UNIQUE RECOGNITION OF ACTIVIN AND INHIBIN BY POLYCLONAL ANTIBODIES TOINHIBIN SUBUNITS

Inhibin-A is a glycoprotein composed of an a subunit containing a glyc osylation site and a beta(A) subunit, whereas activin-A is a homodimer of two inhibin beta(A) subunits, We examined the recognition of activ in-A and inhibin-A by several antisera to the a or beta(A) subunit, an d factors affecting the recognition, A total of six polyclonal antibod ies to inhibin subunits, i.e., two antisera to a peptide fragment of t he alpha subunit [alpha(1-19) and alpha(1-26)], and four antisera to t he beta(A) subunit [beta(A)(1-10), beta(A)(70-79), beta(A)(87-99), and beta(A)(94-105)], was generated, On Western blot analysis, the anti-b eta(A) (87-99) and beta(A)(94-105) sera recognized recombinant human a ctivin-A but not inhibin-A under non-reducing conditions, When inhibin -A was deglycosylated with N-glycosidase-F, inhibin-A could be recogni zed by the anti-beta(A)(87-99) and beta(A)(94-105) sera, In addition, when activin-A bound to a nitrocellulose membrane was pre-incubated wi th recombinant human follistatin, the recognition of activin-A by the anti-beta(A)(87-99) and beta(A)(94-105) sera was decreased, These resu lts suggested that the lower affinity of follistatin to inhibin-A than to activin-A might be likely explained as reflecting a site associate d with the glycosylation of inhibin-A. However, the exposure of amino acids 87-105 of the inhibin beta(A) subunit on the molecular surface t hrough deglycosylation did not increase the affinity of inhibin-A for follistatin but rather resulted in poor binding with follistatin, The present data suggest that (1) amino acids 87-105 of the inhibin/activi n beta(A) subunit are located on the molecular surface, although this region of inhibin-A. is concealed by the carbohydrate chain of the ct subunit, (2) the region responsible for follistatin binding within the activin beta(A) subunit is spanned by amino acids 87-105, and (3) the mode of binding of inhibin-il to follistatin is quite different from that of activin-A to follistatin, and the former may be influenced by glycosylation.