Mol J A, van den Berg T P, Visser T J
Department of Internal Medicine III, Erasmus University Medical School, Rotterdam, The Netherlands.
Mol Cell Endocrinol. 1988 Feb;55(2-3):149-57. doi: 10.1016/0303-7207(88)90129-3.
Rat liver microsomal fraction was treated with several non-ionic, anionic or zwitterionic detergents in order to investigate which is most suitable for subsequent purification of the iodothyronine deiodinase. Criteria for effective solubilization were (a) no or little inhibitory effect of the detergent on deiodinase activity, (b) non-sedimentable activity by centrifugation at 105,000 X g, and (c) a low molecular weight of the soluble complex as determined by Sephacryl S-300 gel filtration in the presence of detergent. Optimal solubilization was obtained by treatment of the microsomes with cholate and subsequent precipitation of dispersed protein with 30% ammonium sulfate, resulting in the removal of adhering phospholipids. Enzyme was resolubilized best with the non-ionic detergents Brij 56 or Emulgen 911 in the presence of 0.5 M NaCl. This deiodinase preparation had an isoelectric point at pH 9.3 and was further purified by subsequent chromatography on DEAE-Sephacel and CM-Sepharose. Only the Emulgen 911-dispersed enzyme was retained by the CM-Sepharose column. Further purification was investigated by chromatofocusing. This resulted in a rapid inactivation of the Emulgen 911 preparation whereas the Brij 56-soluble enzyme was ultimately purified 400 times after DEAE-Sephacel and chromatofocusing.
用几种非离子型、阴离子型或两性离子型去污剂处理大鼠肝脏微粒体部分，以研究哪种去污剂最适合随后纯化碘甲腺原氨酸脱碘酶。有效增溶的标准为：(a) 去污剂对脱碘酶活性无抑制作用或抑制作用很小；(b) 在105,000×g离心时活性不可沉降；(c) 在去污剂存在下通过Sephacryl S - 300凝胶过滤测定的可溶性复合物分子量较低。用胆酸盐处理微粒体，随后用30%硫酸铵沉淀分散的蛋白质，从而去除附着的磷脂，可获得最佳增溶效果。在0.5 M氯化钠存在下，用非离子型去污剂Brij 56或乳化剂911能使酶最佳地重新溶解。这种脱碘酶制剂的等电点为pH 9.3，随后通过DEAE - Sephacel和CM - Sepharose柱色谱进一步纯化。只有乳化剂911分散的酶被CM - Sepharose柱保留。通过聚焦层析研究进一步纯化。这导致乳化剂911制剂迅速失活，而Brij 56溶解的酶在经过DEAE - Sephacel和聚焦层析后最终纯化了400倍。
