Isolation of nine different biologically and immunologically active molecular variants of bovine follicular inhibin.
A combination of immunoaffinity chromatography, SDS-PAGE, and electroelution was used to simultaneously isolate 0.36-4.65 mg of nine different molecular forms of inhibin (pro alpha C-29 kDa; fully processed 34 kDa; and large inhibin forms 49, 53, 58, 77, 88, 110, and > 160 kDa) from 0.675 L of bovine follicular fluid (bFF). Each inhibin form, except pro alpha C, cross-reacted with inhibin alpha C 1-26-and beta A 82-114-subunit-directed antibodies during immunoblot analysis. Pro alpha C cross-reacted only with alpha-subunit antibodies. The inhibin forms consisted of 22-, 29-, 49-, or 58-kDa alpha subunits and 17- or 58-kDa beta subunits. During cultures of ovine pituitary cells, a 5-ng/ml dose of each inhibin form (except pro alpha C) suppressed basal accumulation of FSH 30% to 50% but increased GnRH-induced LH release 40% to 248%. The various inhibin forms cross-reacted in parallel fashion with standard curves generated during homologous and heterologous RIAs but with markedly different relative immunopotencies. In the RIAs, pro alpha cross-reacted 3- to 18-fold more than the fully processed inhibin form. The fully processed and the seven different large forms of inhibin cross-reacted with different relative immunopotencies in a two-site dimer-specific ELISA. We concluded that 1) a combination of immunoaffinity extraction, SDS-PAGE, and electroelution simultaneously isolated relatively large amounts of highly enriched preparations of nine different molecular forms of immunologically and biologically active inhibin from bFF; 2) eight different dimeric forms of bovine inhibin may regulate both basal FSH and GnRH-induced LH secretion by the pituitary gland, and 3) eight or nine different molecular forms of inhibin cross-react with different relative immunopotencies in the two-site dimer-specific assay or RIAs.
Good
T E
TE
Molecular Reproductive Endocrinology Laboratory, Department of Animal Science, East Lansing, Michigan 48824, USA.
Weber
P S
PS
Ireland
J L
JL
Pulaski
J
J
Padmanabhan
V
V
Schneyer
A L
AL
Lambert-Messerlian
G
G
Ghosh
B R
BR
Miller
W L
WL
Groome
N
N
D000818:Animals; D001681:Biological Assay; D002417:Cattle; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015151:Immunoblotting; D007158:Immunologic Techniques; D007265:Inhibins / Q000737:chemistry / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008970:Molecular Weight; D006080:Ovarian Follicle / Q000737:chemistry*; D011863:Radioimmunoassay; D012756:Sheep
D046911:Macromolecular Substances; D007265:Inhibins