Boye N, Laurberg P, Jørgensen P L
2nd University Clinic of Internal Medicine, Kommunehospitalet, Aarhus C., Denmark.
Mol Cell Endocrinol. 1988 Mar;56(1-2):99-106. doi: 10.1016/0303-7207(88)90013-5.
A rapid and sensitive assay of iodothyronine-5'-monodeiodinase (5'-D) was developed using Sephadex column chromatography for separation of substrate 125I-rT3 from the product free 125I-. The distribution of 5'-D activity on rat kidney cortex cell membranes was examined in isopycnic zonal centrifugation experiments using Na,K-ATPase and NADH-cytochrome C reductase as markers for basolateral and intracellular membranes. 5'-D was mainly distributed on fractions containing endoplasmatic reticulum although some association with basolateral membranes could not be excluded. The isopycnic zonal centrifugation of a microsomal fraction prepared by differential centrifugation purified the 5'-D 8-9 times, 80-90% of membrane-bound 5'-D could be solubilized in fully active form with the detergents CHAPS and C12E8. Solubilization led to a further 2- to 3-fold purification of the enzyme. The soluble preparation was used to characterize 5'-D and as antigens in preparation of monoclonal antibodies for further purification and characterization of 5'-D.
利用葡聚糖凝胶柱色谱法从产物游离的¹²⁵I⁻中分离底物¹²⁵I-rT₃，开发了一种快速灵敏的碘甲状腺原氨酸-5'-单脱碘酶（5'-D）检测方法。在等密度区带离心实验中，以Na,K-ATP酶和NADH-细胞色素C还原酶作为基底外侧膜和细胞内膜的标志物，检测了5'-D在大鼠肾皮质细胞膜上的分布情况。5'-D主要分布在内质网组分中，不过也不能排除其与基底外侧膜存在一定关联。通过差速离心制备的微粒体组分进行等密度区带离心，可使5'-D纯化8至9倍，80%至90%与膜结合的5'-D能用去污剂CHAPS和C12E8以完全活性形式溶解。溶解过程使该酶进一步纯化了2至3倍。可溶性制剂用于对5'-D进行特性分析，并作为抗原用于制备单克隆抗体，以进一步纯化和鉴定5'-D。
