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F. Felden, J. L. Guéant, A. Ennya, A. Gérard, S. Frémont, J. P. Nicolas, and H. Gérard


A specific receptor with high affinity for rat androgen-binding protein (rABP) was identified in isolated adult rat germ cells and in the corresponding plasma membrane-enriched preparations. Binding was reversible and time-dependent, with maximum relative binding after 40 min at 4 °C; it was pH-dependent, with maximum binding at pH 6–8. Unlabelled rABP and human sex steroid-binding protein (hSBP), but not lactotransferrin, serotransferrin, asialofetuin, fetuin or bovine serum albumin, competed with labelled rABP for binding sites on isolated germ cells. Scatchard analysis revealed a single class of binding site with apparent dissociation constant (K d) values of 0·78±0·04 nm and 0·97 ± 0·05 nm in intact germ cells and plasma membrane preparations respectively. A K d of 1·72±0·12 nm for hSBP showed that the receptor binding site was effective for both androgen-carrier molecules. Labelled rABP incubated with solubilized germ cell membrane fractions at pH 7 formed a complex excluded from Superose 6B mini-gels; this complex was not formed at pH 3. The receptor complex was also abolished in the presence of a 100fold excess of either unlabelled rABP or unlabelled hSBP, or in the presence of 20 mm EDTA.

These results suggest that the plasma membrane of rat germ cells contains a receptor which selectively binds rABP and hSBP.

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J.-L. Guéant, S. Fremont, F. Felden, J.-P. Nicolas, A. Gerard, B. Leheup, H. Gerard, and G. Grignon


We have studied the binding of [ I-iodo]androgen-binding protein (ABP) and of [3H]Δ6-testosterone photoaffinity-labelled ABP to receptors in the plasma membrane of rat epididymal cells in three ways: ABP binding to a Triton X-100-solubilized membrane extract, ABP binding to isolated epithelial cells in suspension and autoradiography of segments of dissected epididymides after in-vitro intraluminal injection of labelled ABP. The binding of iodinated ABP to the receptor was similar to that of photoaffinity-labelled ABP in gel filtration. The ABP-receptor complex was eluted from Superose 6 gels as an aggregate, with a molecular mass of 2000 kDa. It was separated into two peaks by sucrose gradient ultracentrifugation, with respective sedimentation coefficients of 18.4 and 9.0s. The activity of the receptor (ABP-binding capacity/mg protein) was tenfold higher in the caput than in the cauda. The binding of ABP to the receptor was pH dependent, being almost abolished at pH <4. The binding at 4°C of photoaffinity-labelled ABP to epithelial cells corresponded to two types of binding sites. The numbers of high-affinity and low-affinity sites per cell were 1600 and 7700 respectively; the association constants of these sites were 67.9 and 2.8litres/ nm respectively. The binding was decreased by treatment of the cells with trypsin or incubation in the presence of ED TA. The binding in vitro of labelled ABP to the epididymis epithelium reached a maximum after about 20 min at 4°C. In the autoradiographic study the tracer was found to be closely associated with coated pits, coated vesicles, endosomes and pale multivesicular bodies. Treatment of rats with cycloheximide significantly reduced the uptake of the tracer. Perfusion in vitro of epididymides with chloroquine produced a fourfold increase of the tracer in endosomes and multivesicular bodies.