct contacts between the phosphates and the protein.
Asunto(s)
Operón Lac , Modelos Genéticos , Mutación , Regiones Operadoras Genéticas , Secuencia de Bases , Fenómenos Químicos , Química Física , ADN , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , OligonucleótidosRESUMEN
Circular dichroism and two-dimensional NMR spectra indicate that a peptide fragment consisting of the first 28 residues from the N-terminus of human growth hormone (hGH 1-28) has considerable alpha-helical structure. The peptide, (1) H-Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-Hi s-Arg- Leu-His-Gln-Leu-Ala-Phe-Asp-Thr-Tyr-OH (28), was synthesized on an automated peptide synthesizer using the Merrifield solid-phase method. The peptide can be modeled as an amphiphilic helix, and the unusual stability of the alpha-helix in aqueous solution is suggested to be attributable to formation of a dimer of alpha-helices. Most of the 1H NMR signals were assigned through pure absorption phase COSY/NOESY and single- and double-relay COSY 2D NMR spectra by using the sequential assignment methodology. The NOEs were large and negative, suggesting that the peptide was not a random coil and that it existed in solution primarily as a large, fairly rigid macromolecule, consistent with the dimer structure. A network of N alpha Hi-N alpha Hi+1 NOESY crosspeaks is observed from residues 13 to 18 as are several other crosspeaks which indicate that the peptide has considerable alpha-helical structure between residues 8 and 24. In addition, gel filtration of the peptide is consistent with a dimer structure, presumably involving packing of the two hydrophobic faces of the amphiphilic alpha-helices.
Asunto(s)
Hormona del Crecimiento , Fragmentos de Péptidos , Secuencia de Aminoácidos , Hormona del Crecimiento/síntesis química , Humanos , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Fragmentos de Péptidos/síntesis química , Conformación ProteicaRESUMEN
It is now possible to unambiguously assign all 31P resonances in the 31P NMR spectra of oligonucleotides by either two-dimensional NMR techniques or site-specific 17O labeling of the phosphoryl groups. Assignment of 31P signals in tetradecamer duplexes, (dTGTGAGCGCTCACA)2, (dTAT-GAGCGCTCATA)2, (dTCTGAGCGCTCAGA)2, and (dTGTGTGCGCACACA)2, and the dodecamer duplex d(CGTGAATTCGCG)2 containing one base-pair mismatch, combined with additional assignments in the literature, has allowed an analysis of the origin of the sequence-specific variation in 31P chemical shifts of DNA. The 31P chemical shifts of duplex B-DNA phosphates correlate reasonably well with some aspects of the Dickerson/Calladine sum function for variation in the helical twist of the oligonucleotides. Correlations between experimentally measured P-O and C-O torsional angles and results from molecular mechanics energy minimization calculations show that these results are consistent with the hypothesis that sequence-specific variations in 31P chemical shifts are attributable to sequence-specific changes in the deoxyribose phosphate backbone. The major structural variation responsible for these 31P shift perturbations appears to be P-O and C-O backbone torsional angles which respond to changes in the local helical structure. Furthermore, 31P chemical shifts and JH3'-P coupling constants both indicate that these backbone torsional angle variations are more permissive at the ends of the double helix than in the middle. Thus 31P NMR spectroscopy and molecular mechanics energy minimization calculations appear to be able to support sequence-specific structural variations along the backbone of the DNA in solution.