RESUMEN
The x-ray crystal structures of the cyanide derivative of Lucina pectinata monomeric hemoglobin I (L. pectinata HbI) and sperm whale (Physeter catodon) myoglobin (Mb), generally taken as reference models for monomeric hemoproteins carrying hydrogen sulfide and oxygen, respectively, have been determined at 1.9 A (R-factor = 0. 184), and 1.8 A (R-factor = 0.181) resolution, respectively, at room temperature (lambda = 1.542 A). Moreover, the x-ray crystal structure of the L. pectinata HbI:cyanide derivative has been studied at 1.4-A resolution (R-factor = 0.118) and 100 K (on a synchrotron source lambda = 0.998 A). At room temperature, the cyanide ligand is roughly parallel to the heme plane of L. pectinata HbI, being located approximately 2.5 A from the iron atom. On the other hand, the crystal structure of the L. pectinata HbI:cyanide derivative at 100 K shows that the diatomic ligand is coordinated to the iron atom in an orientation almost perpendicular to the heme (the Fe-C distance being 1.95 A), adopting a coordination geometry strictly reminescent of that observed in sperm whale Mb, at room temperature. The unusual cyanide distal site orientation observed in L. pectinata HbI, at room temperature, may reflect reduction of the heme Fe(III) atom induced by free radical species during x-ray data collection using Cu Kalpha radiation.
Asunto(s)
Cianuros/metabolismo , Hemoglobinas Anormales/química , Hemoglobinas Anormales/metabolismo , Hemoglobinas , Mioglobina/química , Mioglobina/metabolismo , Animales , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Bivalvos , Cristalografía por Rayos X , Cianuros/química , Radicales Libres/química , Hemo/química , Técnicas In Vitro , Modelos Moleculares , Unión Proteica , Conformación Proteica , BallenasRESUMEN
The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3-->Glu, Lys9--> Glu, Arg26-->Asp and Arg124-->Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (approximately 5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.
Asunto(s)
Avidina/química , Escherichia coli/genética , Proteínas Recombinantes/química , Animales , Anticuerpos/sangre , Antígenos/inmunología , Avidina/análisis , Avidina/genética , Unión Competitiva , Biotina/metabolismo , Biotinilación , Pollos , Cristalografía por Rayos X , Expresión Génica/genética , Punto Isoeléctrico , Espectrometría de Masas , Modelos Moleculares , Mutación/genética , Unión Proteica/genética , Desnaturalización Proteica , Proteínas Recombinantes/inmunología , Estreptavidina/análisisRESUMEN
The adiposogenital syndrome develops during the puberal period starting from a particular family constellation, that is described. The origins of the pathological alteration are found at the first object relationships, where feeding is felt as self-feeding. Food normally plays the role of transitional object in child. In these cases, this role fails and ends by helping separation denial. Fat is laid on the intermediate space as a fetish object, a witness of the omnipotent fantasy of antistic feeding. The child develops a great ability to maintain this balance, making eating a highly pleasant activity. Difficulties encountered during treatment are commented.