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We investigated the nature of the complex ATP activation kinetics of plant H+-ATPases. To this aim we analyzed that activation in three isolated isoforms (AHA1, AHA2, and AHA3) of H+-ATPase from Arabidopsis fhaliana. The isoforms were obtained by heterologous expression in endoplasmic reticulum of yeast. ATP stimulation was always with low affinity (/& between 500 and 1800 p~) . In addition, the curves were not Michaelian and displayed positive cooperativity. Detailed studies with AHA2 showed that (a) enzyme solubilized with lysophosphatidylcholine exhibited Michaelian behavior even in the presence of soybean lecithin liposomes free of enzyme, (b) solubilized enzyme incorporated into the same liposomes displayed two-site kinetics with negative cooperativity, and (c) enzyme partially digested with trypsin lost the C-terminal portion of the molecule. Under this condition the ATP activation kinetics was Michaelian or had a slight negative cooperativity and the K0.5ATP was reduced 3-fold. These data suggest that the functional unit of the H+-ATPase has two catalytic ATP sites with variable cooperativity and kinetics competence of the E(ATP) and E(ATP)Z complexes. Such variability is likely modulated by the association of the enzyme with membrane structures and by a regulatory domain in the C terminus of the enzyme molecule.

The complex ATP-activation kinetics of plant H+-ATPase requires two ATP effects on the enzyme. They may result from the simultaneous existence of two ATP sites or a single site that consecutively changes its properties. We describe here three main models for ATP binding to the plant H+-ATPase. Considering the experimental data there are some restrictions in their application. A system with two simultaneous catalytic sites with cooperative binding is possible provided the substrate exerts regulatory properties, and when ES (or SE) leads to a slower velocity path than SES ( u , < 0,). In other words, simple cooperativity does not work. A system with two substrate sites, one of which is catalytic and the other is always and only regulatory (i.e. it affects the overall reaction rate), offers two alternatives: one with potential cooperative binding (the system does not discriminate between binding to the catalytic and regulatory sites ; and the other with intrinsically different affinities of catalytic and regulatory sites (i.e. the system discriminates between binding to the two binding sites). Here it is also obligatory that ES (or SE) leads to a slower-velocity path than SES (v, < u,). Thirdly, a system is possible with single ATP domain that is consecutively catalytic and regulatory as the cycle proceeds. These three mechanisms give rise to equivalent rate equations. Therefore, there is no way to distinguish between them on the basis of kinetic studies. Another conclusion drawn from modeling these schemes is that the form of the plots might resemble but not correspond to certain cooperativity type. For instance, for two substrate sites, a true negative cooperativity for substrate binding can mimic positive cooperativity if the N, velocity pathway is much slower than the uz one.Keywords : plant ; proton transport ; active transport ; adenosinetriphosphatase ; membrane transport.Mammalian cation transport ATPases (Na+/K+-exchanging, Ca2' -transporting, H+/K+-exchanging ATPases) display negative cooperativity towards ATP. These kinetics have been explained as responding to two roles of the substrate: a catalytic phosphorylating role, which acts with very high affinity (Kin below 1 pM); and a regulatory non-phosphorylating role by which, with low affinity ( K , 100-300 pM), it accelerates some ratelimiting step [I -31. However, these models does not explicitly indicate whether the two sites where ATP acts result from a single site that changes its properties during the cycle, or corresponds responds to two different loci for ATP binding. In this regard the experimental evidence is ambiguous, as there is support for the two mechanisms.A puzzling feature of the higher-plant H+-ATPase is that, depending on the enzyme structure and the chemical environment, dramatic changes in the ATP-activation kinetics are obtained. These are evidenced by resemblances to a range of cooperative behaviors towards the substrate, i.e. positive/negative, or Enzymes. Na+/K ' -exchanging ATPase (EC 3.6.1.37) ; Ca2+-transporting ATPase (EC 3.6...

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Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na(+)/Ca(2+) exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far-UV circular dichroism and infrared spectra suggest a majority of beta-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten beta-sheets and two short alpha-helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.