|Budget Amount *help
¥7,800,000 (Direct Cost : ¥7,800,000)
Fiscal Year 1996 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1995 : ¥6,200,000 (Direct Cost : ¥6,200,000)
Inhibin is a glycoprotein consisting of two dissimilar subunits, termed alpha and beta, that are linked by disulfide bounds. Activin is a beta-subunit dimer and consists of inhibin betaA and betaB subunits that have been termed activin A,activin AB,and activin B.It is impossible to isolate and characterize the inhibin in small experimental animals such as the rat that is the most useful for biological and immunological experiments on inhibin. Therefore, we improved the methods for purifying inhibin, activin and follistatin. Immunoaffinity chromatography is the most useful applied method for the purification of inhibin from follicular fluid.
Since inhibin B was not detected in human follicular fluid, we tried to produce recombinant human inhibin B.cDNA clones encoding human inhibin alpha- and betaB subunits were isolated from a human testicular cDNA library. The cDNAs were linked to an expression vector and transfected into Chinese hamster ovarian cells. The cells transfected with the alpha-subunit and betaB-subunit cDNAs secreted inhibin B and activin B.Immunoaffinity chromatography with polyclonal antibodies was used to purify the low levels of inhibin B contained in the culture medium as described above.
In the process of purifying bovine inhibin by immunoaffinity chromatography, the flow through fraction from the immunoaffinity column was collected. The activin/FSP complex was then purified through successive steps of affinity chromatography with sulfated cellulose and heparin Sepharose, and FPLC gel permeation on a Superdex pg S20 column. Activin/FSP complex, was further subjected to reverse-phase HPLC providing six major fractions. The first three fractions had FSH inhibiting activity. The other fractions had FSH stimulating activity with cultured pituitary cells that correspond to activin-A,-AB,and B,respectively.