NADPH-dependent generation of a cytosolic dithiol which activates hepatic iodothyronine 5'-deiodinase. Demonstration by alkylation with iodoacetamide.
We have assessed a previously proposed mechanism mediating 5'-deiodinase activation involving enzymic reduction of disulphides to thiols in non-glutathione cytosolic components of Mr approx. 13,000 (Fraction B) catalysed by NADPH in the presence of other cytosolic components of Mr greater than 60,000 (Fraction A). The extent of Fraction B reduction under various experimental conditions was monitored by determining the amount of 14C incorporated into chromatographically isolated Fractions B and A after their alkylation with iodo[14C]acetamide. Incorporation of 14C into B was found to require the simultaneous presence of NADPH and A, to be directly proportional to the concentration of NADPH added, and to be unaffected by either propylthiouracil or iopanoate. Activation of 5'-deiodinase attainable using B after its partial reduction by various concentrations of NADPH and subsequent alkylation with non-radioactive iodoacetamide was inversely proportional to the previously added concentration of NADPH. Fraction B was stable at 100 degrees C for 5 min, while similar heat treatment of Fraction A or omission of NADPH resulted in a complete loss of 14C incorporation. A greater than 90% reduction in iodo[14C]acetamide incorporation was revealed when 0.2 mM-sodium arsenite was added after enzymic reduction of B, as well as when NADPH was replaced by NADH. Fraction B could be labelled more extensively after reduction non-specifically, with dithiothreitol or NaBH4, but not by GSH. These observations provide strong evidence for the presence in vivo of a cytosolic disulphide (DFBS2) in Fraction B which can be reduced enzymically to a dithiol [DFB(SH)2] by NADPH and cytosolic components in Fraction A. The degree of activation of hepatic 5'-deiodinase correlated with the amount of available (unalkylated) Fraction B.
D000478:Alkylation; D000818:Animals; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D007453:Iodide Peroxidase / Q000378:metabolism*; D007460:Iodoacetamide / Q000494:pharmacology*; D007461:Iodoacetates / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D009249:NADP / Q000494:pharmacology*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D014050:Toluene / Q000031:analogs & derivatives* / Q000378:metabolism
D007461:Iodoacetates; D014050:Toluene; D009249:NADP; D007453:Iodide Peroxidase; C004848:dithiol; D007460:Iodoacetamide