K. J. Catt

Radioimmunoassay of plasma testosterone

Abstract Radioimmunoassay of testosterone in plasma extracts purified by thin layer chromatography has been applied to the development of a highly sensitive and specific method for measurement of plasma testosterone. Antibodies to testosterone coupled to bovine gamma globulin via the 3 position were raised in sheep, and an antiserum of high association constant ( K = 4.5 × 10 9 M −1 ) was employed for assay. Isolation of plasma testosterone prior to assay was performed by thin layer chromatography, and extraction losses were corrected for recovery of tritiated testosterone indicator. For assay, antiserum (1:2500) and tritiated testosterone (40 000 dpm) were incubated at 4° for 16 h, followed by separation of bound and free tracer with 500 μg dextrancharcoal. The sensitivity of the assay was 10 pg, between-assay precision was ± 12% and testosterone recovery was 95–100%. Plasma testosterone levels in individual samples were similar to those obtained by competitive binding assay, and the levels observed in males (709 ± 143; 450–1100 ng/100 ml) and females 54 ± 22; 25–80 ng /100 ml ) were in agreement with those reported by other methods. The combination of radioimmunoassay and thin layer chromatography provides a sensitive and reliable method for the specific determination of testosterone in small plasma samples.

Interaction of glycoprotein hormones with agarose-concanavalin A

Abstract The ability of agarose-coupled concanavalin A to bind carbohydrate and glycoprotein molecules has been applied to chromatography of glycoprotein hormones. Human chorionic gonadotrophin and subunits were bound strongly by Sepharose-concanavalin A, and could be displaced by 0.2 M methyl α- d -glucopyranoside. Desialylated human chorionic gonadotrophin was eluted more slowly than the intact hormone, and asialo-agalacto-human chorionic gonadotrophin was not readily dissociable from combination with concanavalin A. Human luteinizing hormone and follicle stimulating hormone were also adsorbed by Sepharose-concanavalin A columns, and eluted with methyl α- d -glucopyranoside. Prior combination with specific antibody did not prevent 125I-labeled human chorionic gonadotrophin binding to concanavalin A, and subsequent elution with methyl glucopyranoside released the antigen-antibody complex in high yield. Chromatography of human chorionic gonadotrophin on Sepharose-concanavalin A was applied to preparation of 125I-labeled human chorionic gonadotrophin with improved binding affinity for antibody and gonadal receptor sites, and to purification and isolation of human chorionic gonadotrophin from crude urine extracts and human pregnancy plasma. Comparison of plasma and urinary human chorionic gonadotrophin revealed an elution profile consistent with partial desialylation of the urinary hormone. These experiments have shown that the group-specific interactions of glycoprotein hormones with agarose-concanavalin A are of potential value for the purification and isolation of such hormones and their derivatives, and for the preparation of radioiodinated gonadotrophins of improved binding characteristics.

MEDIATION OF CYCLIC AMP SIGNALING BY THE FIRST INTRACELLULAR LOOP OF THE GONADOTROPIN-RELEASING HORMONE RECEPTOR

The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway.

Characterization of angiotensin II receptors in the anterior pituitary gland

Specific and high affinity binding sites for angiotensin II were demonstrated in the anterior pituitary gland by binding studies with [125I] iodoangiotensin II. The binding properties of the pituitary receptors were similar to those of angiotensin II receptors present in the adrenal gland. The concentration of binding sites in rat anterior pituitary (293 +/- 50 fmoles/mg protein) was less than in the adrenal gland, but was much greater than in smooth muscle. Angiotensin II receptors were identified in the anterior pituitary tissue of mature and immature animals of both sexes, and in species including rat, rabbit and dog. No binding of angiotensin II was detected in posterior pituitary homogenates, or in GH3 pituitary tumor cells. Collagenase-dispersed anterior pituitary cells also contained specific binding sites for angiotensin II, with equilibrium binding constant (Ka) of 3.6 x 10(9) M-1. The presence of specific high-affinity angiotensin II receptor in the anterior pituitary gland provides a mechanism by which angiotensin-like peptides could modulate the process of pituitary hormone secretion.

Gonadotrophin binding sites of the rat testis

Abstract Gonadotrophin binding sites with high affinity and specificity for human chorionic gonadotrophin and luteinizing hormone have been demonstrated in rat testis homogenates and subcellular fractions. The binding of 125I-labeled chorionic gonadotrophin to the rat testis homogenate is temperature dependent, with limited capacity (10−12 mole/g) and high affinity (Ka = 2.4·1010 M−1) for chorionic gonadothrophin at 24°C. Gonadotrophin binding is not significantly affected by variations in calcium concentration within the physiological range, and is associated with membrane fragments during differential centrifugation of testis homogenates labeled with 125I-chorionic gonadotrophin. These properties of the chorionic gonadotrophin/luteinizing hormone binding sites present in rat testis homogenate are consistent with those of a specific hormone receptor for gonadotrophins which activate testicular steroidogenesis.

Gonadotroping binding and activation of the interstitial cells of the testis.

