Searchable abstracts of presentations at key conferences on calcified tissues
Bone Abstracts (2013) 1 PP170 | DOI: 10.1530/boneabs.1.PP170

ECTS2013 Poster Presentations Cell biology: osteoblasts and bone formation (50 abstracts)

Calcium transport and phosphomonohydrolase activity by proteoliposomes harboring annexin V and alkaline phosphatase

Maytê Bolean 1, , Ana Maria Simao 1, , Tina Kiffer-Moreira 2 , Marc Hoylaerts 3 , José Luis Millán 2 & Pietro Ciancaglini 1,

1FFCLRP-USP, Ribeirao Preto, São Paulo, Brazil; 2Sanford-Burnham Medical Research Institute, La Jolla, California, USA; 3University of Leuven, Leuven, Belgium.

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 PPi 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 PPi, ATP, and ADP by TNAP. When both proteins are present, the Vm for PPi hydrolysis decreased by around 19 times and K0.5 was not affected significantly. For ATP, Vm decreased around seven times and K0.5 also decreased (nine times). Finally, Vm for ADP decreased two times and K0.5 was not affected. These studies will help us in the development of mineralization-competent MV biomimetics.

Volume 1

European Calcified Tissue Society Congress 2013

Lisbon, Portugal
18 May 2013 - 22 May 2013

European Calcified Tissue Society 

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