125I-labeled Hormone Research Articles

Photoaffinity labeling of the follitropin receptor.

A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the 125I-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the 125I-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed.

Specific non-saturable binding of insulin by human platelets

Studies on the nature and characterization of the specific binding of 125I-insulin to intact human platelets have been undertaken. Although under conditions of physiological buffer osmolality, a binding equilibrium was attained in 4–6 h at 4°C, at higher temperatures (17°C or 24°C) equilibrium was reached only in the presence of very high buffer osmolality or 25 mM NaF. Under conditions of normal osmolality and in the absence of NaF, binding at 17°C was not saturable. This phenomenon was specific for insulin or insulin-like hormones (the insulin-like growth factors) and did not occur with 125I-labelled growth hormone, ACTH or β-endorphin. The non-saturable uptake of insulin appeared due to an energy-dependent specific sequestration or internalization of insulin by mechanisms probably involving the microtubule system. This study emphasizes the need to restrict this non-saturable uptake of insulin if one wishes to adequately study the platelet plasma membrane receptors for insulin. These data also indicate that there is a major interaction of insulin and insulin-like hormones with normal human platelets and support previous demonstrations of major insulin effects on platelet physiology in both normal and diabetic states.

Insulin, via Its Own Receptor, Regulates Growth and Amylase Synthesis in Pancreatic Acinar AR42J Cells

Previous in vivo studies have suggested a long-term regulatory role for insulin in the exocrine pancreas. To directly study the long-term effects of insulin on the pancreas in vitro, we have used cultured AR42J cells, a rat cell line that is derived from a transplantable tumor of the acinar pancreas. Hormone-binding experiments with 125I-labeled hormones indicated that AR42J cells have insulin receptors, relatively fewer receptors for insulin-like growth factor II (IGF-II), and no detectable receptors for insulin-like growth factor I (IGF-I). Insulin at concentrations as low as 1 nM stimulated the growth of these cells, as measured by an increase in DNA and protein content, and in cell number. At 100 nM, where insulin had a maximal effect, the growth of AR42J cells was stimulated by 46.1 +/- 10.9% (mean +/- SEM, N = 11). Insulin increased the amylase activity of AR42J cells over the same concentration range that it stimulated growth; at 100 nM, insulin increased amylase by 91.0 +/- 15.4% (mean +/- SEM, N = 23). Immunoprecipitation of [35S]methionine-labeled proteins revealed that insulin induced a selective increase of amylase synthesis over general protein synthesis. These studies indicate, therefore, that insulin stimulates both growth and amylase synthesis of AR42J cells.

Characterization of the receptors for insulin and the insulin-like growth factors on micro- and macrovascular tissues.

Insulin and insulin-like growth factors (IGFs) have been implicated in the pathogenesis of diabetic retinopathy and peripheral vascular complications. Previously, we have shown that retinal capillary endothelial cells responded to insulin and IGFs for metabolic and growth effects, whereas aortic endothelial cells were not responsive. In contrast, vascular supporting cells from both retinal capillaries (i.e. pericytes) and aorta (i.e. smooth muscle cells) responded equally to insulin, IGF-I, and IGF-II. The structure and ligand specificities of the receptor for these peptides were studied by covalently cross-linking 125I-labeled peptide hormones to their respective receptors using disuccinimidyl suberate, followed by polyacrylamide gel electrophoresis and autoradiography. The binding subunit of the insulin receptor, alpha-subunit, for all cell types was found to have a mol wt 145,000 under reduced conditions. Labeling of this band was inhibited by 10(-9) M insulin, antiinsulin receptor antibodies, and 10(-8) M IGF-I, but not by multiplication-stimulating activity (IGF-II). The beta-subunit of the insulin receptor in endothelial cells was identified by its ability to be autophosphorylated when stimulated by insulin and was found to have a mol wt of 99,000. Covalent cross-linking of IGF-I to its receptor revealed a mol wt of 145,000, similar to that of insulin receptor, except that IGF-I was 100-fold more potent than insulin in competing with [125I]IGF-I for binding. [125I]IGF-II in all cells was cross-linked to receptor with mol wt of 260,000 and 230,000 under reduced and nonreduced conditions, respectively. IGF-I competed weakly with [125I]IGF-II, whereas insulin was ineffective. [125I]IGF-II also bound to the band with alpha mol wt of 135,000, which was inhibited by insulin, IGF-I, and IGF-II. In summary, receptors for insulin, IGF-I, and IGF-II on cells from micro- and macrovessels are biochemically similar to those in other cells. Interestingly, the finding of large numbers of IGF-I and IGF-II receptors on endothelial cells suggests that these growth factors play a physiological role and are involved in vascular complications associated with diabetes.

