Demonstration of membrane-linked iodoprotein in hepatic microsomes following metabolism of the thyroid hormones

The subcellular distribution ot nonextractable ¹²⁵I (NE¹²⁵I) was examined in rat livers 40 hr following the injection of ¹²⁵I-triiodo-L-thyronine (¹²⁵I-T₃). After conventional differential centrifugation of liver homogenates, the distribution of NE¹²⁵I was determined with appropriate corrections for incomplete extraction of residual ¹²⁵I-T₃. NE¹²⁵I occurred in all the subcellular fractions. On this basis of the distribution of glucose-6-phosphatase activity, an additional correction was performed to take into account microsomal NE¹²⁵I trapped in other centrifugal fractions. The final estimate of the distribution of NE¹²⁵I among conventional subcellular fractions was “nuclear” = 3.1%; “mitochondria!” = 2.0%; “microsomal” =70.4%; “supernatant” =24.5%. The submicrosomal distribution of NE¹²⁵I after ¹²⁵I-T₃ (40 hr) and ¹²⁵I-Lthyroxine metabolism (96 hr) was determined by centrifugation of the 105, 000 Xg pellet following treatment with sodium deoxycholate. The major portion of the microsomal NE¹²⁵I was associated with fractions containing solubilized membranes and small and large vesicles. Only a small fraction (0.9-4.2%) of microsomal NE¹²⁵I was associated with relatively pure ribosomes as judged by the RNA/protein ratio, but a decreasing specific NE¹²⁵I activity (% dose/mg RNA or protein) which was found with progressive ribosomal purification suggests membrane contamination. This was documented by the dissociation of the ribosomal peak and NE¹²⁵I after centrifugation of ribosomes in a continuous sucrose density gradient. The comparatively small percentage of microsomal NE¹²⁵I associated with the ribosomal fraction was confirmed in studies conducted 24 and 72 hr after injection of ¹²⁵I-T₃. Thus, microsomal NE¹²⁵I, like iodothyronine deiodinase, appears to be associated predominantly with membrane components. This finding, in conjunction with previous studies which showed increased plasma and membrane iodoprotein formation under the influence of phenobarbital, suggests the possibility that the intracellular iodoprotein is formed as a result of an interaction of intracellular protein or its precursors with a membrane component.