RESUMEN
Previous experiments have shown that acidosis enhances isoproterenol-induced phospholamban (PHL) phosphorylation (Mundiña-Weilenmann, C., Vittone, L., Cingolani, H. E., Orchard, C. H. (1996) Am. J. Physiol. 270, C107-C114). In the present experiments, performed in isolated Langendorff perfused rat hearts, phosphorylation site-specific antibodies to PHL combined with the quantitative measurement of 32P incorporation into PHL were used as experimental tools to gain further insight into the mechanism involved in this effect. At all isoproterenol concentrations tested (3-300 nM), phosphorylation of Thr17 of PHL was significantly higher at pHo 6.80 than at pHo 7.40, without significant changes in Ser16 phosphorylation. This increase in Thr17 phosphorylation was associated with an enhancement of the isoproterenol-induced relaxant effect. In the absence of isoproterenol, the increase in [Ca]o at pHo 6.80 (but not at pHo 7.40) evoked an increase in PHL phosphorylation that was exclusively due to an increase in Thr17 phosphorylation and that was also associated with a significant relaxant effect. This effect and the phosphorylation of Thr17 evoked by acidosis were both offset by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. In the presence of isoproterenol, either the increase in [Ca]o or the addition of a 1 microM concentration of the phosphatase inhibitor okadaic acid was able to mimic the increase in isoproterenol-induced Thr17 phosphorylation produced by acidosis. In contrast, these two interventions have opposite effects on phosphorylation of Ser16. Whereas the increase in [Ca]o significantly decreased phosphorylation of Ser16, the addition of okadaic acid significantly increased the phosphorylation of this residue. The results are consistent with the hypothesis that the increase in phospholamban phosphorylation produced by acidosis in the presence of isoproterenol is the consequence of two different mechanisms triggered by acidosis: an increase in [Ca2+]i and an inhibition of phosphatases.
Asunto(s)
Acidosis , Proteínas de Unión al Calcio/metabolismo , Corazón/efectos de los fármacos , Isoproterenol/farmacología , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Calcio/farmacología , Proteínas de Unión al Calcio/efectos de los fármacos , Corazón/fisiología , Técnicas In Vitro , Masculino , Contracción Miocárdica/efectos de los fármacos , Fosfatos/metabolismo , Fosforilación , Fosfoserina/análisis , Fosfotreonina/análisis , Ratas , Ratas Wistar , Retículo Sarcoplasmático/metabolismoAsunto(s)
Aorta/metabolismo , Proteínas de Unión al Calcio/metabolismo , Microsomas/metabolismo , Músculo Liso Vascular/metabolismo , Nitroprusiato/farmacología , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/aislamiento & purificación , Gatos , Immunoblotting , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Contracción Miocárdica , Fosforilación , Fosfoserina/análisis , Fosfotreonina/análisis , Ratas , Retículo Sarcoplasmático/metabolismoRESUMEN
The 5S rRNA-ribosomal protein YL3 ribonucleoprotein particle was isolated from yeast cells labeled with [32P]orthophosphate. The protein moiety was purified and found to be radioactive. Labeled phosphoserine was detected after partial hydrolysis of the protein. Up to two phosphate residues are sterified per mole of YL3 ribosomal protein.
Asunto(s)
Proteínas Fúngicas/metabolismo , ARN Ribosómico 5S/metabolismo , Proteínas de Unión al ARN , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Fosforilación , Fosfoserina/análisis , Saccharomyces cerevisiae/genéticaRESUMEN
The effect of undernutrition during suckling on phosphoryl serine levels was investigated in rats. Young rats were undernourished by suckling their lactating mothers fed an 8% (by weight) protein diet. Mothers of normal rats were fed a 25% protein diet. After weaning (21 d), normal and undernourished rats were fed a 25% protein diet until 90-120 d of age. Previously we observed that undernutrition during suckling decreases the levels of phosphoryl serine in nuclear proteins from the whole brain of adult rats. In this paper we report more specifically on the effect of undernutrition during this period on the levels of phosphoryl serine in nuclear and synaptosomal proteins from the cerebral cortex of young and nutritionally rehabilitated adult rats. In young, undernourished rats the levels of phosphoryl serine in nuclear proteins were significantly lower (28%) than those in control animals. This alteration persisted in nutritionally rehabilitated adult rats. Serine and protein levels in cerebral cortex were the same in all groups. No alteration in the levels of phosphoryl serine in the synaptosomal fraction was observed.