ATP ACTIVATION OF PLASMA-MEMBRANE YEAST H+-ATPASE SHOWS COMPLEX KINETICS INDEPENDENTLY OF THE DEGREE OF PURIFICATION

ATP stimulation of plasma membrane H+-ATPase activity from a wild baker's yeast (Saccharomyces cerevisiae) was followed under conditions of progressive degrees of purification. A particular emphasis was put to cover a wide range of concentrations which went from 2 mu M up to 3000 mu M ATP. The preparations used were (i) crude membrane fraction, (ii) untreated plasma membrane fraction obtained by differential centrifugation, (iii) residual plasma membrane treated with Triton X-100, (iv) enzyme solubilized with either Zwittergent 3-14 alone or after Triton X-100 treatment. Under all conditions the fitting of the dose-response curves required an equation composed by the sum of two Michaelian terms. Depending on the treatment, the K-m values and V-max values varied. The fitted curves displayed a high affinity-low V-max (K-m values of 7-60 mu M and V-max values of 0.03-0.50 mu mol P-i/mg per min) and a low affinity-high V-max component (K-m values of 408-1960 mu M and V-max values of 0.26-5.82 mu mol P-i/mg per min). The complex ATP activation curve of the yeast plasma membrane H+-ATPase is in line with similar behavior found for the H+-ATPase of higher plants and all known animal cation transport ATPases.