Abstract
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.
Original language | English |
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Journal | Molecular and Cellular Endocrinology |
Volume | 56 |
Issue number | 1-2 |
Pages (from-to) | 99-106 |
Number of pages | 8 |
ISSN | 0303-7207 |
Publication status | Published - Mar 1988 |
Externally published | Yes |
Keywords
- Animals
- Centrifugation, Zonal
- Detergents
- Iodide Peroxidase
- Kidney
- Rats
- Rats, Inbred Strains