Aggregation behavioor and inactivation activity of the solubilized and of the reconstituted Na,K-ATPase in liposomes.

CAROLINA FORTES RIGOS; ROBERTO PUBLIO; HERICA DE LIMA SANTOS; BRUNO MAGGIO; GUILLERMO G. MONTICH; PIETRO CIANCAGLINI

Rosario

Congreso; XXXV Reunión anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

The Na,K-ATPase consists of protomers containing one a (~100 kDa) and one b (~50 kDa) subunit. The ab protomers may be organized into dimers (solubilized form) or higher oligomers. We have studied the thermal unfolding of detergent solubilized and DPPC:DPPE-liposome reconstituted forms of the Na,K-ATPase by circular dichroism spectroscopy, pNPPase activity and FTIR. The obtention of the thermodynamics parameters – activation energy (Ea), enthalpy (∆H), entropy (∆S) and Gibbs energy (∆G) – provides a better comprehension of the stability of the membrane protein and structural changes that occur in high temperatures. 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 as described by Santos et al. [1]. The pNPPase activity of the enzyme was assayed discontinuously at 37oC by monitoring the liberation of p-nitrophenolate ion. The DPPC:DPPE-proteoliposome was prepared in a weight ratio of 1:1 lipid:lipid and 1:3 lipid:protein [2]. The enzyme reconstituted in DPPC:DPPE-liposome was treated with trypsin 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. Using studies of pNPP hydrolysis kinetics it was found that Na,K-ATPase presented higher ÄH values (108.85 kcal/mol) and smaller ∆G values (28.94 kcal/mol) for the solubilized enzyme in comparison to the protein incorporated into liposomes (ÄH=75.68kcal/mol and ∆G=108.80kcal/mol). That occurs because the elements of the secondary structure which are located in the transmembrane region when the protein is incorporated into liposome, are highly stable and resist to thermal denaturation [3]. The extramembrane protein regions need lower energy to be broken and occur with both enzyme forms. Furthermore, the results obtained with the FTIR studies (when comparing liposome incorporated enzyme with that trypnized) show that what occurs is a possible aggregation process of the enzyme due to subunit a interactions.