Differences in the behavior of luteinizing hormones of various species at the rat gonadal cell receptor site.

The ability of different LH-like hormones, such as hCG, PMSG/equine (e) CG, ovine (o) LH, eLH, and rat (r) LH, to bind to and stimulate steroidogenesis in two types of rat gonadal cells was studied under the same experimental conditions. In both Leydig and granulosa cells, the maximal steroidogenic responses elicited by optimal doses of different LHs present during a 2-h incubation were comparable. However, if the cells were exposed to the different LHs for a brief period and then subjected to interference with hormone action by removing the unbound hormone from the medium by washing or adding specific antisera, differences were observed in the amount of steroid produced during subsequent incubation in hormone-free medium. Thus, in the case of hCG, either of these procedures carried out at 15 or 30 min of incubation had little inhibitory effect on the amount of steroid produced at 2 h, the latter being similar to that produced by cells incubated in the continued presence of hCG for 2 h. With eCG and rLH, the effect was dramatic, in that there was a total inhibition of subsequent steroidogenic response. In cells exposed to eLH and oLH, inhibition of subsequent steroidogenesis due to either removal of the free-hormone or addition of specific antisera at 15 or 30 min was only partial. Although all of the antisera used were equally effective in inhibiting the steroidogenic response to respective gonadotropins when added along with hormones at the beginning of incubation, differences were observed in the degree of inhibition of this response when the same antisera were added at later times of incubation. Thus, when antisera were added 60 min after the hormone, the inhibition of steroidogenesis was total (100%) for eCG, partial (10-40%) for eLH and oLH, and totally lacking in cells treated with hCG. From this, it appears that hCG bound to the receptor probably becomes unavailable for binding to its antibody with time, while in the case of eCG and other LHs used, the antibody can still inhibit the biological activity of the hormone. Studies with 125I-labeled hormones further supported the conclusion that hCG differs from all other LHs in being most tightly bound and, hence, least dissociable, while eCG and rLH dissociate most readily; oLH and eLH can be placed in between these hormones in the extent of their dissociability.

Luteinizing hormone receptor binding inhibitor in aqueous extract of human corpus luteum.

Aqueous extracts of human luteal tissue significantly inhibited binding of 125I-labelled human luteinizing hormone (LH) to the 2,000-g subcellular fraction of human corpora lutea. In contrast, aqueous extract of nonluteal tissue of the human ovary did not show a comparable activity to inhibit LH binding. Extracts of human corpus luteum had little or no ability to inhibit LH binding to porcine luteal homogenates. The inhibitory effect of the aqueous extract on LH binding to human luteal homogenate was demonstrated to be dose-related. The inhibitory effect of aqueous extracts of human corpora lutea on LH binding increased from the early to mid and late luteal phase of the menstrual cycle. The results of the present study suggest that there is an LH receptor binding inhibitor (LHRBI) in the 30,000-g aqueous extract of the human corpus luteum and the increase in LHRBI in the luteal tissue as the human corpus luteum ages may explain the means whereby the human corpus luteum regulates its own life span.

Homologous somatotropin radioreceptor assay utilizing recombinant bovine growth hormone

A homologous radioreceptor assay using recombinant bovine growth hormone and bovine liver membranes is described. The total specific binding of 125I-labeled recombinant bovine growth hormone to the 100000 × g pellet was 48% in 24 h at 25 °C. Hormone binding was partially reversible, with 40% of the radiolabeled hormone being irreversibly bound. The amount of specific binding varied with assay pH, with the optimum occurring at pH 7.8. Specific binding was temperature-dependent, with greater specific binding occurring at 25°C than at 5°C or 37°C during a 24 h period. Recombinant bovine growth hormone, human growth hormone, ovine growth hormone and recombinant porcine growth hormone competed effectively with 125I-labeled recombinant bovine growth hormone for binding sites, while bovine prolactin and ovine prolactin were needed in amounts 10 6-fold the concentration of recombinant bovine growth hormone to displace the radiolabeled hormone. Surprisingly, human placental lactogen did not displace the radiolabeled hormone.

