RESUMEN
Using the immunoblotting method, the synthesis of two copper-transporting P1-type ATPases, ATP7A (a candidate for the product of the Menkes disease gene) and ATP7B (presumed product of the Wilson disease gene), in the yolk sac cells of rat embryos at days 11 and 20 of embryogenesis was demonstrated. Concomitantly, yolk sac cells produce ceruloplasmin, a soluble copper-transporting glycoprotein, a proportion of which in secreted proteins progressively diminishes, attaining 5.2% at day 11 and 3.1% at day 20 of development. At different stages of embryogenesis, yolk sac cells synthesize two molecular forms of [14]C-ceruloplasmin, one of which is secreted towards the embryo, whereas the other, towards the decidual membrane. Two forms of ceruloplasmin secreted in polar directions differ in the rate of secretion. The role of the yolk sac as a key organ controlling the delivery and secretion of copper in the embryo during the postimplantation period is discussed.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión/metabolismo , Ceruloplasmina/metabolismo , Cobre/metabolismo , Proteínas Recombinantes de Fusión , Saco Vitelino/metabolismo , Secuencia de Aminoácidos , Animales , ATPasas Transportadoras de Cobre , Femenino , Datos de Secuencia Molecular , Embarazo , RatasRESUMEN
The interaction was studied of ceruloplasmin (Cp, EC 1.16.3.1), a copper-containing plasma protein, with two synthetic peptides P15 and P16 whose structures correlate with those of the noncytosolic regions of the copper transfer P1 type ATPase (ATP7A), apparently encoded by the Menkes disease gene (Atp7a). Pentadecapeptide P15 and hexadecapeptide P16 were synthesized using the solid phase method. They correspond to fragments of two extracellular loops ATP7A, of which one loop is apparently involved in the copper ion transfer (P16) whereas the other is not (P15). The protein footprinting showed that P16 binds to a fragment of the ceruloplasmin domain 6. Kinetics of the ceruloplasmin-P16 binding was studied by affinity chromatography on P16 immobilized on a macroporous disk, and the Kd value (1.5 x 10(-6) M) of this interaction was determined. The ATP7A involvement in the copper ion transfer to nonhepatocyte cells is discussed.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Ceruloplasmina/metabolismo , Cobre/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión , Secuencia de Aminoácidos , Ceruloplasmina/química , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , ATPasas Transportadoras de Cobre , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Huella de ProteínaRESUMEN
The oxidase activity of ceruloplasmin (Cp, EC 1.16.3.1), the content of immunoreactive Cp and copper ion concentration were measured in the serum of eight day-old rats receiving either breast feeding (control group) or commercial nutritive mixture which has been recommended for the newborn children beginning from zero age (experimental group). It was shown that the artificial feeding caused almost 3-fold increase of Cp oxidase activity and copper content in the serum when compared to age-matched controls. No changes in the copper content per Cp molecule were observed. Dot-hybridization of the total liver polyribosomal RNA with Cp [32P]cDNA showed that the increased Cp level in the blood of the rats of experimental group correlated well with the level of expression of Cp gene. The copper content in the liver of experimental rats was two times lower that in control animals while no differences was found in the brain copper content between two groups of rats. The role of the regulation of Cp gene expression in the lactating mammary gland and of milk Cp in the copper homeostasis in the newborn body is discussed.
Asunto(s)
Cobre/metabolismo , Alimentos Infantiles , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Ceruloplasmina/genética , Ceruloplasmina/inmunología , Ceruloplasmina/metabolismo , Cobre/sangre , Expresión Génica , Hígado/metabolismo , Oxidorreductasas/metabolismo , Polirribosomas/genética , ARN/análisis , RatasAsunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Cobre/metabolismo , Síndrome del Pelo Ensortijado/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/metabolismo , Proteínas Recombinantes de Fusión , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ATPasas Transportadoras de Cobre , Mamíferos , Síndrome del Pelo Ensortijado/enzimología , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
The content of ceruloplasmin (Cp) was determined in 96 samples of human breast milk using rocket immunoelectrophoresis and measurement of Cp oxidase activity. The concentration of immunoreactive Cp in milk decreased about 9 times during the first 20 days of lactation while the specific oxidase activity decreased only 4 times. Two-dimensional electrophoresis of purified milk Cp before and after its treatment with chelating agents showed that copper atoms in milk Cp are more sensitive to EDTA treatment that those in blood Cp. The comparison of the different lectin-binding ability of blood and milk Cp's revealed a difference in the composition of their carbohydrate chains. The mechanisms controlling the uptake of copper ions by newborns at the level of the expression of the Cp-encoding gene in the mammary gland of the mother are discussed.