Permeability characteristics of erythrocyte ghosts prepared under isoionic conditions by a glycol-induced osmotic lysis

A detailed study has been made of the permeability characteristics of human erythrocyte ghosts prepared under isoionic conditions by a glycol-induced lysis (Billah, M.M., Finean, J.B., Coleman, R. and Michell, R.H. (1976) Biochim. Biophys. Acta 433, 45-54). Impermeability to large molecules such as dextran (average molecular weight 70 000) was restored immediately and spontaneously after each of the 5-7 lyses that were required to remove all of the haemoglobin. Permeabilities to smaller molecules such as MgATP^2-, [³H]inositol and [¹⁴C]choline were initially high but could be greatly reduced by incubation at 37°C for an hour. The extent of such resealing decreased as the number of lyses to which the ghosts had been subjected increased. Both removal of haemoglobin and permeabilities to small molecules were affected significantly by pH, Ca²⁺ concentrations and divalent cation chelators. Maximum resealing was achieved in ghosts prepared in the basic ionic medium (130 mM KCl, 10 mM NaCl, 2 mM MgCl₂, 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES)) at pH 7.0 (0°C) and with a calcium level around 10^-5 M. Acidic pH facilitated the removal of haemoglobin whilst the presence of divalent cation chelators slowed down its release. Retention of K⁺ by ghosts loaded with K⁺ during the first lysis and subsequently incubated at 37° C was substantial but little K⁺ could be retained within the haemoglobin-free ghosts. Permeability of the ghosts to K⁺ after one lysis was affected by temperature, pH, Ca²⁺ concentrations and by the presence of divalent cation chelators.