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Title: 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3編: ヒト前立腺アンドロゲン・レセプターの精製および精製アンドロゲン・レセプターの定性
Other Titles: Purification of the human prostate androgen receptor. III. Purification of the androgen receptor and characterization of the purified androgen receptor
Authors: 中原, 満  KAKEN_name
Author's alias: NAKAHARA, Mitsuru
Keywords: Prostate
Androgen receptor
Purification
Issue Date: Nov-1986
Publisher: 泌尿器科紀要刊行会
Journal title: 泌尿器科紀要
Volume: 32
Issue: 11
Start page: 1701
End page: 1711
Abstract: ヒト前立腺肥大症の腺腫組織からaffinity chromatographyを用いてアンドロゲン・レセプター(AR)を精製した.この精製ARについて物理化学的性質を検討し,さらにARに対する抗体作製を試みた.ヒト前立腺ARの精製については,前立腺組織855gからheparin Sepharose, R1881 affinity chromatographyなどを使用して最終精製分画100 μgを得た.最終精製分画は100倍量triamcinolone acetonide (TA)存在下に3H-R1881と高結合親和性,一定結合部位数の結合を認め,その解離定数は2.4 nMでありARの結合能を残していた.精製ARの諸種ステロイドに対する比親和性を検討すると,3H-R1881と精製ARの結合はR1881で強く抑制され,TAやestracdiolでは抑制されず,ARとしての高い結合特異性を示した.精製倍率を蛋白量あたりの結合部位数で計算すると,cytosolの約2,360倍となった.7.5%polyacrylamide gel electrophoresisによる検討では,精製AR中にalbumin, testosterone-estradiol binding globulin (TeBG)などの混入を認めなかった.Sephacryl S-300 column chromatographyではvoid volumeに一致した3H-R1881のピークが得られた.これに対して高速液体クロマトグラフィーによる検討では分子量は約32,000と考えられた.両実験における成績の不一致の原因として前者ではARの凝集のため,後者の結果はARの一部が解離したためなどの可能性が考えられた.精製ARに対する抗体の作製としては,精製ARにより家兎を免疫し,抗血清を作製した.この血清は精製ARのみと反応が認められ,抗AR抗体作製に成功したと考えられた
With 855 g of benign prostatic hypertrophic tissue as starting material, the androgen receptor (AR) was purified by combining the affinity chromatography of heparin Sepharose CL-6B and of R1881-carboxymethyloxime-albumin Sepharose 4B. The final purificationfraction had high-affinity, low-capacity binding to 3H-R1881 in the presence of 1,000-fold molar excess of triamicinolone acetonide (TA) with a dissociation constant of 2.4 nM. When the relative binding affinity was being assessed, the binding of the final purification fraction to 3H-R 1881 was not suppressed by estradiol or TA and revealed the binding specificity of the AR. Polyacrylamide gel electrophoresis showed no albumin or TeBG mixed in the final purification fraction. These results give evidence that the AR had been purified and that, in comparison with the original cytosol, the degree of purification was approximately 2,360 fold. The molecular weight of the purified AR was 32,000 as calculated by high performance liquid chromatography. The fact the antiserum from rabbit immunized with the purified AR showed a reaction against the purified AR leads us to believe that antibodies for the AR were successfully produced. Further immunological study on AR measurement is considered to be of great importance.
URI: http://hdl.handle.net/2433/118956
PubMed ID: 3825818
Appears in Collections:Vol.32 No.11

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