FIGURE 1.

BMP-5 pd interacts with N-terminal polypeptides of fibrillin-1 and -2. A, schematic representation of the recombinant fibrillin polypeptides used in this study. B, Coomassie Blue-stained gel of the bacterially expressed, purified, and alkylated BMP-5 pd, after SDS-PAGE in a nonreducing 12.5% gel, demonstrates purity of the sample. In addition, a Coomassie Blue-stained gel of the newly described fibrillin-2 polypeptide rF70 is shown. C, selected sensorgrams from SPR experiments with immobilized BMP-5 pd and soluble ligands rF23 or rF86 show interactions. The fibrillin polypeptides where diluted from 80 to 0 nm in HBS-EP buffer. D, SPR sensorgram with immobilized BMP-5 pd and titrated concentrations of rF92. E, BMP-5 and BMP-7 pds compete for the same binding site in fibrillin-1. Left, sensorgram shows concentration-dependent decrease of the BMP-5 pd-rF23 interaction signal, when BMP-7 pd is preincubated with rF23. BMP-5 pd was immobilized, and the fibrillin-1 N-terminal polypeptide rF23 was injected at a constant concentration of 20 nm in the presence of increasing concentrations (0–100 nm) of BMP-7 pd. Right, inhibition curve shows competition of the BMP-7 pd for the BMP-5 pd-binding site in fibrillin-1. The signal in RU obtained for rF23 at 20 nm without competitor was set as 100%. The decrease of the 100% rF23 signal was graphed against the inhibitor concentration to determine the inhibition constant (I₅₀) for the competition reaction. To account for variations of the rF23 signal because of the buffer change caused by the addition of different amounts of competitor, a buffer matched control sensorgram without competitor was generated for each BMP-7 concentration. This control signal served as the 100% reference signal for each competitor concentration.