Bone Abstracts

The biomineralization process is initiated inside matrix vesicles (MVs), with phosphate and calcium ions crystallizing as hydroxyapatite. This process is accomplished by the activities of several proteins, such as annexins (e.g. AnxV) that mediates Ca²⁺ influx into MVs and tissue-nonspecific alkaline phosphatase (TNAP), a phosphomonohydrolase that uses ATP and PP_i as substrates. Dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) are found in MVs membranes and play a crucial role in the biomineralization process, regulating both Ca²⁺ entry into the MVs and formation of hydroxyapatite crystals. We studied the incorporation of AnxV and TNAP into DPPC and DPPC:DPPS (10% molar ratio) liposomes and their ability to transport Ca²⁺. Proteoliposomes harboring AnxV were reconstituted using 1:100 protein:lipid (molar ratio). When DPPS was used, we had 80% of increase in protein incorporation. Proteoliposomes containing TNAP and AnxV were reconstituted using a 1:15 000 and 1:100 protein:lipid (molar ratio), respectively. The presence of both (70% AnxV and 30% TNAP) into proteoliposomes was confirmed by western blots. The proteoliposomes (10 μg protein) were incubated with a fixed ⁴⁵Ca²⁺concentration (5.5 μCi/ml) and increasing Ca²⁺concentrations (from 1 to 5 mM), resulting in a linear increased uptake, reaching a maximum with 2 mM Ca²⁺. Around 0.8 μmol Ca²⁺ was incorporated, with a similar profile for all proteoliposomes curves. The presence of TNAP in the proteoliposomes containing both proteins did not affect significantly AnxV-mediated Ca⁺² transport. However, the presence of AnxV affected significantly the hydrolysis of PP_i, ATP, and ADP by TNAP. When both proteins are present, the V_m for PP_i hydrolysis decreased by around 19 times and K_0.5 was not affected significantly. For ATP, V_m decreased around seven times and K_0.5 also decreased (nine times). Finally, V_m for ADP decreased two times and K_0.5 was not affected. These studies will help us in the development of mineralization-competent MV biomimetics.