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. 2013 Sep;93(3):222-32.
doi: 10.1007/s00223-013-9745-3. Epub 2013 May 31.

Effects of pH on the production of phosphate and pyrophosphate by matrix vesicles' biomimetics

Ana Maria S Simão  1 , Maytê Bolean, Marc F Hoylaerts, José Luis Millán, Pietro Ciancaglini
Affiliations

Effects of pH on the production of phosphate and pyrophosphate by matrix vesicles' biomimetics

Ana Maria S Simão et al. Calcif Tissue Int. 2013 Sep.

Abstract

During endochondral bone formation, chondrocytes and osteoblasts synthesize and mineralize the extracellular matrix through a process that initiates within matrix vesicles (MVs) and ends with bone mineral propagation onto the collagenous scaffold. pH gradients have been identified in the growth plate of long bones, but how pH changes affect the initiation of skeletal mineralization is not known. Tissue-nonspecific alkaline phosphatase (TNAP) degrades extracellular inorganic pyrophosphate (PPi), a mineralization inhibitor produced by ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1), while contributing Pi from ATP to initiate mineralization. TNAP and NPP1, alone or combined, were reconstituted in dipalmitoylphosphatidylcholine liposomes to mimic the microenvironment of MVs. The hydrolysis of ATP, ADP, AMP, and PPi was studied at pH 8 and 9 and compared to the data determined at pH 7.4. While catalytic efficiencies in general were higher at alkaline pH, PPi hydrolysis was maximal at pH 8 and indicated a preferential utilization of PPi over ATP at pH 8 versus 9. In addition, all proteoliposomes induced mineral formation when incubated in a synthetic cartilage lymph containing 1 mM ATP as substrate and amorphous calcium phosphate or calcium-phosphate-phosphatidylserine complexes as nucleators. Propagation of mineralization was significantly more efficient at pH 7.5 and 8 than at pH 9. Since a slight pH elevation from 7.4 to 8 promotes considerably more hydrolysis of ATP, ADP, and AMP primarily by TNAP, this small pH change facilitates mineralization, especially via upregulated PPi hydrolysis by both NPP1 and TNAP, further elevating the Pi/PPi ratio, thus enhancing bone mineralization.

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Figures

Fig 1
Fig 1
Effect of increasing concentrations of (a) ATP, (b) ADP, (c) AMP and (d) PPi on the Pi-generating activity of DPPC proteoliposomes containing: (●) TNAP, (■) NPP1 or (○) TNAP plus NPP1. Assays were done at 37 °C and buffered with 50 mM AMPOL, pH 8, containing 2 mM MgCl2 and substrate and released Pi measured
Fig 2
Fig 2
Effect of increasing concentrations of (a) ATP, (b) ADP, (c) AMP and (d) PPi on the Pi-generating activity of DPPC proteoliposomes containing: (●) TNAP, (■) NPP1 or (○) TNAP plus NPP1. Assays were done at 37 °C and buffered with 50 mM AMPOL, pH 9, containing 2 mM MgCl2 and substrate and released Pi measured
Fig 3
Fig 3
Progression of the formation of (a) ADP, (b) AMP and (c) adenosine during hydrolysis of 100 nmol of ATP by (●) TNAP-proteoliposomes, (■) NPP1-proteoliposomes and (■) TNAP plus NPP1-proteoliposomes. Hydrolysis was determined in 50 mM AMPOL buffer, pH 9, containing 2 mM MgCl2 and 1 mM ATP. Nucleotide concentrations were monitored by HPLC
Fig 4
Fig 4
Progression of the formation of (a) AMP and (b) adenosine during hydrolysis of 100 nmol of ADP by (●) TNAP-proteoliposomes, (■) NPP1-proteoliposomes and (○) TNAP plus NPP1-proteoliposomes. Hydrolysis was determined in 50 mM AMPOL buffer, pH 9, containing 2 mM MgCl2 and 1 mM ADP. Nucleotide concentrations were monitored by HPLC
Fig 5
Fig 5
Representation of all the possible simultaneous enzymatic reactions during catalysis of substrates by TNAP and NPP1 at alkaline pH. Indicated in black and gray are the enzymes with higher and lower catalytic efficiencies, respectively, for the indicated substrates
Fig 6
Fig 6
Effect of TNAP-, NPP1-, and TNAP plus NPP1-proteoliposomes on mineralization of ACP and PS-CPLX-seeded SCL, at pH 7.5 (A), 8 (B) and 9 (C), in the presence of 1 mM ATP, after 48 h of incubation at 37°C. White bars: PS-CPLX-seeded SCL. Black bars: ACP-seeded SCL. The ACP and PS-CPLX nucleators were prepared and mineralization was monitored in the microplate, as described in Materials and Methods. Empty liposomes without incorporated enzymes were used as control, and the bars show the increment in absorbencies over 48h.
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