Partial purification of rat kidney iodothyronine-5'-deiodinase by zonal centrifugation.
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.
Boye
N
N
2nd University Clinic of Internal Medicine, Kommunehospitalet, Aarhus C., Denmark.
Laurberg
P
P
Jørgensen
P L
PL
D000818:Animals; D002501:Centrifugation, Zonal; D003902:Detergents; D007453:Iodide Peroxidase / Q000302:isolation & purification*; D007668:Kidney / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains
D003902:Detergents; D007453:Iodide Peroxidase