Article ID Journal Published Year Pages File Type
1924872 Archives of Biochemistry and Biophysics 2015 11 Pages PDF
Abstract

•Ca2+ transport mediated by AnxA5 was observed in DPPC and DPPC:DPPS 10%-liposomes.•AnxA5 and DPPS can affect TNAP specificity during hydrolysis of substrates.•The best catalytic efficiency was found for DPPC:DPPS 10%-proteoliposomes.•Studies confirmed the functional reconstitution of AnxA5 in the mimetic systems.•The presence of TNAP affect Ca2+ transport just at lower Ca2+ concentrations.

We describe the production of stable DPPC and DPPC:DPPS-proteoliposomes harboring annexin V (AnxA5) and tissue-nonspecific alkaline phosphatase (TNAP) and their use to investigate whether the presence of AnxA5 impacts the kinetic parameters for hydrolysis of TNAP substrates at physiological pH. The best catalytic efficiency was achieved in DPPS 10%-proteoliposomes (molar ratio), conditions that also increased the specificity of TNAP hydrolysis of PPi. Melting behavior of liposomes and proteoliposomes was analyzed via differential scanning calorimetry. The presence of 10% DPPS in DPPC-liposomes causes a broadening of the transition peaks, with AnxA5 and TNAP promoting a decrease in ΔH values. AnxA5 was able to mediate Ca2+-influx into the DPPC and DPPC:DPPS 10%-vesicles at physiological Ca2+ concentrations (∼2 mM). This process was not affected by the presence of TNAP in the proteoliposomes. However, AnxA5 significantly affects the hydrolysis of TNAP substrates. Studies with GUVs confirmed the functional reconstitution of AnxA5 in the mimetic systems.These proteoliposomes are useful as mimetics of mineralizing cell-derived matrix vesicles, known to be responsible for the initiation of endochondral ossification, as they successfully transport Ca2+ and possess the ability to hydrolyze phosphosubstrates in the lipid–water interface.

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