Tissue receptor sites with high affinity for luteinizing hormone (LH) and human chorionic gonadotropin (hCG) have been defined in the interstitial cells of the testis, and also in the normal and gonadotropin-luteinized rat ovary (1–8). Comparable binding sites have also been demonstrated in the bovine corpus luteum, and in porcine granulosa cells during maturation in vivo and in vitro (9). The common receptor site for LH and hCG in these tissues is highly specific, with binding affinity only for molecules with the conformation characteristics of LH and chorionic gonadotropin. The biological specificity of hormone binding is demonstrable with LH from a wide variety of species, and with the placental gonadotropins of man, primates and the horse (2). The properties of the testis LH/hCG receptors, and their applications to radioligand-receptor assay, have been previously described in detail (2). In this chapter, the characteristics and application of gonadotropin receptors will be briefly reviewed, and more recent studies on the analysis of hormone-receptor interactions and the target cell responses to gonadotropins will be described.

Properties of detergent-solubilized adenylate cyclase and gonadotropin receptors of testis and ovary

The relationship between solubilized hormone-binding sites and adenylate cyclase was examined in detergent extracts of particulate testis and ovarian fractions. Both basal and fluoride-stimulated activities of the particulate enzyme were markedly increased in the presence of detergents, and about 60% of the enzyme activity was recovered as the soluble form in the 300,000 g supernatant. Enhancement of adenylate cyclase activity was more marked with Lubrol PX and WX than with Triton X-100, and the highest recovery and activation of adenylate cyclase were obtained with 0.5% Lubrol PX. The particulate and solubilized testicular enzymes were more active in the presence of Mn2+, and the detergent-extracted soluble ovarian cyclase showed a small and inconstant response to gonadotropin. Fractionation of Lubrol-solubilized testis and ovarian preparations on Sepharose 6B showed two peaks of free gonadotropin receptors. The binding activity eluted with Kav of 0.32 corresponded to the receptor sites previously characterized in detergent-solubilized gonadal particles, and was coincident with the elution profile of adenylate cyclase activity. An additional peak of binding activity with Kav of 0.56 was not accompanied by detectable adenylate cyclase activity. These observations suggest that the peak of larger molecular size could represent dissociated receptors or binding sites which were not coupled to adenylate cyclase.

Development of heterologous down-regulation of lactogen receptors in the rat testis

Gonadotropin-induced loss (down-regulation) of testicular lactogen receptors was studied in 5-60-day-old rats. An i.m. injection of 600 IU/kg of hCG elicited in 5-day-old animals a 38-56% increase in testicular lactogen binding, measurable between 6 and 72 h after the hormone injection. In contrast, at the age of 60 days the same hCG dose decreased lactogen receptors by 75% at 24 h, which loss recovered in 2-3 days. When the amount of lactogen receptors was measured 24 h after the hCG injection (600 IU/kg) at different ages, the binding increased on average by 50% between ages 5 and 20 days, no effect on binding was seen at day 30, and a loss of binding by 50% was evident in animals of 40 days of age and older. The present results indicate that the plasma-membrane events elicited by gonadotropin binding to neonatal Leydig cells are clearly different from those occurring in the adult, and that down-regulation and functional coupling of LH and lactogen receptors are accompaniments of functional differentiation of the Leydig cell during pubertal maturation.

Computer analysis of the binding reaction between hCG and gonadotropin receptors of the rat testis.

The presence of specific high-affinity binding sites for luteinizng hormone (LH) and human chorionic gonadotropin (hCG) has been established in the Leydig cells of the rat testis (1–3). The binding of hCG to these receptors has been demonstrated to be a saturable process, and the velocity of binding shows marked temperature dependence (3). Analysis of gonadotropin binding data by Scatchard plots previously indicated the presence of a single class of binding sites (3). To define more accurately the association constant and the rate constants of this hormone-receptor interaction, more detailed equilibrium and kinetic experiments were performed, and the data analyzed with an interactive non-linear curve fitting program. This analytical system (MLAB) has been previously described (4, 5), and runs on a PDP-10 digital time-sharing computer. This approach has the advantage of permitting the use of complex models while performing simultaneous fits to multiple sets of data, without a significant increase in the complexity of the computation procedure. In particular, data expressing the total bound hormone and the non-specifically bound hormone as functions of the initial hormone concentration could be analyzed simultaneously, with computation of specific binding from the results of the simultaneous fitting process.

Prolonged retention of high specific activity by 125I-labeled angiotensin II — a consequence of ‘decay catastrophe’

Abstract Angiotensin II labeled with a single atom of 125 I per moleculed had been shown to retain 80% of the original specific activity of the radioiodinated peptide during storage for up to 5 months. This phenomenon is attributable to destruction of the angiotensin II molecule during the radioactive decay process, so that the immunoreactive peptide effectively desappears at the same rate as the incorporated 125 I atom. For this reason, monoiodinated angiotensin II can be employed for prolonged periods in radioimmunoassay and binding studies, with retention of high specific activity in the absence of formation of free iodide or interfering peptide fragments.