Na,K-ATPase reconstituted in ternary liposome: the presence of cholesterol affects protein activity and thermal stability.

Differential scanning calorimetry (DSC) was applied to investigate the effect of cholesterol on the thermotropic properties of the lipid membrane (DPPC and DPPE). The thermostability and unfolding of solubilized and reconstituted Na,K-ATPase in DPPC:DPPE:cholesterol-liposomes was also studied to gain insight into the role of cholesterol in the Na,K-ATPase modulation of enzyme function and activity. The tertiary system (DPPC:DPPE:cholesterol) (molar ratio DPPC:DPPE equal 1:1) when cholesterol content was increased from 0% up to 40% results in a slight decrease in the temperature of transition and enthalpy, and an increase in width. We observed that, without heating treatment, at 37°C, the activity was higher for 20mol% cholesterol. However, thermal inactivation experiments showed that the enzyme activity loss time depends on the cholesterol membrane content. The unfolding of the enzyme incorporated to liposomes of DPPC:DPPE (1:1mol) with different cholesterol contents, ranging from 0% to 40% mol was also studied by DSC. Some differences between the thermograms indicate that the presence of lipids promotes a conformational change in protein structure and this change is enough to change the way Na,K-ATPase thermally unfolds.

Addition of subunit γ, K⁺ ions, and lipid restores the thermal stability of solubilized Na,K-ATPase.

Differential scanning calorimetry (DSC) was applied to ascertain the effect caused by K⁺, Na⁺, ATP, detergent, DPPC, DPPE, and subunit γ on the thermostability of Na,K-ATPase. The enthalpy variation (ΔH) for the thermal denaturation of the membrane-bound is twice the ΔH value obtained for solubilized Na,K-ATPase. Denaturation occurs in five steps for membrane-bound against three steps for the solubilized enzyme, therefore a multi-step unfolding process. In the presence of Na⁺, the melting temperature is 61.6°C, and the ΔH is lower as compared with the ΔH obtained in the presence or in the absence of K⁺. Addition of ATP does not alter the transition temperatures significantly, but the shape of the curve is modified. Subunit γ probably stabilizes Na,K-ATPase in the beginning of thermal unfolding, and different amounts of detergents in the solubilized sample change the protein stability. Reconstitution of Na,K-ATPase into a liposome shows that lipids exert a protector effect. These results reveal differences on the thermostability depending on the conformation of Na,K-ATPase. They are relevant because it allows a comparison with future studies, e.g. how the composition of the membrane interferes on the stability of Na, K-ATPase, elucidating the importance of the lipid type contained in cell membrane.

Unraveling the Na,K-ATPase alpha(4) subunit assembling induced by large amounts of C(12)E(8) by means of small-angle X-ray scattering.

In the current work, we studied the effect of the nonionic detergent dodecyloctaethyleneglycol, C(12)E(8), on the structure and oligomeric form of the Na,K-ATPase membrane enzyme (sodium-potassium pump) in aqueous suspension, by means of small-angle X-ray scattering (SAXS). Samples composed of 2 mg/mL of Na,K-ATPase, extracted from rabbit kidney medulla, in the presence of a small amount of C(12)E(8) (0.005 mg/mL) and in larger concentrations ranging from 2.7 to 27 mg/mL did not present catalytic activity. Under this condition, an oligomerization of the alpha subunits is expected. SAXS data were analyzed by means of a global fitting procedure supposing that the scattering is due to two independent contributions: one coming from the enzyme and the other one from C(12)E(8) micelles. In the small detergent content (0.005 mg/mL), the SAXS results evidenced that Na,K-ATPase is associated into aggregates larger than (alphabeta)(2) form. When 2.7 mg/mL of C(12)E(8) is added, the data analysis revealed the presence of alpha(4) aggregates in the solution and some free micelles. Increasing the detergent amount up to 27 mg/mL does not disturb the alpha(4) aggregate: just more micelles of the same size and shape are proportionally formed in solution. We believe that our results shed light on a better understanding of how nonionic detergents induce subunit dissociation and reassembling to minimize the exposure of hydrophobic residues to the aqueous solvent.

Cytoplasmatic domain of Na,K-ATPase alpha-subunit is responsible for the aggregation of the enzyme in proteoliposomes.

