Senescence-Dependent Increase in the Permeability of Liposomes Prepared from Bean Cotyledon Membranes

Abstract

Liposomes containing 79 mM Tris-acetate and 50 mM KCl were prepared from the total lipid extracts of smooth microsomal membranes isolated from 2, 4, 7 and 9 d old bean cotyledons. Permeability to glycerol was determined by spectrophotometric measurements of osmotic swelling when the liposomes were placed in either isotonic or slightly hypotonic glycerol. For liposomes from 2 and 4 d old membrane there was a time-dependent decrease in absorbance at 450 nm from which initial swelling rates reflecting the influx of glycerol and water were calculated. At 25 °C these rates were not significantly different For liposomes from 7 and 9 d old membrane there was no change in absorbance with time at 450 nm signifying that these older liposomes were equally permeable and non-electrolytes and non-electrolytes, and therefore incapable of swelling. Permeability to glucose was determined by preparing the liposomes in a solution of the sugar, passing them through a Sephadex column to eliminate unsequestered glucose, and quantifying sugar leaked from the liposomes over time by measuring NADPH formation through the tandem actions of hexokinase and glucose-6-phosphate dehydrogenase. The rate constants for glucose leakage from 2 and 7 d old liposomes were 0.55 and 3.94 respectively, again indicating a dramatic increase in permeability with advancing age. These changes in permeability correlate temporally with the appearance of gel phase lipid in both liposomes and the membranes from which they were derived, suggesting that the coexistence of discrete liquid-crystalline and gel phase lipid domains renders membranes leaky and contributes to loss of intracellular compartmentation in senescing tissue.