RESUMEN
An RT-PCR based strategy to clone the membrane-associated steroid binding protein ratp28 additionally amplified a novel sequence-related PCR product termed HC5. The HC5 PCR product was cloned and sequenced and showed 94% nucleotide sequence similarity to ratp28. The HC5 cDNA sequence open reading frame encodes a predicted 75 amino acid (8.0kDa) protein, and is therefore truncated compared to ratp28 (195 amino acids, 21.6kDa). In vitro transcription and translation of the HC5 cDNA resulted in the production of 2 proteins of approximately 8 and 6kDa. Restriction digests from various tissues demonstrated that liver and heart expressed primarily ratp28 mRNA whereas kidney and blood contained both ratp28 and HC5 transcripts. Phage display was employed to generate an antibody fragment to a peptide sequence conserved in ratp28 and HC5. Western blotting identified a 10kDa protein in cytosolic fractions of rat kidney. The function of HC5 remains to be determined.
Asunto(s)
Proteínas Portadoras/química , Riñón/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Globulina de Unión a Progesterona/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Proteínas Portadoras/biosíntesis , Clonación Molecular , Secuencia Conservada , Citosol/metabolismo , ADN Complementario/metabolismo , Ensayo de Inmunoadsorción Enzimática , Péptidos y Proteínas de Señalización Intracelular , Masculino , Microsomas/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Biblioteca de Péptidos , Péptidos/química , Perfusión , Plásmidos/metabolismo , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Transcripción GenéticaRESUMEN
Progesterone (P4) inhibits both granulosa cells and spontaneously immortalized granulosa cells (SIGCs) from undergoing apoptosis. P4 does so through a plasma membrane-initiated event. It appears that P4's membrane-initiated actions are mediated by a 60-kDa P4 binding protein (P4BP), which is detected by an antibody directed against the ligand binding domain of the nuclear P4 receptor (i.e., C-262). Immunohistochemical analysis revealed that a C-262-detectable protein was first observed in the periphery of a few granulosa cells within early antral-stage follicles. In nonatretic antral follicles, this protein was detected at the periphery of virtually all granulosa cells. In contrast, granulosa cells of atretic follicles lost the distinct peripheral localization of this C-262-detectable protein. This reduction in the membrane localization was also observed by Western blot analysis. To assess the temporal changes in this 60-kDa P4BP during apoptosis, studies were conducted using SIGCs. That this 60-kDa protein is important in mediating P4's action was confirmed by the observation that C-262 but not IgG attenuated P4's antiapoptotic action. Interestingly, the membrane localization of this 60-kDa P4BP was maintained but the ability of P4 to prevent apoptosis was lost within 20 min of initiating the apoptotic cascade. In addition, Erk-1 and -2 phosphorylation (i.e., activity) increased within 20 min of P4 withdrawal. Further, P4 suppressed the increase in the Erk-1 phosphorylation if administered within 5 but not 20 min of initiating the apoptotic cascade. Moreover, the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, reduced the percentage of SIGCs undergoing apoptosis in the absence of P4. Because MEK phosphorylates Erk, these observations suggests that 1) the increase in Erk-1 activity is an important part of the apoptotic cascade, 2) P4 promotes granulosa cell viability by modulating the activity of Erk-1, and 3) P4 becomes "uncoupled" from its antiapoptotic signal transduction mechanism within 20 min of initiating apoptosis, even though the membrane localization of the 60-kDa P4BP is maintained.
Asunto(s)
Apoptosis/fisiología , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Sistema de Señalización de MAP Quinasas , Globulina de Unión a Progesterona/metabolismo , Animales , Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Flavonoides/farmacología , Células de la Granulosa/efectos de los fármacos , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Peso Molecular , Progesterona/metabolismo , Progesterona/farmacología , Globulina de Unión a Progesterona/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas WistarRESUMEN
The isolation of cortisol and progesterone binding globulin (CBG) from pregnant women serum was performed using affinity and hydrophobic chromatography. The purity and specificity of isolated transcortin was tested by agarose gel electrophoresis using racket and cross immunoelectrophoresis and specific CBG antibodies. High purity and immunoreactivity of the isolated globulin destitute of other proteins contamination, were obtained.
Asunto(s)
Cromatografía/métodos , Embarazo/sangre , Transcortina/aislamiento & purificación , Cromatografía de Afinidad , Electroforesis en Gel de Agar , Femenino , Humanos , Globulina de Unión a Progesterona/química , Globulina de Unión a Progesterona/aislamiento & purificación , Sensibilidad y Especificidad , Transcortina/químicaRESUMEN
A progesterone- and testosterone-binding globulin, has been isolated from a pool of pregnant guinea pig serum, using DEAE-Sephadex chromatography, gel filtration on Sephadex G-200 and preparative polyacrylamide gel electrophoresis. This globulin appeared to be heterogeneous on ion exchange chromatography, isoelectric focusing and equilibrium ultracentrifugation, indicating a quarternary structure sensitive to concentration and to ionic environment. In line with the steroid-binding serum proteins so far described progesterone-binding globulin is a glycoprotein with a carbohydrate content of 42%. Extrapolated sedimentation and diffusion coefficients were 4.52 S and 5.1.x 10(-7) cm2/s, respectively. A partial specific volume of 0.678 cm3/g has been calculated from the chemical composition. A molecular weight of 82,00 was obtained from equilibrium centrifugation experiments in the presence of o0I1 sodium dodecyl sulfate. The electrophloretic mobility at pH 8.6 corresponds to that of an a,lpha1globulin; isoelectric points were 4.4 and 3.5; Stokes radius, 4.9 nm; frictional ratio, 1.74; extinction coefficient at 280 nm, 7.3. Pure progesterone-binding globulin showed an association constant at 4.0 degrees of 14 x 10(9)M-(1) for progesterone. The affinity for testosterone was lower being 8.2 x 10(7) M(-1). The number of high-affinity binding sites was determined to 1.0 or both steroids.