3.2. Enzyme Purification

The plasma membranes were isolated as follows: The K. lactis strain NCYC 416 (the National Collection of Yeast Cultures; Norwich, UK.) was grown in YPD at 30 ºC by 20 hr. The cells were harvested at the mid-log phase and suspended in 1 M sorbitol pH 7.0, containing Zymolyase-20T (20 units/g wet-weight) at 30 °C by 1 to 2 h. The yeast spheroplasts were disrupted by sonication at 4 °C, and plasma membranes were isolated by differential centrifugation, briefly: the cell homogenate was centrifuged at 2,080× g to remove unbroken cells and cell walls, then the supernatant was centrifuged at 18,500× g to remove organelles such as mitochondria, after that, the supernatant was centrifuged at 38,400× g; plasma membranes were found in the pellet. The H⁺-ATPase was purified from the plasma membranes upon solubilization with Zwittergent 3,14 and then by centrifugation on a trehalose concentration gradient as described by Sampedro et al. (2007) [22]. The fractions containing the H⁺-ATPase were centrifuged at 100,000× g by 3 h, and the pellets were suspended in a small volume of 2 mM EGTA, 10 mM MOPS, pH 7.2, and kept at −70 °C until used. On SDS-PAGE, the ~100,000 M_r band corresponding to the plasma membrane H⁺-ATPase was ~90% (as determined by densitometry) from total protein yield. The protein concentration was determined by using the Lowry assay and bovine serum albumin as standard [47].