RESUMO
1,25 Dihydroxyvitamin D3 has been shown to stimulate calcium fluxes across skeletal muscle membranes. The involvement of calmodulin in the effects of the metabolite was investigated. Primary cultures of chick embryo skeletal muscle myoblasts and soleus muscles from vitamin D-deficient or 1,25 (OH)2D3-treated chicks were used. Culture of myoblasts and vitamin D-deficient soleus with 1,25 (OH)2D3 (0.05 ng/ml) for 24 and 1 hour, respectively, significantly increased 45Ca uptake by the preparations. In the presence of the calmodulin antagonists flufenazine or compound 48/80 in the uptake medium, no differences between control and treated cultures were observed. The calmodulin content of myoblasts and soleus homogenates and subcellular fractions derived therefrom was estimated by measuring their capacity to stimulate calmodulin-depleted cAMP phosphodiesterase. No changes in total calmodulin cellular content could be detected in response to 1,25(OH)2D3. However, the sterol produced an increase in calmodulin levels of microsomes, mitochondria, and crude myofibrillar fraction and a proportional decrease in cytosolic calmodulin concentration. The 1,25(OH)2D3-dependent changes in calmodulin distribution among subcellular fractions of soleus muscle were observed either in vivo or in vitro. The effects in vitro were already detectable after 5 minutes of treatment with the sterol and parallel 1,25(OH)2D3-dependent changes in tissue Ca uptake. The results suggest that changes in calmodulin intracellular distribution may underly part of the mechanism by which 1,25(OH)2D3 affects muscle calcium transport.
Assuntos
Calcitriol/farmacologia , Calmodulina/análise , Músculos/análise , Animais , Cálcio/farmacocinética , Calmodulina/antagonistas & inibidores , Galinhas , Músculos/ultraestrutura , Frações Subcelulares/análise , Frações Subcelulares/efeitos dos fármacosRESUMO
Rat brain proteins able to react with anti-myelin basic protein antiserum, raised under conditions to induce experimental allergic encephalomyelitis in rabbits, were examined by immunoblot methods after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Apart from the four forms of myelin basic protein present in rat brain, the antiserum detected other proteins of higher molecular weight. Subcellular fractionation shows that these high-molecular-weight proteins are relatively concentrated in a synaptosome-enriched fraction compared to a myelin fraction. A major protein fraction immunorelated to myelin basic protein migrated in the gels as a doublet with apparent molecular weights of approximately 80K and 86K; these proteins were tentatively identified as synapsin Ia and Ib. A purified synapsin preparation analyzed by immunoblot after two-dimensional gel electrophoresis also reacted with anti-myelin basic protein antisera. When the serum was purified by affinity chromatography on a myelin basic protein-conjugated Sepharose column the nonadsorbed material lost this activity whereas the eluted antibodies reacted with myelin basic protein and synapsin. In addition, sequence amino acid comparison of decapeptides showed some homology between these two proteins. A possible implication of immunological agents against myelin basic protein cross-reacting with extra-myelin proteins in the process of experimental allergic encephalomyelitis is considered.
Assuntos
Soros Imunes , Proteína Básica da Mielina/imunologia , Proteínas do Tecido Nervoso/análise , Sequência de Aminoácidos , Animais , Química Encefálica , Eletroforese em Gel de Poliacrilamida , Encefalomielite Autoimune Experimental/metabolismo , Técnicas de Imunoadsorção , Peso Molecular , Ratos , Frações Subcelulares/análise , SinapsinasAssuntos
Membrana Celular/fisiologia , Canais Iônicos/análise , Neurônios/fisiologia , Animais , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Humanos , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , Lipossomos/metabolismo , Glicoproteínas de Membrana/análise , Modelos Moleculares , Neurônios/metabolismo , Receptores de Neurotransmissores/análise , Frações Subcelulares/análiseRESUMO
The rapid restoration of liver protein mass observed in protein-depleted mice when they are fed with an adequate diet is quantitatively explained by a large decrease in the average rate of breakdown of total liver proteins. This study was performed in order to know whether this inhibition of breakdown affects in the same way all the protein constituents of the tissue, or only affects a group of these proteins belonging to a particular subcellular fraction. Subcellular fractions were obtained by differential centrifugation. The relative rates of breakdown of their proteins were estimated by the conservation of radioactivity in these proteins previously labelled by the administration of NaH14CO3 to mice. The results obtained indicated: 1) a general decrease in the rate of breakdown of proteins of subcellular fractions from re-fed livers compared with livers of protein-depleted mice; 2) a decrease of breakdown of proteins from cytosol in re-fed mice which is higher as lower is the molecular weight of the proteins subunits.