Incorporation of [1-14c]galactose, [1-14c]mannose and [4,5-3h2]leucine into Soluble Proteins by Rat Thyroids in Vitro

1. Rat thyroid lobes were incubated for various periods of time in Krebs-Ringer bicarbonate containing [3H]leucine and either [1-14C]galactose or [1-14C]mannose. Radioactivity in soluble proteins was determined after their separation by sucrosegradient centrifugation. 2. The time-course of incorporation of label from [14C]mannose into soluble thyroid proteins was parallel to that observed for [3H]leucine. There was a lag of at least 30min. before either label appeared in non-iodinated thyroglobulin (protein 17-18s). During this time both labels were detected in two fractions known to contain subunit precursors of thyroglobulin (fractions 12s and 3-8s). Radioactivity from double-labelled fractions 12s and 3-8s was transferred to protein 17-18s during subsequent incubation in an unlabelled medium. 3. In contrast, most ofthe [14C]galactose was immediately incorporated into protein 17-18s. 4. During the first hour of incubation, puromycin almost completely inhibited the incorporation of label from [3H]leucine and [14C]mannose into all protein fractions, but had little effect on the incorporation of [14C]galactose into protein 17-18s. 5. These results indicate that mannose is incorporated into the carbohydrate groups of protein 17-18s at an earlier stage in its formation than galactose. It is suggested that the synthesis of the carbohydrate groups of thyroglobulin begins soon after formation of the polypeptide components, more than 30min. before these are aggregated to protein 17-18s; carbohydrate synthesis then proceeds in a stepwise manner, galactose being incorporated at about the time of aggregation of subunits to protein 17-18s. Most, if not all, the carbohydrate is added to thyroglobulin before it is iodinated.