Studies on protein synthesis in the guinea pig thyroid: I. In vivo incorporation of H 3-leucine into the proteins of subcellular fractions

Abstract

Male guinea pigs, stimulated with TSH 24 hours before sacrifice, were given H 3 -leucine by intracardiac injection, and the in vivo incorporation of the leucine into the proteins of thyroid subcellular fractions was followed over a period of 3 hours. In the main series of experiments the thyroid homogenate was divided into nuclear, mitochondrial, microsomal, and supernatant fractions by a relatively simple differential centrifugation technique. In a second series of experiments a mitochondrial fraction of high purity and a fraction mainly consisting of ribosome-studded vesicular formations were produced by centrifugation in a sucrose density gradient. A third series of experiments comprised the isolation of a purified nuclear fraction. The proteins of the fractions were determined after trichloroacetic acid precipitation. The radioactivity of the proteins and of the acid-soluble supernatants was determined in a liquid scintillation spectrometer. In some experiments the leucine of the deproteinized supernatant was isolated by column chromatography in order to estimate the share of free H 3 -leucine in the acid-soluble radioactivity. In the main experimental series the specific activity of the microsomal proteins rose more rapidly and reached higher values than that of any other fraction. The specific activity of the supernatant fraction rose more slowly than that of the other fractions, but, in contrast to the specific activity of the particulate fractions, it continued to increase throughout the observation period. Purification of the nuclear fraction resulted in a considerable decrease of its specific activity. The purified mitochondrial fraction showed a higher specific activity than the standard mitochondrial fraction during the first observation hour. The fraction consisting of ribosome-studded vesicular structures reached a higher specific activity than the microsomal fraction of the main series. A tentative interpretation of the labeling pattern is given on the basis of the morphology of the fractions.