Activin-binding proteins in human serum and follicular fluid.

Abstract

Binding proteins that transport and/or modify the biological action of peptide hormones and growth factors have been identified for an increasing number of endocrinologically important substances. Since these binding proteins can mask epitopes critical for recognition in immunoassays and can neutralize the bioactivity of their targets, elucidation of hormonal physiology can be intricately tied to analysis of binding protein structure and function. Therefore, we investigated whether circulating activin- and inhibin-binding proteins exist in human serum by incubating purified recombinant human 125I-activin with serum samples. After gel permeation chromatography, radioactive activin was identified in three peaks, a high molecular wt (mol wt) binding protein peak (230 kDa), a lower mol wt binding protein peak (60 kDa), and free activin (22.5 kDa). Bound activin was displaced from the lower mol wt binding protein with either activin or inhibin, but was not displaced from the high mol wt peak with a 10-fold greater concentration of activin. Since an activin-binding protein, follistatin, has been identified in ovarian and pituitary extracts, these same analytical techniques were applied to analysis of human follicular fluid as well. A large, 60 kDa binding protein peak eluting in a position similar to the lower mol wt peak in serum was observed, consistent with this protein being follistatin. These results demonstrate the presence of at least two activin-binding proteins, distinguishable by size, in human serum that may interfere with attempts to assay activin levels in circulation without prior extraction, and may also be involved in regulating the biological actions of activin.

The Myostatin Propeptide and the Follistatin-related Gene Are Inhibitory Binding Proteins of Myostatin in Normal Serum

“…It is known that follistatin is present in serum and can bind to myostatin (2,10,34,35). Furthermore, transgenic mice overexpressing follistatin under a skeletal muscle-specific promoter show a double muscle phenotype consistent with the negative regulation of myostatin (10).…”

Activin and Follistatin as Local Regulators in the Human Ovary

“…FS has been identified in the human follic ular fluid [39][40][41] and both FS-activin and FS-inhibin complexes have been detected [41]. Messenger RNA encoding FS has also been found in the human ovary by Northern blot [38] and in situ hybridization [6] and FS protein detected by immunocytochemistry [6].…”

Measurement of activin in physiological fluids

“…This observation spurred development of second-generation assays using synthetic peptides to target antibody development to specific sequences on a-or ß-subunits, allowing the formulation of two-site immunoassays with required specificity for dimeric inhibin (8,9). During the development phase, it was discovered that binding proteins for inhibin and activin existed in physiological fluids that interfered with analytical recovery (10)(11)(12), thereby requiring adaptation of assay formats to circumvent these problems (13)(14)(15).…”

“…Follistatin, a 32-35kD singlechain polypeptide, binds and neutralizes activin (19) with high affinity and low reversibility under physiolo¬ gical conditions, including those in serum and human follicular fluid (hFF) (10,20). Furthermore, the binding sites of many peptide-directed anti-activin antibodies overlap with at least one of follistatin's putative activin binding sites (20).…”

“…this problem may be significant. Another potential problem is that this study only evaluates the effects of FS on activin measurement, while other studies have demonstrated that additional serum proteins, such as a2-macrogIobulin, can also bind activin, although at lower affinity (10)(11)(12). Thus, the actual recovery of activin from human serum taken from a variety of physiological conditions would seem warranted as part of the validation.…”