An ultrarapid filtration method adapted to the measurements of water and solute permeability of synthetic and biological vesicles

Abstract

An ultrarapid filtration method was adapted to the determination of water and solute permeability of membrane vesicles. This method consisted of measuring substance washout from vesicles first loaded with 3H 2O or labeled solutes, placed on filters, and rinsed at high rates for short periods. The retention of the vesicles on the filters was analyzed and was found to be a function of the nature and porosity of the filters as well as of the vesicle origin. Washing buffer flow rate and washing duration did not affect vesicle retention. The diffusional water permeability of cholesterol-free liposomes was determined at 16°C. Its value was reduced by a factor of 2.5 when the liposomes were prepared with 20% cholesterol and a threefold increase was noted when the liposomes were preincubated with gramicidin (6 mg/g lipid). Water permeability of liposomes was strongly temperature-dependent: E a = 15.3 kcal/mol. Diffusional water permeability of pink ghosts was also measured: a value of (4.4 ± 0.2) × 10 −3 cm/s ( n = 3) was obtained at 13°C. This permeability was reduced by 45.2% with 0.4 m m HgCl 2. The urea permeability of intestinal and renal brush-border membrane vesicles was (1.15 ± 0.18) × 10 −6 cm/s ( n = 7) and (1.67 ± 0.08) × 10 −6 cm/s ( n = 9), respectively. The renal value was reduced by a factor of 4.4 by 100 m m thiourea. This ultrarapid filtration technique provides an accurate method of transport measurement in sealed membranes such as liposomes and plasma membrane vesicles.