ヒト前立腺アンドロゲン・レセプター精製に関する研究 第2編: ヒト前立腺アンドロゲン・レセプターの定性

Abstract

ヒト前立腺cytosolのアンドロゲン・レセプター(AR)の結合能に及ぼすmolybdate, NaSGN, heparinなどの影響を検討した.1) ARに及ぼすmolybdateの影響:最終濃度10 mMのmolybdateをcytosolに加えると, 3H-R1881とARの蛋白量あたりの最大結合部位数はcontrolに比較して約60%増加し, 解離定数はやや高くなった.遮糖密度勾配遠心法による検討でmolybdateの添加によりcytosolでは8～9 Sの部位に再現性のピークを得たが, molybdate非添加cytosolでは有意のピークを得なかった.これらはmolybdateのAR安定化作用を証明するものである.さらに, 比親和性relative binding affinityによる検討では, molybdateはステロイド特異性には影響しなかった.2) ARに及ぼすNaSCNの影響:0.1～0.5 M NaSCNをcytosolに添加すると3H-R1881・AR複合体の解離および形成はともに促進傾向を示した.しかし, 0.3 M以上のNaSCNによりARの結合能は急速に失われ, ゲル濾過でNaSCNを除去するとARの結合能は部分的に回復した.3) ARに及ぼすheparinの影響:4 mg/ml heparinは3H-R1881・AR複合体の解離を促進させ, 4 mg/ml heparinの添加でARの最大結合部位数は56%減少した

For improved purification of human prostate androgen receptors (AR), the effect of molybdate, NaSCN and heparin on the binding capacity of human prostate cytosol AR was investigated. When 10 mM of molybdate in final concentration was added to cytosol, the increase in the maximum binding sites of AR to 3H-R1881 was approximately 60%, while the dissociation constant was rather increased. With sucrose density gradient centrifugation, the cytosol with molybdate added showed a constant peak of 8 approximately 9 S. However, cytosol not enriched with molybdate did not show any significant peak. These results constitute evidence that molybdate has a stabilizing effect of the AR. When 0.1 approximately 0.5 M of NaSCN was added to the cytosol, the dissociation as well as the association of the 3H-R1881-AR complex showed a tendency to accelerate. However, at a concentration of 0.3M and over NaSCN, the binding capacity of the AR rapidly disappeared. When the NaSCN was removed with gel filtration, the binding capacity of the AR recovered partially. The effect of NaSCN on the AR was therefore different from that on the estrogen receptors. Four mg/ml of heparin accelerated the dissociation of the 3H-R1881-AR complex. Furthermore, with the addition of 4 mg/ml of heparin, the maximum binding sites of AR decreased to 56%. The above results make it clear that molybdate, NaSCN and heparin have various effects on human prostate cytosol AR, which might be applied to the purification of AR.