ヒト前立腺肥大組織のアンドロゲン結合蛋白に関する実験的研究

Abstract

ヒト前立腺肥大組織を用いて細胞質アンドロゲン結合蛋白について基礎的検討を行なった.Scatchard plotにより前立腺サイトゾールの反応を解析したところ, DHTに対する解離定数(Kd)=9.4×10-10M, 最大結合部位数(B max)=55 fmoles/mg protein, R1881に対してはKd=5.1×10-9M, B max=25.5 fmoles/mg protein, とこのサイトゾール中に高親和性で飽和性のある結合蛋白を認めた.この結合蛋白を3H-DHTまたは3H-R1881の結合下に, 100倍量の非標識ステロイドと競合させた結果, DHTあるいはR1881に特異性を有することが証明された.前立腺サイトゾールと3H-DHTとの結合体はゲル濾過法でVoおよびIgG分画付近の2ヵ所に溶出されたが, 3H-R1881との結合体はVoに溶出された.結合蛋白と3H-DHTとの結合は, Ca2+およびMg2+イオン濃度の上昇に伴い増加し, 3H-R1881との結合は, 両イオン濃度の増加に伴って減少傾向を示した.3H-DHTとTBGとの結合でも両イオンはその結合量を増加させたが, 両イオンによる差はほとんどなかった.3H-R1881とTBGとの結合はG-200カラムでIgG分画にわずかのピークとして溶出されたが, 非特異的な結合と考えられた.3H-DHTあるいは3H-R1881と結合蛋白との結合に対するCa2+またはMg2+の影響をG-200カラムにより分析したところ, Mg2+よりもCa2+の影響が大きかった.これらの変化は二価金属イオンの影響によるものとも考えられるが, サイトゾール中にはCa2+あるいはMg2+の存在下で結合蛋白分子に作用をおよぼす物質が存在する可能性も考えられた

Androgen-binding proteins in the cytosol of human benign prostatic hypertrophic tissue were studied with 3H-dihydrotestosterone (DHT) and 3H-methyltrienolone (R 1881) as ligands. In the prostatic cytosol, saturable binding proteins with high affinity to 3H-DHT or 3H-R1881 were found by Scatchard analysis. The dissociation constant (Kd) for 3H-DHT was 9.4 X 10(-10)M, and the maximum number of binding sites (B max) was 55 fmoles/mg protein; the Kd for 3H-R1881 was 5.1 X 10(-9)M, and B max was 25.5 fmoles/mg protein. Competitive binding assay for each binding protein with various non-labeled steroids, demonstrated a specificity for DHT or R1881. The binding of prostatic cytosol and 3H-DHT was eluted by Sephadex G-200 column chromatography in 2 places, one in the void volume and one near the IgG fraction. The binding with 3H-R1881 was eluted in the void volume, the elution near the IgG fraction being very small, which suggests a non-specific binding. The binding of prostatic cytosol and 3H-DHT increased with increase in the concentration of Ca2+ or Mg2+ (0-12 mM), but the binding with 3H-R1881 decreased conversely. This change was studied with Sephadex G-200 column chromatography; the binding with 3H-DHT showed a decrease of the peak in the void volume and marked increase of the peak near the IgG fraction with increase in the concentration of both ions, whereas the binding with 3H-R1881 only showed a decrease of the peak in the void volume. Ca2+ had a greater influence than Mg2+ on the change of these bindings. These results indicate a change of binding protein due to ionic action, suggesting also the possibility of the presence of a substance in the cytosol which changes the properties of the binding protein dependent on Ca2+.