Isolation of mRNA from Bovine Pituitary. The Cell-Free Synthesis of the alpha and beta Subunits of Luteinizing Hormone

Abstract

RNA derived from bovine steer pituitary was translated in wheat germ cell-free extracts containing [35S]methionine. Antisera generated against purified denatured a and fi subunits of lutropin were used to demonstrate the synthesis of both proteins in vitro. The immunoprecipitated products of the cell-free system were resolved on sodium dodecyl sulfate/polyacrylamide gels and it was observed that the molecular weight of the immunoprecipitated a subunit protein was approximately 14000, while that of the p protein was estimated to be 16000. Since the molecular weights of authentic a and p subunits are 10600 and 14000 respectively, the cell-free products presumably represented their pre-protein forms.The ratio of the immunoprecipitated subunit pre-proteins was dependent on the magnesium concentration in the translation mixtures; at 2.1 mM, translation of lutropin a and p mRNAs was comparable. RNA isolated from cow pituitary tissue directed the synthesis of fivefold less of the a and immunoprecipitated proteins than did steer RNA. Since the blood levels of gonadal steroids are higher in the cow, the results supported the hypothesis that lutropin a and fl mRNA biosynthesis is repressed by these steroids. The data also suggest that synthesis of lutropin a and p subunits is coordinately expressed in certain physiological situations.The adenohypophysis is the source of a variety of polypeptide hormones, each possessing a unique biological function. It exerts a major influence on the control of gonadal function and reproduction in most mammalian species through its secretion of the glycoprotein hormones follitropin and lutropin. The pituitary elaborates an additional glycoprotein hormone, thyrotropin, which controls the biosynthesis of thyroxine. The elaboration of gonadotropins and thyrotropin is controlled by their respective releasing hormones produced in the hypothalamus, and, in addition, gonadotropin release is under gonadal control via sex steroid hormones.The pituitary glycoprotein hormones and human choriogonadotropin are composed of two non-identical glycosylated subunits, designated a and p. The amino acid sequence of all the a subunits is nearly identical [I -81, and it is apparent that the structural determinants for the biological specificity of these hormones reside in the sequence of the corresponding

Regulation of rat luteinizing hormone subunit messenger ribonucleic acids by gonadal steroid hormones.

“…In addition, cell-free translation studies have shown that both pituitary a-subunit (17) and LH,3-subunit (21) mRNA activity levels are lower in gonadal-steroid hormonetreated OVX ewes than in untreated GDX controls (7). Using (16)(17)(18)(19). This hypothesis was based upon the repeated observation that serum and intracellular levels of pituitary and placental free a-subunit exceed the serum and intracellular levels of intact glycoprotein hormones (14,15,40,41).…”

“…The excess production of a-subunits over LHf-subunits (14,15) has suggested that the rate of LHfl-subunit synthesis determines the amount of intact LH produced (16)(17)(18)(19). Several studies at the pretranslational level using cell-free translational analyses have indicated that the levels of a-and LHfl-subunit messenger RNAs (mRNAs) are elevated in castrate animals (17)(18)(19)(20), and that they decrease with gonadal steroid replacement (7,9,21).…”

“…The excess production of a-subunits over LHf-subunits (14,15) has suggested that the rate of LHfl-subunit synthesis determines the amount of intact LH produced (16)(17)(18)(19). Several studies at the pretranslational level using cell-free translational analyses have indicated that the levels of a-and LHfl-subunit messenger RNAs (mRNAs) are elevated in castrate animals (17)(18)(19)(20), and that they decrease with gonadal steroid replacement (7,9,21). In a detailed study examining the time course of the gonadectomy-induced increase in translatable a, LHfl, and follicle stimulating hormone-f precursors, Corbani et al (20) found that maximum levels of subunit precursors were reached -3 wk after castration, but that significant increases were noted as early as 1 wk after castration.…”

Cell‐free translation of the rat pituitary messenger RNA coding for the precursors of α and β subunits of lutropin

“…17000 was found. Asomewhat loweri21, (16000-18 000) than that determined by us for pre-rLqO has been determined by others for bovine pre-Lt1/3 [9,24].…”

“…An app. M, 14 000 has been reported for bovine pre-LHa [8,24] and for pre-TSHa synthesized by mRNA from mouse thyrotropic tumor [25,26]. However, comparing prea subunits synthesized in cell-free conditions by RNA from bovine pituitaries and from mouse pituitary tumor 1271, in both cases IV,.…”