The Oligomeric State of the Plasma Membrane H⁺-ATPase from Kluyveromyces lactis.

The plasma membrane H⁺-ATPase was purified from the yeast K. lactis. The oligomeric state of the H⁺-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H⁺-ATPase hexamer in both MASs as the sole/main oligomeric state-in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H⁺-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H⁺-ATPase. It is concluded that the quaternary structure of the H⁺-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.

Mapping the ATP binding site in the plasma membrane H(+)-ATPase from Kluyveromyces lactis.

The plasma membrane H(+)-ATPase from Kluyveromyces lactis contains 14 tryptophan residues. Binding a nucleotide or unfolding with Gnd-HCl quenched intrinsic fluorescence by ≈60% suggesting that in the H(+)-ATPase-Nucleotide complex there is solvent-mediated collisional quenching of W505 fluorescence. N-bromosuccinimide (NBS) treatment of H(+)-ATPase modified a single W residue in both native and Gnd-HCl-unfolded H(+)-ATPase. Denaturing the H(+)-ATPase with 1% SDS led to expose six tryptophan residues while requiring 17 NBS/H(+)-ATPase. The remaining eight tryptophan residues kept buried indicating a highly stable TM domain. Acrylamide generated static quenching of fluorescence; partial in the native enzyme (V = 0.43 M(-1)) and complete in the Gnd-HCl-unfolded H(+)-ATPase (V = 0.81 M(-1)). Collisional quenching (K sv) increased from 3.12 to 7.45 M(-1) upon H(+)-ATPase unfolding. W505 fluorescence titration with NBS yielded a molar ratio of 6 NBS/H(+)-ATPase and quenched ≈ 60% fluorescence. In the recombinant N-domain, the distance between W505 and MantATP was estimated to be 21 Å by FRET. The amino acid residues involved in nucleotide binding were identified by N-domain molecular modelling and docking with ATP. In the N-domain/ATP complex model, the distance between W505 and ATP was 20.5 Å. ATP binding leads to a conformational change in the N-domain of H(+)-ATPase that exposes W505 to the environment.

Trehalose-mediated thermal stabilization of glucose oxidase from Aspergillus niger.

Thermal inactivation and enzyme kinetics of glucose oxidase (a FAD dependent enzyme) were studied in the absence and presence of trehalose. The inactivation rate constant decreased by up to 50% at temperatures between 50 and 70 degrees C in the presence of 0.6M trehalose; as a consequence the glucose oxidase half-life increased. Intrinsic fluorescence spectra showed a maximum center of spectral mass (CSM) red shift of 6.5nm. Therefore, major structural changes seem to be related to glucose oxidase thermal inactivation. Trehalose decreased the rate constant for unfolding as monitored by CSM red shift kinetics indicating that this disaccharide favors the most compact folded state. The E(a) for unfolding was increased from 204 to 221kJ mol(-1). It is proposed that FAD dissociation is preceded by the exposition of hydrophobic regions, while the presence of trehalose was able to hinder the release of FAD. Enzyme kinetics analysis showed that trehalose does not affect V(max) but instead decreases K(m); as a result enzyme efficiency was increased. The stabilizing effect of trehalose in a cofactor-dependent enzyme has not been tested to date. In addition, glucose oxidase has an enormous commercial importance and therefore, the use of trehalose to stabilize glucose oxidase in its multiple applications seems to be promising.

Fluorescence quenching by nucleotides of the plasma membrane H+-ATPase from Kluyveromyces lactis.

The yeast plasma membrane H+-ATPase isolation procedure was improved; a highly pure enzyme (90-95%) was obtained after centrifugation on a trehalose concentration gradient. H+-ATPase kinetics was slightly cooperative: Hill number = 1.5, S0.5 = 800 microM ATP, and turnover number = 36 s-1. In contrast to those of other P-type ATPases, H+-ATPase fluorescence was highly sensitive to nucleotide binding; the fluorescence decreased 60% in the presence of both 5 mM ADP and AMP-PNP. Fluorescence titration with nucleotides allowed calculation of dissociation constants (Kd) from the binding site; Kd values for ATP and ADP were 700 and 800 microM, respectively. On the basis of amino acid sequence and homology model analysis, we propose that binding of the nucleotide to the N-domain is coupled to the movement of a loop beta structure and to the exposure of the Trp505 residue located in the loop. The recombinant N-domain also displayed a large hyperbolic fluorescence quenching when ATP binds; however, it displayed a higher affinity for ATP (Kd = 100 microM). We propose for P-type ATPases that structural movements during nucleotide binding could be followed if a Trp residue is properly located in the N-domain. Further, we propose the use of trehalose in enzyme purification protocols to increase the purity and quality of the isolated protein and to perform structural studies.