ALPHA-2-MACROGLOBULIN IS A BINDING-PROTEIN OF INHIBIN AND ACTIVIN

The development of a two-site immunoassay for dimeric inhibin resulted in the detection of higher mol wt substances in follicular fluid that interfere with the measurement of dimeric inhibin by this assay, but not by a previously characterized inhibin alpha-subunit RIA. Therefore, we initiated a study to identify inhibin- and activin-binding proteins in biological fluids using I-125-labeled recombinant human inhibin-A and activin-A. By gel permeation chromatography, we found that [I-125] inhibin A or [I-125]activin-A incubated with either plasma or follicular fluid became associated with several higher mol wt proteins. The lowest mol wt complex eluted with a retention time similar to that of [I-125] inhibin or [I-125]activin bound to porcine follistatin, a known inhibin- and activin-binding protein. The largest mol wt complex eluted near the void volume, coincident with human alpha2-macroglobulin (halpha2M) incubated with [I-125]inhibin or [I-125]activin. Human plasma was incubated with [I-125]inhibin or [I-125]activin, and putative binding proteins were chemically cross-linked and analyzed by polyacrylamide gel electrophoresis. Cross-linked I-125-labeled inhibin or activin plasma binding protein complexes comigrated with [I-125] inhibin or [I-125]activin cross-linked to halpha2M. Antiserum to halpha2M precipitated I-125-labeled inhibin- and activin-binding proteins in human plasma that migrated identically in polyacrylamide gel electrophoresis to complexes formed between [I-125]inhibin or [I-125]activin cross-linked to halpha2M. Excess unlabeled inhibin or activin decreased the labeling of halpha2M and high mol wt human plasma binding proteins by [I-125]inhibin or [I-125] activin. These results demonstrate that in addition to follistatin, alpha2M is a binding protein of both inhibin and activin. Although the physiological relevance of the inhibin-alpha2M and activin-alpha2M interaction is unknown, alpha2M may play a role in the delivery or clearance of inhibin and activin similar to that proposed for transforming growth factor-beta and a number of other growth factors.