Journal
BRAZILIAN JOURNAL OF MEDICAL AND BIOLOGICAL RESEARCH
Volume 39, Issue 5, Pages 603-610Publisher
ASSOC BRAS DIVULG CIENTIFICA
DOI: 10.1590/S0100-879X2006000500006
Keywords
matrix vesicles; endochondral ossification; osseous plate; alkaline phosphatase; ectopic mineralization; calcification
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Funding
- NIAMS NIH HHS [AR 47908] Funding Source: Medline
- NIDCR NIH HHS [DE 12889] Funding Source: Medline
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Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 103 run. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PP), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNTP, ATP and PPi as substrates. While the enzymatic efficiency (k(cat)/K-m) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k(cat)/K-m for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.
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