| Abstract: | The specific androgen receptors for testosterone (T) and 5alpha-dihydrotestosterone (DHT) in the cytosol fraction of the anterior pituitary of rats have been further characterized using electrophoresis and isoelectric focusing in polyacrylamide gels. After labeling of the cytosol fraction in vivo and in vitro, we were able to demonstrate androgen-protein complexes moving with an electrophoretic mobility (Rf) of 0.5 in 3.25% acrylamide gels containing 0.5% agarose and 10% glycerol. This method was used for quantitative measurements of pituitary androgen receptors, allowing multiple samples to be run simultaneously with little or no non-specific binding. There was no measurable dissociation of the androgen-receptor complexes during electrophoresis. When radioactive testosterone (1 nM) was added to pituitary cytosol fractions in vitro, there was an increase in the binding up to 4 hours of incubation at 0 C and little or no increase between 4 and 24 hours. All the binding studies therefore were done by incubation overnight at 0 C. When cytosol fractions were incubated with increasing concentrations of radioactive testosterone, a typical saturation curve was found. Scatchard plot analysis showed a binding capacity of 12.0 femtomoles/mg protein and the equilibrium constant of dissociation was estimated to be 3.4 +/- 0.7 (SD) X 10(-10)M. Like other androgen-receptor complexes, the testosterone-receptor complex in the anterior pituitary gland had an extremely slow rate of dissociation at 0 C (t1/2 greater than 4 days). The steroid specificity of the cytoplasmic androgen receptors was tested in vitro by the competing efficiency of different unlabeled steroids for [3H] testosterone binding. T and DHT caused the same inhibition of [3H]T to the receptors. However, since metabolism, of DHT to 5alpha-androstane-3alpha,17beta-diol occurred even at 0 C, the affinity of DHT for the receptor is probably somewhat underestimated. Cyproterone acetate had approximately half the affinity for the receptor compared with T, whereas lower affinities were found for progesterone and 17beta-estradiol. Cortisol did not appear to have any affinity for the receptors. Isoelectric focusing in polyacrylamide gels showed a peak of bound radioactivity with an isoelectric point of 5.8. Thus, the characteristics of the cytoplasmic androgen receptors of the anterior pituitary gland are very similar to those of the androgen receptors described in the ventral prostate, epididymis, and testis. |
