Subcellular Distribution of Thyroxine 5'-Monodeiodinase Activity in Rabbit Kidney

Summary

Thyroxine 5'-monodeiodinase is located in the proximal tubules of the rabbit kidney. To estimate the subcellular distribution of 5'-monodeiodinase activity, we prepared subcellular fractions, a basolateral membrane fraction and a brush border membrane fraction, from kidneys of Japanese white rabbits. Each fraction (0.5 mg protein) was incubated at 37 °C for 60 min with 0.5 μg T₄ in the presence of 5 mM DTT. The T₃ generated in the reaction mixture was extracted with cold ethanol and measured by RIA. For analysis of propylthiouracil-insensitive thyroxine 5'-monodeiodinase, we examined its kinetic behavior at nanomolar concentrations of the substrate, T₄, in the presence of 100 μM propylthiouracil. In order of decreasing activity, basolateral membrane, microsomal fraction, mitochondrial fraction, cytosolic fraction, brush border membrane and nuclear fraction were capable of converting T₄ to T₃. Upon addition of 10_-5 M propylthiouracil to the reaction mixture, 5'-monodeiodinase activities of basolateral membrane and brush border membrane were inhibited by more than 90%, but that of microsomes was inhibited by only about 50%. In addition, kinetic analysis of microsomal 5'-monodeiodinase activity at nanomolar T₄ concentrations in the presence of 10_-4 M propylthiouracil suggested on apparent Km of 3.8 nmol. These results indicate that there is high-Km 5'-monodeiodinase activity (PTU-sensitive) in the basolateral and brush border membranes and also high-Km and low-Km 5'-monodeiodinase (PTU-insensitive) in the microsomes of rabbit kidney.

Key words

Thyroxine - 3,5,3'-Triiodothyronine - Proximal Tubules - Basolateral Membrane - Brush Border Membrane - Microsome