RESUMEN
The S100 subfamily of EF-hand proteins is distinguished by the binding of Zn(2+) in addition to Ca(2+). In an effort to understand the role of Zn(2+) in modulating the activity of S100 proteins, we have carried out heteronuclear NMR studies of Zn(2+)-bound S100A2 and obtained near complete resonance assignments. This analysis revealed an equilibrium between multiple isoforms due to cis-trans isomerism of proline residues in flexible regions of the protein. The secondary structure of S100A2 has been determined based on the NMR chemical shift index (CSI) technique. The protein is found to possess essentially the same secondary structure found in other S100 proteins such as S100A6 and S100B. Homology models have been built based on the high resolution three-dimensional structures of other S100 proteins. The models predict two Zn(2+) binding clusters, one involving residues His17-Cys21-Cys93 and the other Cys2-His39, and with Cys86 participating in either the N-terminal or the C-terminal binding site.
Asunto(s)
Proteínas de Ciclo Celular , Factores Quimiotácticos/química , Proteínas S100/química , Zinc/química , Proteínas de Unión al Calcio/química , Proteínas Portadoras/química , Humanos , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Factores de Crecimiento Nervioso/química , Conformación Proteica , Reproducibilidad de los Resultados , Proteína A6 de Unión a Calcio de la Familia S100 , Proteína A7 de Unión a Calcio de la Familia S100 , Subunidad beta de la Proteína de Unión al Calcio S100 , Homología de Secuencia de AminoácidoRESUMEN
The Ca(2+)-binding S100A1 protein displays a specific and differential expression in the human myocardium and is considered to be an important regulator of heart function. Because of its high expression levels in the heart we tested the performance of S100A1 as a diagnostic indicator of acute myocardial damage. Therefore, we established a S100A1-specific sandwich ELISA and determined the S100A1 plasma levels in patients with signs of acute myocardial ischemia. The concentration-time course of S100A1 was distinct from that of the "classical" biochemical markers, CK, CKMB, and troponin I, showing an early rise and a fast decline in plasma after the ischemic event. We suggest that S100A1 should be included in combinatorial measurements as an early diagnostic marker for ischemic coronary diseases.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Isquemia Miocárdica/metabolismo , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Proteínas de Unión al Calcio/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/fisiopatología , Valor Predictivo de las Pruebas , Pronóstico , Proteínas S100RESUMEN
PURPOSE: Determine the effect of PEGylation on in-vitro degradation for recombinant human Megakaryocyte Growth and Development Factor (rHuMGDF) in the neutral pH range. METHODS: Degradation products were characterized by cation-exchange HPLC, N-terminal sequencing and mass spectrometry. RESULTS: The main route of degradation was through non-enzymatic cyclization of the first two amino acids and subsequent cleavage to form a diketopiperazine and des(Ser, Pro)rHuMGDE This reaction was prevented by alkylation of the N-terminus by polyethylene glycol (PEG). CONCLUSIONS: PEGylation of proteins is commonly performed to achieve increased in-vivo circulation half-lives. For rHuMGDF, an additional advantage of PEGylation was enhanced in-vitro shelf-life stability.
Asunto(s)
Polietilenglicoles/química , Polietilenglicoles/metabolismo , Trombopoyetina/química , Trombopoyetina/metabolismo , Alquilación , Antibacterianos , Cromatografía Líquida de Alta Presión , Dicetopiperazinas , Almacenaje de Medicamentos , Humanos , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Piperazinas , Polietilenglicoles/análisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Trombopoyetina/análisisRESUMEN
Disulfide linkages in peptides or proteins were analyzed by automated gas-phase Edman sequencing performed in minimized reducing agents. If cystine linkage was regulated at the same position in two peptides during peptide preparation, the diphenylthiohydantoin derivative of cystine was significantly recovered by Edman reaction. In contrast, when the crosslinked half cystines were present at different positions in the peptides, the derivative could be poorly detected. Upon direct sequence analysis of intact bovine insulin, the PTH derivatives of cystine from both Cys-A7 and Cys-B7 were significantly released after Edman cycle 7 and gave approximately 20% recovery of common PTH amino acids. However, Cys-A11 linked to Cys-A6 was poorly detectable after Edman cycle 11. For general use of this method, proteins need to be subjected to several sets of proteolytic or chemical cleavages in the hope that at least one of the fragments will have cystine linkage at the same position. This method was applied to several fragments of platelet-derived growth factor B chain and brain-derived neurotrophic factor.
Asunto(s)
Disulfuros/química , Fragmentos de Péptidos/química , Acetilación , Secuencia de Aminoácidos , Animales , Factor Neurotrófico Derivado del Encéfalo , Bovinos , Cromatografía Líquida de Alta Presión , Cistina/química , Ditiotreitol/farmacología , Insulina/química , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Pepsina A/metabolismo , Feniltiohidantoína , Factor de Crecimiento Derivado de Plaquetas/química , Análisis de Secuencia , Termolisina/metabolismoRESUMEN
Three disulfide linkages of recombinant human brain-derived neurotrophic factor (BDNF) were determined by peptide sequence analysis and characterized by mass spectrometry. The three disulfide bonds for BDNF expressed in Chinese hamster ovary cells include Cys-13-Cys-80, Cys-58-Cys-109 and Cys-68-Cys-111, and the disulfide structure was homologous to that of nerve growth factor.