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 microM up to 3000 microM 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 Km values and Vmax values varied. The fitted curves displayed a high affinity-low Vmax (Km values of 7-60 microM and Vmax values of 0.03-0.50 mumol P(i)/mg per min) and a low affinity-high Vmax component (Km values of 408-1960 microM and Vmax values of 0.26-5.82 mumol 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.
Berberián
G
G
Instituto de Investigación Médica, Mercedes y Martín Ferreyra, Córdoba, Argentina.
Helguera
G
G
Beaugé
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D017830:Octoxynol; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003902:Detergents; D000255:Adenosine Triphosphate; D017830:Octoxynol; D006180:Proton-Translocating ATPases