Studies on the protein synthesis in the guinea pig thyroid: II. In vivo labeling of thyroglobulin with 3H-leucine

Abstract

Guinea pigs were given ~H-leucine at different intervals, ranging from 10 minutes to 6 hours, before sacrifice. The thyroids were excised and homogenized, and the homogenate was divided into nuclear, mitochondrial, microsomal, and supernatant fractions by differential centrifugation. The soluble proteins of the supernatant and of the particulate fractions, from the latter released by vigorous rehomogenization of the pellets in buffered saline, were analyzed by centrifugation in a 10-25 % sucrose gradient. About 40 fractions were collected from each gradient, and their optical density at 280 m/~ and radioactivity were determined. Similar analyses were performed on the material solubilized by digi tonin from the particulate fractions after the extraction of soluble proteins with buffered saline. The results show that the 19 S thyroglobulin of the microsomal fraction is labeled before that of the supernatant fraction. At early observation times the microsomal 12 S and 19 S proteins have a higher specific activity than the supernatant 12 S and 19 S, respectively. During the subsequent hours there is a shift in the relation between microsomal and supernatant 12 S and 19 S specific activities. In principle, the same results are obtained from TSHstimulated and nonstimulated thyroids with the exception that in the latter case there is a lag period before the appearance of any labeling of the microsomal 19 S prote in . In the course of the last observation hours the supernatant 12 S labeling decreases while the supernatant 19 S labeling increases. T h e soluble proteins released from the nuclear and mitochondrial fractions show no labeling or only traces of labeling in the 12 S and 19 S regions. Digitonin treatment solubilizes from all particulate fractions labeled components of low sedimentation rate but no labeled 12 S or 19 S proteins. The principal conclusions drawn from these data are: (i) The formation of 12 S protein and 19 S thyroglobulin is microsome associated. No significant labeling of these components occurs in the nuclear or mitochondrial fractions. (ii) The 12 S protein is probably a subunit precursor of 19 S thyroglobulin. (iii) A large proport ion of the labeled substances found in the thyroid after administration of ~H-leucine represents other processes than thyroglobulin synthesis.