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Thermal stability of Na,K-ATPase reconstituted in DPPC:DPPE liposome: an infrared spectroscopy (FTIR) study
aRigos, C.F.; aPúblio, R.; aSantos H.L.; bMontich G.G.; bMaggio B. and aCiancaglini, P.
aDepto. de Química- Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, -FFCLRP-USP, Brasil.
bDepto. de Química Biológica-CIQUIBIC, Facultad de Ciencias Químicas, UNC, Córdoba, Argentina.
We have studied the thermal unfolding of DPPC:DPPE-liposome reconstituted forms of the Na,K-ATPase intact or trypsinized by infrared spectroscopy (FTIR). The (ab)2 form of Na,K-ATPase rabbit kidney outer medulla was purified by gel filtration at 4°C on a Sepharose 6-B column and DPPC:DPPE-proteoliposome was prepared in a weight ratio of 1:1 lipid:lipid and 1:3 lipid:protein (Santos et al., Coll. Surf. B:Biointerfaces, 41(4):237, 2005). The enzyme reconstituted in DPPC:DPPE-liposome (0.6 mg/mL) was treated with 0.2 mg/mL of typsin during 30 minutes at 37oC. Proteoliposome and trypsinized proteoliposome were ultracentrifuged for 1 hr, at 100,000 x g and 4oC and the pellets were washed with D2O containing 0.1 M KCl. FTIR spectra were recorded on a IR spectrometer using CaF2 cells with 50 µm Teflon spacer in a metal cell housing thermostatted to within 0.1oC. Interferograms were accumulated over the spectral range 1750–1550 cm-1 with a normal resolution of 2 cm-1.
The intensity graphics of the amide I bands correspondent to the a-helix (1650 cm-1) and b-sheet (1625 cm-1) structures against temperature show slight variations of curve inclination of the spectra around 50°C, but do not follow the expected form for cooperative conformational variations. It is probable that this discrepancy is due to the aggregation process, made evident by the appearance of band 1618 cm-1 and favored by the high concentration of the sample used in the experiment. The analysis of the transmembrane domain presented characteristic spectra for a-helix at low temperatures, and an increase of b-sheet structure with the increase of temperature was observed. On the other hand, the analysis of the signal correspondent to the vibration of the CH2- groups of the hydrocarbon chains showed that the presence of the protein produces a sensible decrease in the transition temperature of the lipid.
Recently, Rigos et al. (Cell Biochemistry and Biophysics, 44(1): 438-445, 2006) demonstrated the protective role of the phospholipid bilayer in the preservation of the catalytic activity as compared to the detergent solubilized enzyme. These studies of FTIR, together with the results from other techniques such as fluorescence and calorimetry, will contribute to an improved understanding of the mechanism of subunit association of the Na,K-ATPases and the mechanisms of regulation in the cell membrane.
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