Purification and partial characterization of iodothyronine 5'-deiodinase from rat liver microsomes

We have isolated and purified iodothyronine 5'-deiodinase from rat liver microsomes to homogeneity as judged by PAGE and analytical HPLC. The enzyme progressively lost activity after solubilization, and specific activity enhancement was a modest 22-fold, but the final preparation still had substantial activity and was used for molecular characterization. The enzyme had an Mr of 56,000 with a single band in SDS-PAGE, suggesting absence of subunit structure. The high Km, and the GSH-responsive low Km, activities were co-purified, but the low Km enzyme lost GSH-responsiveness upon pretreatment with dithiothreitol (DTT) and urea. The enzyme was strongly inhibited by the iron chelator, alpha,alpha'-dipyridyl and showed a broad absorbance band at 410 nm. Spectral analysis with diethylpyrocarbonate (DEPC) revealed 5 histidine residues/mol enzyme, while enzyme activity was inhibited by DEPC in a pseudo-first order process with modification of 1 histidine residue/mol.