Fig. 8: Kinetics and interaction analyses.

a Kinetic analysis of inhibition of PAPP-A cleavage of IGFBP-4 by STC2(C120A). STC2(C120A) cannot bind covalently to PAPP-A. Intact radiolabeled IGFBP-4 was used, and relative initial velocities (v/v₀) of the cleavage reaction were determined by quantification of IGFBP-4 and cleavage products following separation by SDS-PAGE for each concentration of inhibitor (the inset shows an example). The concentrations of PAPP-A, IGFBP-4, and IGF2 were 50 pM, 10 nM, and 100 nM, respectively. The inhibition constant (K_i) was determined to be 47.3 ± 1.1 × 10⁻⁹ M by fitting the Morrison K_i equation (competitive inhibition) to the data. The experiment was repeated three times with similar results. b Assessment of the ability of PAPP-A variants as indicated above the lanes to form a covalent complex with wild-type STC2. Following incubation (16 h) of separately synthesized proteins, the reaction mixtures were separated by non-reducing SDS-PAGE and visualized by Western blotting for PAPP-A. Only wild-type PAPP-A and PAPP-A(dLNR1-2) show increased molecular weight following incubation with STC2 and thus the ability to form a covalent complex. The experiment was repeated three times with similar results. c Binding of PAPP-A monoclonal antibodies to PAPP-A (left) or PAPP-A·STC2 (right) assessed by surface plasmon resonance (SPR). The control antibody (mAb PA1A) binds both PAPP-A and PAPP-A·STC2, while the antibody mAb PA141, which inhibits PAPP-A activity toward IGFBP-4, shows diminished interaction with PAPP-A·STC2. d Complex formation between separately synthesized PAPP-A and STC2 over time assessed by an immunoassay specific for the PAPP-A·STC2 complex. PAPP-A mAb PA141 prevents complex formation. Average values with error bars (SD) are plotted. e Binding to a 415-residue monomeric C-terminal PAPP-A fragment, PA(T1213-G1627), assessed by SPR. PAPP-A mAb PA141 prevents binding of STC2(C120A). Average values with error bars (SD) are plotted. n = 4 independent experiments. f Similar to e, but carried out in the absence or presence of EDTA. The loss of bound Ca²⁺ weakens the interactions between STC2 and the C domain. g Similar to e, but with IGFBP-4 as the analyte in the absence or presence of EDTA to disrupt binding of Ca²⁺ to LNR3. The loss of bound Ca²⁺ weakens the interactions between IGFBP-4 and the C domain. The sensorgrams of c, e, f, g are representative of at three independent experiments. Source data of a–g are provided as a Source data file.