Chloride transport in human erythrocytes and ghosts: a quantitative comparison.
1. Homogeneous preparations of resealed ghosts with intracellular KCl concentrations between 15 and 900 mM could be prepared. Virtually all ghosts sealed to chloride. The chloride transport system was found not to be damaged: a quantitative comparison of the self-exchange of 36Cl- across intact and resealed membranes showed that both the transport capacity and a number of characteristic properties were identical (saturation kinetics, temperature dependence and the effect of inhibitors). 2. Due to the absence of intracellular titratable buffers intracellular chloride concentration in ghosts vary only slightly between pH5 and 11. The unidirectional exchange flux was constant between pH 7 and 11, showing that the transport system does not have a functionally important titratable group in the alkaline range, as previously assumed. The decrease of transport below pH 7 is similar in intact erythrocytes and ghosts. 3. Mean cellular volume of the resealed ghosts was a function of the amount of KCl added at 'reversal', before the ghosts are sealed. The ghosts shrank by osmosis when KCl was added to the suspension of 'unsealed' ghosts. The reflexion coefficient of sucrose (and therefore the osmotic effect) is larger than that of KCl. It was, therefore, possible to demonstrate that volume changes do not affect the chloride transport across the human red cell membrane. Unidirectional chloride fluxes at a KCl concentration of 165 mM were independent of ghost volume (100-40 mum3).
Chloride transport in human erythrocytes and ghosts: a quantitative comparison. 1. Homogeneous preparations of resealed ghosts with intracellular KCl concentrations between 15 and 900 mM could be prepared. Virtually all ghosts sealed to chloride. The chloride transport system was found not to be damaged: a quantitative comparison of the self-exchange of 36Cl- across intact and resealed membranes showed that both the transport capacity and a number of characteristic properties were identical (saturation kinetics, temperature dependence and the effect of inhibitors). 2. Due to the absence of intracellular titratable buffers intracellular chloride concentration in ghosts vary only slightly between pH5 and 11. The unidirectional exchange flux was constant between pH 7 and 11, showing that the transport system does not have a functionally important titratable group in the alkaline range, as previously assumed. The decrease of transport below pH 7 is similar in intact erythrocytes and ghosts. 3. Mean cellular volume of the resealed ghosts was a function of the amount of KCl added at 'reversal', before the ghosts are sealed. The ghosts shrank by osmosis when KCl was added to the suspension of 'unsealed' ghosts. The reflexion coefficient of sucrose (and therefore the osmotic effect) is larger than that of KCl. It was, therefore, possible to demonstrate that volume changes do not affect the chloride transport across the human red cell membrane. Unidirectional chloride fluxes at a KCl concentration of 165 mM were independent of ghost volume (100-40 mum3).