CHARACTERIZATION OF UNIQUE BINDING-KINETICS OF FOLLISTATIN AND ACTIVIN OR INHIBIN IN SERUM

Serum binding proteins (BPs) have been identified for several peptide and protein hormones, and their presence has significant implications for the biological action of the hormone. Follistatin (FS) has been id entified as an activin- and inhibin-BP in tissues, serum, and follicul ar fluid of several species, including humans. In this study, the bind ing kinetics of FS for activin and inhibin were characterized in human serum using gel filtration chromatography and compared to those of pu re recombinant hormones using chromatography and a new solid phase ass ay. When complexed with radiolabeled activin or inhibin, FS eluted at a volume corresponding to a mol wt range of 67,000-150,000, an elution volume identical to the lower mol wt BP peak observed in serum. Furth ermore, kinetic analyses of recombinant FS binding to activin using a solid phase assay revealed that 1) the FS-activin interaction is of hi gh affinity, similar to or exceeding that estimated for activin bindin g to its receptor; 2) binding to activin is essentially irreversible a t physiological pH; and 3) the potency of inhibin is approximately 500 - to 1000-fold lower than that of activin in the FS binding assay. The lack of FS-[I-125]activin complex reversibility observed in the solid phase assay was confirmed using a modified gel filtration chromatogra phy protocol. Thus, preincubation of pure FS or serum with unlabeled a ctivin for 2 h eliminated all binding of subsequently added labeled ac tivin despite a much longer incubation period. However, when labeled a ctivin was incubated with FS for 2 h, subsequent addition of unlabeled activin or inhibin was unable to displace labeled activin from FS, ag ain demonstrating a lack of reversibility. Finally, to map this high a ffinity interaction, overlapping synthetic peptides were used to compe te with labeled activin for FS binding. Two potential contact sites be tween FS and activin were identified, one near the N-terminus (amino a cids 15-29) and the other near the C-terminus (amino acids 99-116). Gi ven its apparently irreversible nature, high affinity, and ability to neutralize activin's biological activity, FS is quite different from t he typical hormone-BP. These unique properties of FS undoubtedly attes t to the potency of activin in many physiological and developmental se ttings and, therefore, to the importance of BPs, such as FS for regula ting activin's bioactivity, distribution, and/or clearance.