H⁺-ATPase and Na⁺/H⁺ antiport activities at plasma membrane vesicles.

<p>Plasma membranes were isolated from mutant cells (AXT3K) and related transgenic strains (SpSOS1, SpAHA1 and SpSOS1+SpAHA1) using the aqueous two-phase system. Fluorescent quenching of quinacrine was used to monitor the acidification of plasma membrane vesicles from these yeast cells. Formation of a pH gradient, indicating that H⁺ was pumped into the vesicles, was initiated with ATP. Once fluorescence was stabilized, 50 mM Na₂SO₄ was added to the cuvette, and fluorescence recovery, indicating Na⁺/H⁺ exchange, was monitored. The reaction was terminated by adding 25 mM (NH₄)₂SO₄, which dissipated the pH gradient. The change in fluorescence is expressed as arbitrary units (A). H⁺-ATPase activity was analyzed by measuring ΔpH across the membrane; it is expressed as the change in fluorescence quenching per minute per milligrams of membrane protein (ΔF/min/mg protein) (B). Na⁺/H⁺ exchange activity is given as the proportion of dissipation of the preformed pH gradient per minute per milligram of membrane protein (ΔF%/ min/mg protein) (C). Values are expressed as the means±SE of three replicates.