Assaying P-Type ATPases Reconstituted in Liposomes.

Reconstitution of P-type ATPases in unilamellar liposomes is a useful technique to study functional properties of these active ion transporters. Experiments with such liposomes provide an easy access to substrate-binding affinities of the ion pumps as well as to the lipid and temperature dependence of the pump current. Here, we describe two reconstitution methods by dialysis and the use of potential-sensitive fluorescence dyes to study transport properties of two P-type ATPases, the Na,K-ATPase from rabbit kidney and the K(+)-transporting KdpFABC complex from E. coli. Several techniques are introduced how the measured fluorescence signals may be analyzed to gain information on properties of the ion pumps.

KdpFABC reconstituted in Escherichia coli lipid vesicles: substrate dependence of the transport rate.

KdpFABC complexes were reconstituted in Escherichia coli lipid vesicles, and ion pumping was activated by addition of ATP to the external medium which corresponds to the cytoplasm under physiological conditions. ATP-driven potassium extrusion was studied in the presence of various substrates potentially influencing transport rate. The pump current was detected as a decrease of the membrane potential by the voltage-sensitive dye DiSC3(5). The results indicate that high cytoplasmic K(+) concentrations have an inhibitory effect on the KdpFABC complex. The pump current decreased to ∼25% of the maximal value at 140 mM K(+) and minimal Mg(2+)concentrations. This effect could be counteracted with increased Mg(2+) concentrations on the cytoplasmic side. This observation may be explained by the Gouy-Chapman effect of two Mg(2+) ions probably bound with a K1/2 of 0.8 mM close to the entrance of the access channel to the binding sites. This factor ensures that under physiological conditions the rate-limiting effect of K(+) release is significantly reduced. Also both ADP and inorganic phosphate are able to reduce the turnover rate of the pump by reversing the phosphorylation step (Ki of 151 µM) and the dephosphorylation step (Ki of 268 µM), respectively. In the case of the DDM-solubilized KdpFABC complex, activation energy under turnover conditions was previously found to be 55 kJ/mol, and the o-vanadate inhibition constant is shown here to be ∼1 µM, which is in agreement with values reported for other P-type ATPases. In the case of the reconstituted enzyme, however, significant differences were observed that have to be assigned to effects of the lipid bilayer environment. The activation energy was increased by a factor of 2, whereas the inhibition by o-vanadate became reduced in a way that only ∼66% of the enzyme could be inhibited and the inhibition constant was increased to a value of ∼60 µM.

Role of protons in the pump cycle of KdpFABC investigated by time-resolved kinetic experiments.

The time-resolved kinetics of the KdpFABC complex solubilized in Aminoxide WS-35 was investigated by ATP concentration jump experiments. ATP was photoreleased from its inactive precursor, caged ATP, and charge movements in the membrane domain of the KdpFABC were detected by the electrochromic dye RH421. At low ATP concentrations, the ATP binding step became rate-limiting with an apparent, pH-independent ATP binding affinity of ~70 µM. At saturating ATP concentrations, the rate-limiting step is the conformational transition (E1-P → P-E2) with a rate constant of ~1.7 s(-1) at 20 °C that was independent of K(+) concentration. This observation together with the detected fluorescence decrease indicates that K(+) (or another positive ion) is bound in the membrane domain after enzyme phosphorylation and the conformational transition to the P-E2 state. pH dependence experiments revealed different roles of H(+) in the transport mechanism. Two different functions of protons for the ion pump must be distinguished. On one hand, there are electrogenically bound "functional" protons, which are not transported but prerequisite for the performance of the ATP-driven half-cycle. On the other hand, protons bind to the transport sites, acting as weak congeners of K(+). There possibly are noncompetitively bound protons, affecting the enzyme activity and/or coupling between KdpA and KdpB subunits. Finally, the recently proposed Post-Albers model for the KdpFABC complex was supplemented with stoichiometry factors of 2 for K(+) and 3 for H(+), and additional inhibitory side reactions controlled by H(+) were introduced, which are relevant at pH <6.5 and/or in the absence of K(+).

