ZINC POTENTIATION OF ANDROGEN RECEPTOR-BINDING TO NUCLEI INVITRO

Zn2+ potentiates binding of the 4.5S [3H]dihydrotestosterone-receptor complex to isolated rat prostate Dunning tumor nuclei in vitro when assayed in the presence of 300 .mu.M ZnCl2, 3 mM MgCl2, 0.25 M sucrose, 5 mM mercaptoethanol, 0.15 M KCl and 50 mM tris(hydroxymethyl)aminomethane, pH 7.5. In the presence of 5 mM mercaptoethanol, the concentration of 50 .mu.M total Zn2+ required to promote half-maximal receptor binding to nuclei corresponds to a free Zn2+ concentration of 50 nM. The receptor-nuclear interaction appears to be selective for Zn2+; other divalent cations when added to a concentration of 1 mM to a buffer containing 5 mM mercaptoethanol are less effective (Ni2+) or have essentially no effect (Ca2+, Mg2+, Mn2+, Co2+, Cu2+ and Cd2+). Zn2+ does not alter the sedimentation rate of the 4.5S [3H]dihydrotestosterone receptor in the presence of mercaptoethanol; in the absence of mercaptoethanol, Zn2+ causes the receptor to aggregate. Zn2+-dependent nuclear binding of the 4.5S [3H]dihydrotestosterone receptor is saturable at 1.4 .times. 10-13 mol of receptor sites/mg of DNA, corresponding to .apprx. 1150 sites/nucleus. In the presence of excess nuclei, up to 60% of added receptor is nuclear bound. An apparent binding constant for the receptor-nuclear interaction of 10-13 M-1 was approximated. Pyridoxal 5''-phosphate (.ltoreq. 10 mM), but not 0.4 M KCl, inhibits Zn2+-dependent nuclear binding of the [3H]dihydrotestosterone receptor. Up to 66% of nuclear-bound receptor can be extracted in buffer containing 3 mM EDTA plus 0.4 M KCl or 10 mM pyridoxal 5''-phosphate. Nuclear receptor extracted in buffer containing the protease inhibitor diisopropyl fluorophosphate (2 mM) sediments at 4.5 S on sucrose gradients, but the receptor sediments at 3 S when extracted without the inhibitor. Zn2+-dependent nuclear binding of the [3H]dihydrotestosterone receptor is temperature dependent, with association rate constants (Ka) at 0.degree., 15.degree. and 25.degree. C of 4.0 .times. 105, 2.8 .times. 106 and 9.7 .times. 106/M per min, respectively. The activation energy of binding is 21 kcal/mol.