Molecular Assembly of rh-FS, rh-Inhibin A, and rh-Activin A Complexes In Vitro
The apparent molecular masses of rh-FS288 analyzed by SDS-PAGE were 39 kDa and 42 kDa under reducing conditions and 32 kDa and 35 kDa under nonreducing conditions (Fig. 1A). The two molecular size forms are accounted for by glycosylation. The increase in apparent mobility when the molecule was reduced may be because the reduced protein occupies more space, which would decrease mobility through the gel. FS contains 36 sulfhyrides that form intramolecular bonds resulting in a compact molecule when intact (i.e., nonreduced). buy yasmin online
FS-inhibin complexes of 66-kDa, 97-kDa, 133-kDa, and > 220-kDa molecular masses were clearly detected when rh-FS was cross-linked to radiolabeled rh-inhibin A (Fig. 1B). When inhibin A was cross-linked to itself, a faint band at 66 kDa was seen, likely secondary to a small amount of aggregation of this 32-kDa protein. The increased intensity of the 66-kDa band after inhibin-FS cross-linking is likely due to a 1:1 ratio of the two proteins, although experiments aimed at analyzing the ratio of inhibin to FS on a molar basis were unsuccessful (with SDS-PAGE and silver staining).
FIG. 1. A) Recombinant human FS, rh-activin, and rh-inhibin were subjected to SDS-PAGE and silver staining under both nonreducing and reducing conditions (heat, р-mercaptoethanol). FS, rh-FS; Act, rh-activin; Inh, rh-inhibin. Note the shift from 32- and 35-kDa molecular size bands of rh-FS under nonreducing conditions to 39- and 42-kDa bands with reducing conditions while activin and inhibin fall apart into their respective subunits (a, 1 8 kDa; and рд 14 kDa). Inhibin under nonreducing conditions shows evidence of aggregation. B) Gel-shift analysis using 125I-labeled inhibin and 125I-labeled activin was performed to identify complex formation upon addition of rh-FS (FS). Each ligand was cross-linked with disuccinimidyl sub-erate (DSS) alone, as well as in the presence of FS. Upon the addition of FS, subsequent bands were seen at 66 kDa, 97 kDa, 1 33-1 35 kDa, and larger molecular sizes. Inhibin cross-linked alone resulted in a faint 66-kDa band; but upon addition of FS and cross-linker, the 66-kDa band intensified. These bands may represent FS and activin/inhibin in a 1:1 ratio (66 kDa), a 2:1 ratio (97 kDa), or multimers of these combinations. C) FS and activin/ inhibin were combined in varying ratios and cross-linked with DSS, then subjected to SDS-PAGE and silver staining under nonreducing and reducing conditions. Ratios are expressed as activin/inhibin:FS. AXF, activin crosslinked to FS; IXF, inhibin cross-linked to FS. A 97-kDa band is prominent upon a 2:1 FS:activin ratio, suggesting that this complex consists of two FS molecules and one activin. In the IXF panel, the lower molecular weight band is consistent with unbound rh-FS. Right panel: The 97-kDa complex (FS-activin) was marginally detected with FS monoclonal antibodies upon Western blotting when compared to FS. The FS-inhibin complex was not well detected by these antibodies and is not shown.