Matrix extracellular phosphoglycoprotein (MEPE) is an inhibitor of mineralization in situ and in cell cultures where altered expression is associated with oncogenic osteomalacia and hypophosphatemic rickets. The purpose of this study was to determine whether the intact protein or the peptide(s) originating from this protein was responsible for the inhibition. The ability of the intact protein and the acidic, serine-and aspartate-rich MEPE-associated motif (ASARM) peptide to promote or inhibit de novo hydroxyapatite formation and growth of hydroxyapatite seed crystals, in both phosphorylated and dephosphorylated forms, was assessed at room temperature in a dynamic gel diffusion system at 3.5 and 5 days. The most effective nucleator concentration was also examined when associated with fibrillar type I collagen. The phosphorylated intact protein was an effective promoter of mineralization in the gelatin gel diffusion system, while the ASARM peptide was an effective inhibitor. When dephosphorylated both the intact protein and the ASARM peptide had no effect on mineralization. Associated with collagen fibrils, some of the effect of the intact protein was lost. This study demonstrates the importance of posttranslational modification for the site-specific activity of MEPE and its ASARM peptide. Based on studies in mice in which MEPE was ablated [5] leading to enhancement of bone formation, MEPE is believed to inhibit mineralization. This was confirmed in cell culture experiments [6,7] as well as in cell-free solution studies [8]. In the cell-free studies, it was the ASARM peptide, a substrate and ligand for PHEX, that provided the inhibition [6,8,9,10]. Similar to the other SIBLING proteins, we hypothesized that posttranslational modification of MEPE would alter its effects on mineralization and that the intact protein would have a distinct effect compared to the peptides. Thus, we tested the intact protein and the ASARM peptide in the dynamic gelatin gel system [11] to validate this hypothesis. We also investigated whether phosphorylation and dephosphorylation would alter the effects of these proteins and peptides on the mineralization process. Materials and Methods Preparation of MEPE and MEPE FragmentsMEPE protein was generated by expression in insect cells using the sf9 system, as previously described [6]. ASARM peptides (phosphorylated and without phosphate) were synthesized and purchased from Neo-MPS (now called PolyPeptide Laboratories, San Diego, CA). Dephosphorylation of the intact protein in Tris buffer was performed with alkaline phosphatase-agarose beads (Sigma, St. Louis, MO) by incubating overnight at room temperature. The Gel SystemHydroxyapatite formation and growth were monitored in the dynamic collagen gel hydroxyapatite growth system [10]. In this double diffusion system, Ca 2+ and HPO 4 2− (Pi) ions circulate at room temperature and diffuse into opposite ends of a 6-cm-long 10% gelatin gel (Bloom 275; Fisher Chemicals, Fair Lawn, NJ). The pH of the 10% gelatin solution, prepared in tris-(hyd...