We have examined the influence of assay conditions on the 
6-n-propyl-2-thiouracil (PTU) sensitivity of the iodothyronine 5'-deiodinase in 
brown adipose tissue (BAT) from hypothyroid rats. These results were compared 
with similar studies of 5'-deiodinase activity in kidney microsomes from 
euthyroid animals. Even though BAT microsomes contain largely type II 
(PTU-insensitive) deiodinase activity, the 5'-deiodination of T4 can be 
inhibited by PTU if the dithiothreitol (DTT) concentration in the assay is 
reduced to 5 mM or less. The apparent Ki for PTU of BAT microsomes was 4.3 mM at 
5.0 mM DTT and 0.41 mM at 0.5 mM DTT. The kinetics of inhibition were 
noncompetitive. With kidney microsomes, PTU inhibition of rT3 5'-deiodination 
was both time and enzyme/substrate ratio dependent. For example, using 1 
microgram microsomal protein, 2 nM rT3, and 5 mM DTT, the inhibitory effect of 
PTU was not maximal until 12 min after PTU addition. At stable reaction 
velocities PTU inhibition was uncompetitive, and the Ki was about 1 microM. 
Deiodination by kidney microsomes was completely inhibited by 50 microM PTU. 
Even though it is possible to inhibit the type II 5'-deiodinase activity with 
high concentrations of PTU (in the presence of low DTT concentrations), the 
deiodinase in kidney is about 1000-fold more sensitive to PTU. By these criteria 
the kidney microsome 5'-deiodinase is type I.