Fig. 2. Biological activity and inhibitory properties of BMP-2 variants. (A) Alkaline phosphatase (ALP) activity was measured after incubation with 250 nM BMP-2 or BMP-2 variant. The response elicited by each variant was calculated as a percentage of the BMP-2 response. Values represent the mean value (±SD) of four measurements. Variants with red symbols are altered in BMPR-II interaction, as shown in Figure 3. Dark or light blue symbols indicate variants with altered association or dissociation rate constants, respectively, for binding to the BMPR-IA receptor chain. (B) Dose-dependent induction of ALP activity in serum-starved C2C12 cells is shown for BMP-2 (circles, black) and for BMP-2 variants D30K (squares, light blue), P50A (triangles, dark blue) and A34D (diamonds, red). (The background absorption at 405 nm of 0.08 ± 0.02 was not subtracted to indicate the signal-to-noise ratio.) (C) The inhibition of induction of ALP activity was determined in serum-starved C2C12 cells after incubation with 250 nM BMP-2 variant in the presence of 10 (circles) or 20 nM (triangles) BMP-2. The response obtained in the presence of BMP-2 alone is indicated by the dotted line and was taken as 100%. Data points represent the mean ± SD of four measurements. Variants with red symbols are altered in BMPR-II interaction. Light or dark blue symbols indicate variants with altered association or dissociation rate constants, respectively for the binding to the BMPR-IA receptor chain. (D) Inhibition of BMP-2 activity (10 nM BMP-2) by increasing doses of putative antagonistic/partial agonistic BMP-2 variants in serum-starved C2C12 cells. Dose–response curves of the variants A34D (circles), H39D (squares), S88A (upright triangles), L90A (inverted triangles) and L100A (diamonds) in the presence of 10 nM BMP-2 were obtained after incubation of the cells for 3 days and analysis of the induced ALP activity.