Photoaffinity labeling of rat type I iodothyronine deiodinase

The photoreactive compound p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PAL) was coupled to [125I]rT3, T4, or T3 and incubated with liver and kidney microsomes of hypo-, hyper-, or euthyroid rats to identify the type I iodothyronine deiodinase. Various substrates or inhibitors of the enzyme, including rT3, T4, T3, 6-n-propylthiouracil (PTU), and iopanoic acid, were used as competitors to establish the specificity of protein labeling. The PAL derivatization enhanced the behavior of T4 and T3 as substrates for the type I enzyme. No specific labeling of microsomal proteins was observed with either rT3 or T4-PAL, presumably due to deiodination of the labeled compound. In contrast, T3-PAL labeled a 27-kDa band, the presence of which paralleled thyroid status. The labeling of only this protein was blocked by either substrates or enzyme inhibitors in a dose-dependent fashion, with a rank order of potency predicted by the activity of such compounds in type I enzyme assays. The specific nature of these competitions provides further evidence that this 27-kDa protein, identified in previous studies using N-bromoacetyl [125I]T3 or -T4, contains the active site of the rat type I deiodinase. This is in agreement with the mol wt of the rat type I deiodinase deduced from the recently identified cDNA coding for this protein.