Fig. 4. The effect of anti-HA antibody on the ATPase activity in yeast expressing the HA-tagged subunits. Bafilomycin A₁-sensitive ATPase activity was measured for solubilized and fractionated vacuolar membranes with or without anti-HA antibody. Relative activity is given for each strain as compared to its specific ATPase activity without the antibody (100% activity). For each assay 50 µl (2 µg protein) of the fraction with most active ATPase (fraction no. 5 out of the 4–6 active fractions for Vma16p-HA and Vma10p-HA; fraction no. 4 out of the 4–7 fractions for Vma7p-HA; and fraction no. 7 out of the 5–8 active fractions for wild type, WT), was preincubated in the presence and absence of 50 µl of 1:1000 (w/v) diluted anti-HA monoclonal antibody. 5 mmol l^–1 MgATP was added, and following 10 min incubation at 30°C, the reaction was stopped and absorbance at 660 nm monitored. Each value represents a one single experiment out of three.