Optimum temperature, time and dose of androgenic steroids (5α-dihydrotestosterone (5α-DHT) and testosterone) required for quantitative estimation of nuclear and cytosol androgen receptor (AR), before and after affecting nuclear translocation of AR, was investigated in normal and cancerous breast tissues of women. Translocation of AR was temperature dependent, being maximum at 37°C. Optimal conditions required for estimation of AR in nuclei and cytosol are described. Three methods, single saturation dose analysis, sucrose gradient analysis and multidose saturation analysis, were used to estimate AR. The results of the three methods had a high coefficient of correlation (≫0.95). Immunoabsorption of plasma proteins significantly reduced population of specific androgen binding sites in cytosol without effecting number of AR translocated to nucleus. 5α-DHT estimated 1.51 times the number of androgen binding sites compared with testosterone. Majority ( $ ̃80%) of specific androgen binding sites in situ were distributed as free sites in cytosol and a significant percentage was occupied by endogenous steroid either in cytosol or nuclear fraction. An important finding was thai only a part and a highly variable percentage (19-100%) of specific androgen binding sites in the cytosol were translocated to the nucleus even after immunoabsorption of plasma proteins from the cytosol. Based on these findings, a new approach, involving the estimation of translocatable androgen receptors/100 μg nuclear DNA. by the easy and simple procedure of single dose saturation analysis, with either 5α-DHT or testosterone, is suggested. This method will help in determining the total population of functionally active receptor sites in a cell. which can be more accurately used as a diagnostic test in the endocrine treatment of the patient.
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