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.
Goswami
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Department of Medicine, Boston University School of Medicine, Massachusetts 02118.
Rosenberg
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D000818:Animals; D002793:Cholic Acids; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D007453:Iodide Peroxidase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats
D002793:Cholic Acids; D003902:Detergents; D007453:Iodide Peroxidase; D005978:Glutathione; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D004229:Dithiothreitol