Identification of high affinity binding sites for inhibin on ovine pituitary cells in culture.
The aim of this study was to identify and characterize binding sites for inhibin in primary cultures of ovine anterior pituitary cells. Recombinant human 31-kDa inhibin A was iodinated by an optimized lactoperoxidase procedure. Fractionation of the labeled protein by gel filtration chromatography on Sephadex G-100 in 0.1 M HCl yielded two immunoactive peak regions, the second of which was bioactive as assessed by in vitro bioassay, with a ratio of bioactivity/immunoactivity of 0.62-0.77 and an iodine incorporation ratio of 1.7-2.0 mol 125I/mol inhibin. The specific binding of purified [125I]inhibin to cultured ovine pituitary cells varied with time, temperature, and cell number. Displacement of the tracer by unlabeled inhibin, as assessed by Scatchard analysis, revealed two binding sites with average Kd values of 0.28 and 3.9 nM and with approximately 250 and 3100 binding sites/anterior pituitary cell, respectively. There was little cross-reaction between inhibin and activin A (<2%), transforming growth factor-beta (<0.2%), or follistatin (<<0.1%). Examination of cell lines that were not expected to have inhibin receptors showed that there was no specific binding of inhibin to human leukemia (Jurkat) cells, whereas the binding to human embryonic kidney (293) cells was displaced by both inhibin and activin with a similar degree of cross-reaction, which suggests binding to an activin receptor. It is concluded that inhibin-binding sites with high affinity and specificity have been identified on ovine pituitary cells, consistent with both inhibin action on the pituitary and the presence of the putative inhibin receptor.
Hertan
R
R
Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia.
Farnworth
P G
PG
Fitzsimmons
K L
KL
Robertson
D M
DM
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D006801:Humans; D007265:Inhibins / Q000378:metabolism*; D007457:Iodine Radioisotopes / Q000378:metabolism; D019169:Jurkat Cells; D007700:Kinetics; D007784:Lactoperoxidase / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000378:metabolism; D012756:Sheep
D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D007265:Inhibins; D007784:Lactoperoxidase