Mechanistic analysis of the pump cycle of the KdpFABC P-type ATPase.

The high-affinity potassium uptake system KdpFABC is a unique type Ia P-type ATPase, because it separates the sites of ATP hydrolysis and ion transport on two different subunits. KdpFABC was expressed in Escherichia coli. It was then isolated and purified to homogeneity to obtain a detergent-solubilized enzyme complex that allowed the analysis of ion binding properties. The electrogenicity and binding affinities of the ion pump for K(+) and H(+) were determined in detergent-solubilized complexes by means of the electrochromic styryl dye RH421. Half-saturating K(+) concentrations and pK values for H(+) binding could be obtained in both the unphosphorylated and phosphorylated conformations of KdpFABC. The interaction of both ions with KdpFABC was studied in detail, and the presence of independent binding sites was ascertained. It is proposed that KdpFABC reconstituted in vesicles translocates protons at a low efficiency opposite from the well-established import of K(+) into the bacteria. On the basis of our results, various mechanistic pump cycle models were derived from the general Post-Albers scheme of P-type ATPases and discussed in the framework of the experimental evidence to propose a possible molecular pump cycle for KdpFABC.

Inhibitory effect of platinum and ruthenium bipyridyl complexes on porcine pancreatic phospholipase A2.

Pancreatic phospholipase A(2) (PLA(2)) plays an important role in cellular homeostasis as well as in the process of carcinogenesis. Effects of metallo-drugs used as chemotherapeutics on the activity of this enzyme are unknown. In this work, the interaction between porcine pancreatic PLA(2) and two selected transition metal complexes--tetrachloro(bipyridine) platinum(IV) ([PtCl(4)(bipy)]) and dichloro (bipyridine) ruthenium(III)chloride ([RuCl(2)(bipy)(2)]Cl)--was studied. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence spectroscopy have been used to analyse the enzyme activity in the absence and presence of metal complexes and to verify potential binding of these drugs to the enzyme. The tested metal complexes decreased the activity of phospholipase A(2) in an uncompetitive inhibition mode. A binding of the ruthenium complex near the active site of the enzyme could be evidenced and possible modes of interaction are discussed.

Laser desorption and ionization time-of-flightversus matrix-assisted laser desorption and ionization time-of-flight mass spectrometry of and metal complexes.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently established as a powerful, "soft" ionization technique for the analysis of both transition metal complexes, which are used as metallo-drugs in the therapy of various types of tumors, and biomolecules. Since some metal complexes absorb light in the UV range, it should be possible to analyse them without additional matrices, i.e. using LDI-TOF MS. In this study, the matrix-free approach was tested for the analysis of [PtCl2(dach)] (dichloride(1,2-diamincyclohexane) platinum(ii)), [RuCl2(en)2]Cl (dichloridobis(ethylenediamine) ruthenium(iii) chloride) and [RuCl2(bipy)2]Cl (bis(bipyridine)dichloridoruthenium(iii) chloride) and the detection limit for these compounds was determined. In summary, the LDI-TOF mass spectra of [PtCl2(dach)] and [RuCl2(en)2]Cl are rather simple, whereas in the presence of 2,5-DHB as a matrix, additional peaks are generated. On the other hand, the standard MALDI-TOF mass spectrum of [RuCl2(bipy)2]Cl exhibits only one peak arising from the complex, in contrast to six peaks detectable in the LDI-TOF mass spectrum. The detection limit in the MALDI-TOF MS analysis of [PtCl2(dach)] and [RuCl2(bipy)2]Cl complexes was lower than that determined in LDI-TOF MS. Taking all into account, in this paper, we have demonstrated some advantages and drawbacks of the matrix-free LDI-TOF mass spectrometric analysis of transition metal complexes.