High-Affinity Binding of 4-Androstene-3,17-dione in Rat Liver

Intraperitoneal administration of [1,2,6,7-³H]testosterone to castrated male rats resulted in the appearance of an [³H]androstenedione-protein complex (“complex A”) in liver cytosol. When analyzed by sucrose density gradient centrifugation this complex had a sedimentation coefficient of 10 S. Complex A was stable toward treatment with dextran-coated charcoal, was unaffected by incubation with DNase and RNase, but was destroyed following incubation with trypsin, chymotrypsin, and protease. Complex A was also formed following incubation of liver cytosol from castrated male rats with [1,2,6,7-³H]testosterone and after incubation of ABF (= the androgen-binding fraction collected at about 0.40 column volume during chromatography of liver cytosol from castrated male rats on Sephadex G-100) with [1,2-³H]androstenedione. Incubation with ABF made it possible to study the binding of androstenedione in vitro without interfering metabolism of this substrate; saturation of the androstenedione-binding protein in ABF was obtained at a substrate concentration of about 1 × 10^-7 M and the concentration of binding sites in ABF was about 8 × 10^-16mol/mg of protein (corresponding to 1.6 × 10^-16 mol/mg of protein in unfractionated cytosol). When castrated male rats were injected intraperitoneally with [1,2,6,7-³H]testosterone, liver nuclei specifically retained [³H]androstenedione. In order to investigate whether androstenedione could be taken up from the blood into the liver cell, pulse-labeling experiments were performed where [1,2,6,7-³H]testosterone was administered intravenously to intact male and female rats. The concentrations of [³H] androstenedione and 5α-[³H]dihydrotestosterone were higher in portal vein than in aortic blood indicating uptake of these metabolites in liver or lung tissue. When the ³H-labeled unconjugated steroids in liver cytosol were measured in the same experiment, 5 -[³H]dihydrotestosterone, 5α-[³H]androstane- 3α,17β-diol, and 5α-[³H]androstane-3β,17β-diol were found to decrease rapidly in concentration just after injection of isotope whereas [³H]androstenedione was present in very constant concentration in both male and female rats from about 2 min after administration of isotope. These results indicate the existence of specific mechanisms in liver cytosol protecting androstenedione from being metabolized. It is suggested that the hepatic androstenedioneprotein complex—that presents several of the characteristics described for steroid-receptor protein complexes—is involved in androgen action in the liver.