Isolation of plasma membranes from mung bean hypocotyl sections by two-phase partitioning: suitability of the technique for ageing tissues

This paper discusses the application of a particular two-phase partitioning system to the isolation of plasma membranes from heterogeneous starting material, differing in physiological age. Plasma membranes were isolated from hypocotyl segments of mung beans ( Vigna radiata L. Wilczek) on four successive days in order to examine the variation caused by ageing of the seedling. Additionally, the segments were cut at different positions of the hypocotyl to measure variation caused by position-related ageing. To assess purity and degree of contamination of the plasma membrane-enriched preparations, a series of membrane enzyme markers were screened for all isolated fractions. Glucan synthetase II activities were enriched in the plasma membrane fractions, but enrichment and recovery became less pronounced with increasing age. Plasma membrane ATPase activity affected by VO₄^3-, Ca²⁺ and K⁺ was similar in all segments throughout the time-course of the experiment (4 days). However, control ATPase activity varied with segment origin: the physiologically oldest segments showed only 75% activity compared to the youngest ones. K_m and V_max values indicated a smaller proportion of active enzyme but higher substrate affinity as the age of the segments increased. Contamination by intracellular membranes was minimal and unrelated to tissue age.