Characterization of Essential Enzyme Sulfhydryl Groups of Thyroxine 5′-Deiodinase from Rat Kidney*

Abstract

T₄ 5′-deiodinase, found in cell membranes of kidney and liver, is a thiol-dependent enzyme inhibited by propylthiouracil (PTU). Pretreatment of enzymatically active kidney membrane preparations (Mx) with PTU (10 μm) and increasing concentrations of L-T₄ under N₂ resulted in increasing degrees of persistent inhibition, as assayed in the absence of pretreatment reagents. Aerobic pretreatment with PTU in the presence or absence of L-T₄ resulted in 80% inhibition of T₄ 5′-deiodination. Sulfhydryl modification of kidney Mx with iodoacetate or Nethylmaleimide irreversibly inactivated T₄ 5′-deiodinase. The formation of a reversible PTU-enzyme complex was found to protect T₄ 5′-deiodination from irreversible inactivation by iodoacetate or N-ethylmaleimide. A dose-dependent increase in the ³⁵S content of kidney Mx was found after injection of [³⁵S]thiouracil (TU) into rats, and the Mx ³⁵S content was reduced 4-fold by simultaneous injection of methimazole. Treatment of kidney Mx, containing bound ;ISS, with 10 mm dithiothreitol released 33% of the bound ³⁵S. Similarly, incubation of a deoxycholate-solubilized active T₄ 5′-deiodinase preparation with [³⁵S]TU, resulted in protein-bound ³⁵S (PB³⁵S), as judged by gel filtration (Sephadex G-25); formation of PB³⁵S was enhanced by L-T₄ and depressed by methimazole. Dithiothreitol treatment of the PB³⁵S fraction released 45% of the bound ³⁵S, which was identified as free thiouxacil. These findings show that 1) substrate utilization as well as aerobic conditions enhance enzyme inhibition by PTU, 2) sulfhydryl modification of Mx preparations results in irreversible inhibition of T₄ 5′-deiodinase and prior formation of a PTU-enzyme complex substantially protects from this inhibition, and 3) thiouracil binds to enzyme preparations, forming an inactive protein-TU mixed disulfide. (Endocrinology106: 444, 1980)