RESUMEN
It has been found that 1 h after rats' irradiation (dose 800 rad) the incorporation of the label 2, 4, 6-trinitrobenzenesulpho-acid into phosphatidylethanolamine of erythrocyte membranes increases and 3 h and 72 h after irradiation it decreases. Concanavalin and calcium ions modify the incorporation of the label into phosphatidylethanolamine.
Asunto(s)
Membrana Eritrocítica/efectos de la radiación , Nitrobencenos/efectos de la radiación , Fosfatidiletanolaminas/efectos de la radiación , Traumatismos Experimentales por Radiación/sangre , Ácido Trinitrobencenosulfónico/efectos de la radiación , Animales , Calcio/sangre , Concanavalina A/sangre , Membrana Eritrocítica/metabolismo , Rayos gamma , Fosfatidiletanolaminas/sangre , Ratas , Ratas Endogámicas , Factores de Tiempo , Ácido Trinitrobencenosulfónico/sangreRESUMEN
The glutathione content of human erythrocytes rapidly diminishes when cells are exposed to 2,4,6-trinitrobenzenesulfonate (20 mumol/l cells) at 37 degrees C. Even at 0 degrees C a slow decrease in glutathione content is observed. The uptake of trinitrobenzenesulfonate by the cells is retarded by inhibitors of the inorganic anion exchange system, indicating that trinitrobenzenesulfonate enters the cells by this pathway. The disappearance of glutathione most probably results from the reaction: 2 GSH + trinitrobenzenesulfonate leads to GSSG + aminodinitrobenzenesulfonate. The reaction of trinitrobenzenesulfonate with glutathione occurs prior to its covalent binding to amino groups of hemoglobin which makes this reaction a more sensitive method of detection of penetration of trinitrobenzenesulfonate into erythrocytes. Results of studies on the asymmetric distribution of phospholipids using trinitrobenzenesulfonate as the only probe should be reconsidered in the light of these new data.
Asunto(s)
Eritrocitos/metabolismo , Nitrobencenos/sangre , Ácido Trinitrobencenosulfónico/sangre , Glutatión/sangre , Humanos , Cinética , Oxidación-Reducción , Fosfolípidos/sangreRESUMEN
The major aims of this study were to determine the degree of phospholipid asymmetry and the neighbor analysis of phospholipids in different types of cell membranes. For this study a penetrating probe (FDNB), a non-penetrating probe (TNBS) and a cross-linking probe (DFDNB) were used. The reaction of hemoglobin, membrane protein and membrane PE and PS of erythrocytes with DFNB and TNBS was studied over a concentration range of 0.5 to 10 mM probe. TNBS reacts to an extremely small extend with hemoglobin over the concentration range 0.4 to 4 mM whereas FDNB reacts with hemoglobin to a very large extent (50 fold more than TNBS). The reaction of membrane protein of intact erythrocytes reaches a sharp plateau at 1 mM TNBS whereas the reaction of membrane protein goes to a much larger extent with FDNB with no plateau seen up to 4 mM FDNB. This data shows that TNBS does not significantly penetrate into the cell under our conditions whereas FDNB does penetrate into the cell. The results show that there are four fold more reactive sites on proteins localized on the inner surface of the erythrocyte membrane as compared to the outer surface. TNBS at 0.5 to 2 mM concentration does not label membrane PS and labels membrane PE to a small extent. The reaction of PE with TNBS shows an initial plateau at 2 mM probe and a second slightly higher plateau between 4 to 10 mM probe. TNBS from 0.5-2.0 mM does not react with PS, but between 3 to 10 mM concentration, a very small amount of PS reacts with TNBS. Hence above 2 mM TNBS or FDNB a perturbation occurs in the membrane such that more PE and PS are exposed and react with these probes. These results demonstrate that essentially no PS is localized on the outer surface of the membrane and only 5% of the total membrane PE is localized on the outer surface of the erythrocyte membrane. TNBS and FDNB were reacted with yeast, E. coli, and Acholeplasma cells. With yeast cells, FDNB reacts to a much larger extent with PE than does TNBS, indicating that FDNB penetrates into the cell and labels more PE molecules. With E. coli, but not with erythrocytes or yeast cells, phospholipase A activity was very pronounced at pH 8.5 giving rise to a large amount of DNP-GPE from DNP-PE. A phosphodiesterase was also present which hydrolyized DNP-GPE to DNP-ethanolamine. The multilayered structure of the E. coli cell envelop did not permit a definitive interpretation of the results. It is clear, however, that TNBS and FDNB react to a different extent with PE in this cell. The Acholeplasma membrane had no detectable PE or PS but contains amino acid esters of phosphatidylglycerol. The reaction of these components with TNBS and FDNB indicate that these aminoacyl-PG are localized on both surfaces of the membrane, with 31% being on the outer surface and 69% on the inner surface...