Alkaline phosphatase and nucleotide pyrophosphatase phosphodiesterase activity in transduced clones. Enzyme activities were measured in transduced clones at day 14 (proliferation), at day 28 (nonmineralizing hypertrophy), and at day 35 (mineralization). Empty, uncloned cells transduced with pLNCX vector; WT, wild-type ank. For alkaline phosphatase (AP) measurements, the units of enzyme were determined by first subtracting the optical density reading of a blank from diluted samples at 2 minutes. The result of this calculation was then subtracted from a similar calculation performed on samples determined at time point 0. Levels of AP are negligible at day 14 of culture and increase at day 28. Consistent with previous studies , AP activity is much higher in cells at day 35 of culture during mineralization. Although AP activity is higher for cells overexpressing WT ank compared with cells transduced with empty vector at days 28 and 35 of culture, cells expressing mutant ank constructs did not demonstrate AP activity that was significantly different from cells transduced with WT ank. Nucleotide pyrophosphatase phosphodiesterase (NPP) activity for each sample were determined by comparison with the standard curve of p-nitrophenol and expressed as units, where one unit is equivalent to 1 μmol substrate hydrolyzed per hour. With the exception of the cell line transduced with the P5L mutant, all transduced lines exhibited NPP activity that was comparable with cells transduced with empty vector only. AP and NPP activities for untransduced cells and empty vector were comparable. n = 9; *P ≤ 0.05. At least three independent clones for each cell line were evaluated; results presented are from single clones and are representative of other clones for each cell line.