Thermodynamic properties and characterization of proteoliposomes rich in microdomains carrying alkaline phosphatase

Tissue-nonspecific alkaline phosphatase (TNAP) is associated to the plasma membrane via a GPI-anchor and plays a key role in the biomineralization process. In plasma membranes, most GPI-anchored proteins are associated with “lipid rafts”, ordered microdomains enriched in sphingolipids, glycosphingolipids and cholesterol. In order to better understand the role of lipids present in rafts and their interactions with GPI-anchored proteins, the insertion of TNAP into different lipid raft models was studied using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), sphingomyelin (SM) and ganglioside (GM1). Thus, the membrane models studied were binary systems (9:1 molar ratio) containing DPPC:Chol, DPPC:SM and DPPC:GM1, ternary systems (8:1:1 molar ratio) containing DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 and finally, a quaternary system (7:1:1:1 molar ratio) containing DPPC:Chol:SM:GM1. Calorimetry analysis of the liposomes and proteoliposomes indicate that lateral phase segregation could be noted only in the presence of cholesterol, with the formation of cholesterol-rich microdomains centered above Tc = 41.5 °C. The presence of GM1 and SM into DPPC-liposomes influenced mainly ΔH and Δt1/2 values. The gradual increase in the complexity of the systems decreased the activity of the enzyme incorporated. The presence of the enzyme also fluidifies the systems, as seen by the intense reduction in ∆H values, but do not alter Tc values significantly. Therefore, the study of different microdomains and its biophysical characterization may contribute to the knowledge of the interactions between the lipids present in MVs and its interactions with TNAP.

DSC thermograms of quaternary systems (10 mg/mL). Differential scanning calorimetry thermograms were processed in excess heat capacity, Cp (kcal·K−1·mol−1) as a function of temperature (°C) of vesicles constituted by DPPC:Chol:SM:GM1 (7:1:1:1), molar ratio: (A) Liposome and (B) Proteoliposome. Dashed curves symbolize deconvolution analysis.Research highlights► The formed liquid-ordered complexes in the liposomes present a great lipid packing. ► A pronounced decrease in ∆H when liposome system is compared with proteoliposome. ► Enzyme incorporation influences the reduction in cooperativity, but no changes in TC. ► TNAP presence resulted in an even greater segregation of the phase transition peaks. ► These systems are excellent to study changes in enzyme activity in model membranes.