CHARACTERIZATION OF THE BINDING OF TRANSFORMING GROWTH FACTOR-BETA-1, FACTOR-BETA-2, AND FACTOR-BETA-3 TO RECOMBINANT BETA-1-LATENCY-ASSOCIATED PEPTIDE

Preprotransforming growth factor-beta-1 (TGF-beta-1) is a 390-amino acid precursor polypeptide that undergoes a number of processing steps to yield mature TGF-beta-1 (amino acid residues 279-390) and a pro portion (residues 30-278) termed beta-1-latency-associated peptide (beta-1LAP). The dimeric form of beta-1LAP has been shown to associate noncovalently with the mature growth factor, resulting in inactivation of biological activity. To further characterize this interaction, the mature TGF-beta-1 was radioiodinated and used to determine dissociation constants. A crosslinking method using the bifunctional covalent crosslinker bis-(sulfosuccinimidyl)suberate was found to be the best approach for measuring the amount of bound growth factor. The efficiency of cross-linking was constant within each experiment and varied between 45-55%. Saturation plots and their associated Scatchard analyses indicate apparent K(d) values between 1. 1-1.8 nm. Competition of TGF-beta-1 binding to beta-1LAP by TGF-beta-2 and TGF-beta-3 (two closely related growth factors) revealed that the latter also bind beta-1LAP tightly, with apparent K(d) values of 1.9 and 0.4 nm, respectively.