Abstract
The size of androgen receptors from rat ventral and dorsal prostate, dorsal prostate (Dunning) tumor, testis, epididymis, and seminal vesicle was determined using Sephadex G-200 chromatogrpahy and sucrose gradient centrifugation. The protease inhibitor diisopropyl fluorophosphate (DFP) was used to minimize receptor breakdown. An 8-9 S, 85 to 106 A receptor (Mr = 280,000 to 365,000; f/fo = 1.9 to 2.4) observed in unfractionated cytosol prepared in low ionic strength buffer with or without DFP is in equilibrium with a 4.5-5 S, 58 A form (Mr = 117,000; f/fo = 1.8) observed at salt concentrations greater than 0.1 M KCl. Receptor partially purified using (NH4)2SO4 or phosphocellulose chromatography in the absence of DFP was present as smaller fragments of 3.6 S, 37 A and 3.0 S, 23 A. Similar fragments could be generated from the 4.5 S or 8 S receptor by mild trypsin treatment. In addition, ventral prostate contains a DFP-insensitive enzyme which specifically converts the 4.5 S, 58 A receptor to the 3.6 S 37 A fragment. The DFP-insensitive enzyme is partially inhibited by rabbit bile and appears similar to the enzyme seminin, a secretory protein of human prostate. Androgen receptor isolated in the presence of DFP from nuclei labeled in vivo is predominantly 4.5 S, 58 A, with smaller forms (37 and 23 A) appearing in the absence of DFP. The 4.5 S, 58 A nuclear receptors were also in equilibrium with a large 8 S form. Receptor breakdown by DFP-insensitive and sensitive proteases appears to be an in vitro phenomenon. Furthermore, the size of the androgen receptor is not significantly changed during receptor migration from cytoplasm to nucleus.
Highlights
North Carolina 27514 and Biochemistry, School of Medicine, University of North Carolina, Chapel Hill, The size of androgen receptors from rat ventral and dorsal prostate, dorsal prostate (Dunning) tumor, testis, epididymis, and seminal vesicle was determined using Sephadex G-200 chromatography and sucrose gradient centrifugation
= 280,000 to 365,000; f/f0 = 1.9 to 2.4) observed in unfractionated cytosol prepared in low ionic strength buffer with or without diisopropyl fluorophosphate (DFP) is in equilibrium with a 4.5
Effect of DFP-The above studies have shown that the androgen receptor in cytosol from epididymis, testis, seminal vesicle, dorsal prostate, and a dorsal prostate tumor exists in two interconvertible forms
Summary
Reagents from the following sources were used: [1,2,4,5,6,7,-3H]- or [1,2,4,5,6,7,16,17-3H]-dihydrotestosterone, 123 and 190 Ci/mmol, respectively, and [1,2,6,7-3H]testosterone (98 Ci/mmol) from New England Nuclear; unlabeled steroids, beef liver catalase, ovalbumin, horse skeletal muscle myoglobin, ammonium sulfate, trypsin (T-1005) type Xl from bovine pancreas, and trypsin inhibitor (T9003) lyophilized from soybean from Sigma; and diisopropyl fluorophosphate from Sigma or Cal Biochem; Sephadex G-200 and G-25, DEAE-Sephadex A-50, and blue dextran 2000 from Pharmacia; cellulose phosphate P 11, potassium form, and DEAE-cellulose DE52 from Whatman; hydroxylapatite (Bio-Gel HTP) were from Bio-Rad; bovine y-globulin, Fraction II, from Miles; Eagle’s minimum essential medium (with Earle’s salts, without glutamine) from Grand Island Biological. The costs of publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked “advertisement”. ’ The abbreviations used are: dibydrotestosterone, 17P-hydroxy5a-androstan-3-one; testosterone, 17/3-hydroxy-4-androsten-3-one; DFP, diisopropyl fluorophosphate.
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