We studied the thermal dependence of amide I' infrared absorption and fluorescence emission of Trp residues in the Na,K-ATPase of rabbit kidney. We studied the whole enzyme solubilized with detergent, the whole enzyme reconstituted in proteoliposomes and the protein fraction that remained in the lipid membrane after the trypsin digestion of the proteoliposomes. Cooperative unfolding and aggregation with increasing temperature were observed in the whole protein, whether solubilized or reconstituted, but not in the fraction remaining after trypsinization. The protein influenced the physical state of the lipid, decreasing the temperature of the gel to liquid-crystalline phase transition and the degree of cooperativity. This study provides new information for the understanding of the processes controlling the association mechanisms that are important for enzyme function in natural membranes.

The association of Na,K-ATPase subunits studied by circular dichroism, surface tension and dilatational elasticity.

Different stoichiometries are observed between alpha and beta subunits of Na,K-ATPase that depend on the method employed to solubilize and purify the enzyme. It is not known whether this variability is due to loss of protein-protein association, or is a result of the replacement of essential phospholipids by detergent molecules. With the aim of understanding the effect of enzyme/surfactant ratio on both the catalytic activity and the enzyme structure, we have investigated the bulk and surface properties of the enzyme. The circular dichroism (CD) spectra, surface tension and dilatational surface elasticity results were compared with the residual ATPase activity of the Na,K-ATPase in different surfactant and protein concentrations. Na,K-ATPase in the (alphabeta)(2) form dissociated to the alphabeta form on dilution, and associated to the (alphabeta)(4) form when concentrated. These different stoichiometries have similar ATPase activities and are in equilibrium at C(12)E(8) concentrations below the CMC (0.053 mg mL(-1)). At detergent concentrations above the CMC the ATPase activity of all forms was abolished, which is concomitant with the dissociation of the alpha and beta subunits.

Biostimulation of Na,K-ATPase by low-energy laser irradiation (685 nm, 35 mW): comparative effects in membrane, solubilized and DPPC:DPPE-liposome reconstituted enzyme.

OBJECTIVE: The aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of the different forms of the Na,K-ATPase. METHODS: Membrane-bound and solubilized (alphabeta)(2) form of Na,K-ATPase was obtained from the dark red outer medulla of the kidney and proteoliposomes of DPPC:DPPE and Na,K-ATPase was prepared by the co-solubilization method. Irradiations were carried out at 685 nm using an InGaAIP diode laser. RESULTS: The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/cm(2). However, with irradiation doses ranging from 32 to 40 J/cm(2), a 28% increase on the ATPase activity was observed while when using up to 50 J/cm(2) no additional enhancement was observed. When biostimulation was done using the solubilized and purified enzyme or the DPPC:DPPE-liposome reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/cm(2). With irradiation above these values (24 J/cm(2)) no additional increase in the activity was observed. These studies revealed that the biostimulation of ATPase activity from different forms of the Na,K-ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes.

Lipid bilayer stabilization of the Na,K-ATPase reconstituted in DPPC/DPPE liposomes.

Different subunit aggregates of the Na,K-ATPase may be formed depending on the method used to solubilize and purify the enzyme. We have studied the thermal unfolding of detergent-solubilized and dipalmitoylphosphatidylcholine/ dipalmitoylphosphatidylethanolamine liposome-reconstituted forms of the Na,K-ATPase by circular dichroism (CD) spectroscopy and p-nitrophenylphosphatase activity. The ellipticity at 222 nm of the solubilized and reconstituted forms showed a sigmoid decrease in the absolute value of the signal of 36 and 31% with T(50%) of 44 and 42 degrees C, respectively. The catalytic activity was reduced in two steps with T(50%) of 32 and 52 degrees C in the detergent-solubilized enzyme and T(50%) of 25 and 53 degrees C in the reconstituted enzyme. The reduction in catalytic activity of the detergent-solubilized enzyme was bi-exponential with t(1/2) of 8.3 and 67.9 min, resulting in the total loss of activity after 120 min. However, under the same conditions, the ATPase activity of the reconstituted enzyme was reduced by approx 35% with a t(1/2) of 145 min. The results suggest that the alpha- and beta-subunits present different thermal stability that may be modulated by the nature of the co-solvent (detergent or lipid) used in the preparations of the Na,K-ATPase. In addition, distinct processes of beta-subunit displacement and alpha-alpha-subunit aggregate formation may also contribute to the changes in both the CD spectra and the enzyme activity. Furthermore, we have demonstrated the protective role of the phospholipid bilayer in the reconstituted enzyme compared with the detergent-solubilized enzyme.