The plasma membrane H⁺ ‐ATPase from yeast

Abstract

The H+-ATPase from the plasma membrane of Saccharnmyces cerevisiae was isolated and purified. The rate of ATP hydrolysis and ATP binding was measured as a function of pH and the effect of the vanadate and erythrosine B inhibitors was investigated.The pH dependence of the rate of ATP hydrolysis forms a bell-shaped curve with a maximum at pH 6 and half-maximal rates at pH 5.0 and 7.4. Only the pH dependence between pH 6 and pH 7.6 is reversible. Above pH 7.6 and below pH 5.5, denaturation of the isolated enzyme is observed. The rate of ATP binding shows the same pH dependency as that of ATP hydrolysis. Both pH dependencies can be described by the dissociation of a monovalent acidic group with a pK of 7.4. It is concluded that the enzyme must be protonated before ATP binding.Vanadate does not inhibit ATP binding, ADP release or Pi release at concentrations where complete inhibition of ATP hydrolysis is observed. It is concluded that vanadate inhibits a step of the reaction cycle which occurs after Pi release. In contrast, erythrosine B inhibits ATP binding and thus affects the first step of the reaction cycle.The plasma membrane Hf-ATPase is an integral membrane protein of yeast cells. The enzyme hydrolyzes ATP and transports protons from the cytosol to the extracellular medium. It was proposed that the H+-ATPase plays an important role in controlling several cellular functions such as nutrient uptake, intracellular pH and cell growth [see reviews by Goffeau and Slayman (1981);Serrano (1988); Goffeau and Green (1990); Serrano (1989)l.The plasma membrane H+-ATPase from yeast belongs to the family of P-type ATPases (Pedersen and Carafoli, 1987). These ATPases are characterized by a covalent phosphorylated intermediate which is formed during the catalytic cycle by transfer of the y-phosphate of ATP to an aspartic acid residue (Amory et al., 1980). The P-type ATPases exist in two major functional states. One has the ability to bind ATP and is phosphorylated by the y-phosphate of the nucleotide. The other does not bind ATP and is phosphorylated by Pi in the presence of magnesium (Amory et al., 1982). ATP hydrolysis, catalyzed by P-type ATPases, is strongly inhibited by vanadate through a non-competitive mechanism (Dufour et al., 1980;Borst-Pauwels and Peters, 1981). It was suggested that vanadate acts as a transition state analogue of phosphate (Cantley et al., 1978; Willsky, 1979). Erythrosine B inhibits ATP hydrolysis catalyzed by the P-type Mg2 + ATPase from radish microsomes (Cocucci and Marre, 1984). Erythrosine B is an eosin derivative and it is assumed to bind at the ATP binding site, i.e. its mechanism is similar to that found for eosin and the Na' /K+-ATPase (Skou and Esmann, 1981).Correspondence to P. Graber, Biologisches Institut, Universitat Stuttgart, Pfaffenwaldring 57, W-7000 Stuttgart 80, Federal Repbulic of GermanyEnzyme. ATPase or adenosine triphosphatase (EC 3.6.1.3).In this work we want to identify parts of the reaction sequence of the catalytic cycle of H+-ATPase, and determine (a) in which step pro...

Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica

“…ATPase (Plesner, 1995). Activities of recombinant MP67 and MpAPY2 were insensitive to azide, an inhibitor of F-type ATPase, although activities of native MP67 and recombinant MP67 were sensitive to vanadate, an inhibitor of P-type ATPase (Borst-Pauwels and Peters, 1981;Wach and Gräber, 1991). Thus, these experiments clearly show that MP67 exhibits unique biochemical characteristics.…”

Structure and mechanism of ATP-dependent phospholipid transporters

“…Generally speaking the mechanism by which both proteins hydrolyze ATP is analogous, as can be inferred by the fact that both types of proteins are sensitive to vanadate [130,131], which mimics the transition state of ATP during hydrolysis [132].…”

Co‐reconstitution of plasma membrane H+‐ATPase from yeast and bacteriorhodopsin into liposomes

“…The rate of ATP hydrolysis increases with increasing pH,,, up to about 6.8 then decreases again. The decrease above pH,,, of 6.8 reflects presumably the same effect as observed in the dark period and for the isolated enzyme (Wach and Graber, 1991), i.e., a functional group of the enzyme accessible from the outside is not protonated and thereby the reaction cycle stops.…”

“…This dependence is similar to that observed with the isolated enzyme. In this case, the functional dependence of the rate on pH,,, for pH,, greater than 6.0 can be described by the protonation of a functional group with an apparent pK of 7.4 (Wach and Graber, 1991). The data from Fig.…”

“…Based on reaction schemes proposed earlier Slayman, 1987;Nakamoto and Slayman, 1989;Goffeau and Green, 1990;Wach and Graber, 1991) the reaction cycle can be described as follows. Protons from the cell interior are bound to species Ei to a group with pKcp of 7.3.…”