Cell-free translation of the messenger RNA coding for the alpha subunit of thyroid-stimulating hormone.

Abstract

SUMMARY Polyadenylated RNA prepared from a murine pitui- tary tumor that produces thyroid-stimulating hormone (TSH) was translated in membrane-free heterologous cell-free systems derived from wheat germ, reticulo- cytes, and Krebs ascites cells. By electrophoretic anal- yses, the predominant labeled product had an apparent molecular weight (&fr) of 14,000. Several tryptic pep- tides of this major translation product matched those obtained from the authentic, secreted (Y subunit of TSH. The protein of M, = 14,000 is the product of the trans- lation of the mRNA coding for the (Y subunit of TSH. In addition, a second protein of M, = 17,000 was detected in the wheat germ system. Both the M, = 14,000 and M, = 17,000 translation products are larger by approxi- mately 3,000 to 4,000 daltons than are the putative core unglycosylated (Y (Mr = 10,600) and p (Mr = 13,000) subunits of TSH, respectively, but are smaller than those of the glycosylated native TSH-cu subunits se- creted by intact tumor cells in vitro (apparent M, = 22,000) and TSH-P (apparent M, = 18,000). The effects of magnesium and potassium on the synthesis of these proteins in the wheat germ system were examined. Increasing concentrations of magnesium (1.7 to 3.7 mM) resulted in a 6-fold decrease in the amount of (Y product and a a-fold increase in the amount of M, = 17,000 product. Increasing concentrations of potassium (40 to 115 mM) had little effect on the relative amounts of the M, = 14,000 (a product) and M, = 17,000 products at low magnesium concentration [1.7 mM], but stimulated the synthesis of both products at high magnesium concen- tration [3.2 mM]. The results suggest that the product of the translation of the mRNA coding for the (Y subunit is probably a precursor form of the subunit with an additional sequence of approximately M, = 3,000, sim- ilar to the precursor forms found in the analogous translation products for other protein hormones. It is likely that the (Y and fl subunits of TSH are coded for by