Low serum somatomedin-C in protein deficiency: relationship with changes in liver somatogenic and lactogenic binding sites

We have investigated whether the low serum levels of somatomedin-C (SM-C) observed in protein malnutrition could be related to changes in liver growth hormone and prolactin binding. Growing female rats were fasted for 3 days and subsequently refed for 14 days with isocaloric diets containing 5% (low) or 25% (normal) protein. Control rats were fed a normal protein diet during the entire study. The numbers and affinity constants of somatogenic (GH) and lactogenic (PRL) binding sites were determined by analysis of saturation curves using liver homogenates incubated with 125I-labelled bovine growth hormone and 125I-labelled ovine prolactin. Serum SM-C and growth hormone concentrations were measured by radioimmunoassay. After fasting for 3 days body weight dropped by 21% ( P < 0.01 vs. controls) and serum SM-C by 53% ( P < 0.01), while GH and PRL binding capacities decreased respectively by 63% ( P < 0.01) and 62% ( P < 0.01). On refeeding with a normal protein diet, body weight, serum SM-C, GH and PRL binding capacities returned to control values. In contrast, with low protein intake, body weight, SM-C, GH and PRL binding capacities remained respectively 16%, 55%, 49% and 81% lower than controls ( P < 0.01). No significant changes in serum growth hormone concentrations occurred with fasting or refeeding. Only in the fasted rats were the affinity constants of GH and PRL binding sites significantly ( P < 0.01) lower ( GH 0.63 ± 0.06 × 10 9 M −1 vs. 0.86 ± 0.04 × 10 9 M −1; PRL 0.90 ± 0.04 × 10 9 M −1 vs. 1.34 ± 0.07 × 10 9 M −1 ). These findings indicate that adequate protein intake is an essential regulator of liver somatogenic and lactogenic binding sites. In addition, the data suggest that the low serum SM-C levels in protein deficiency might be due to the reduction in hepatic GH and PRL binding capacities and that this reduction may, in turn, be responsible for the liver insensitivity to growth hormone.

Specific receptors for epidermal growth factor in human bone tumour cells and its effect on synthesis of prostaglandin E2 by cultured osteosarcoma cell line.

Using tumour cell lines derived from human bone tumours, specific binding sites for epidermal growth factor (EGF), a potent growth stimulator in many tissues, and its effect on synthesis of prostaglandin (PG) E2, a potent bone-resorbing factor, by cultured osteosarcoma cell line were studied. Three tumour cell lines, one osteosarcoma (HOSO) and two giant cell tumours of the bone (G-1 and G-2), all possessed specific binding sites for 125I-labelled EGF: the apparent dissociation constant was approximately 4-10 X 10(-10) M and the maximal binding capacity was 50 000-80 000 sites/cell. EGF had no mitogenic effect in these cell lines. However, these cell lines did not have specific binding sites for 125I-labelled parathyroid hormone (PTH) or calcitonin. HOSO line produced and secreted PGE2 into medium, while no significant amount of PGE2 was demonstrated in G-1 or G-2 line. EGF significantly stimulated PGE2 production in HOSO line in a dose-dependent manner (0.5-50 ng/ml); its stimulatory effect was completely abolished by indomethacin, an inhibitor of PG biosynthesis. Exogenous PGE1 significantly stimulated cyclic AMP formation in HOSO line, whereas PGF2 alpha, PTH, calcitonin, or EGF had no effect. None of these calcium-regulating hormones affected cyclic AMP generation in either G-1 or G-2 line. These data indicate that human bone tumour cells have specific EGF receptors unrelated to cell growth, and suggest that EGF may be involved in bone resorption through a PGE2-mediated process in human osseous tissues.

Interaction of epidermal growth factor with receptors on human and porcine thyroid membranes

Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1.4 X 10(9) l/mol); the density of binding sites was low compared with the TSH receptor. At 37 degrees C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0.5 nmol/l) using 0.5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity.

In vitro modulation of gh secretion from human fetal pituitaries (HFPS) by growth hormone releasing factor (GRF) and somatostatin (SRIF)

The effects of fasting and refeeding on hepatic growth hormone receptors, on insulin receptors and on plasma somatomedin activity were studied. Female rats were either subjected to fasting for 4 days, refed for 3 days after a 4-day fasting, or allowed free access to food (controls). The specific binding of 125I-labelled bovine growth hormone was low in liver microsomal membranes (45% that of controls) and in plasma membranes (52% that of controls) of fasted rats. The number of somatotropic sites rather than the affinity of the binding was decreased. Lactogenic sites as judged by the binding of 125I-labelled human growth hormone were not significantly reduced in liver membranes of fasted rats. 125I-labelled insulin specific binding was enhanced in microsomal (184% that of controls) and plasma membranes (136% that of controls) of fasted rats; these modifications were associated with a decreased insulinaemia. But immunoreactive rat growth hormone levels were not different in plasma of fasted, refed and control animals. Decreased plasma bioassayable somatomedin was associated with the low number of somatotropic binding sites in liver membranes of fasted rats. Somatomedin activity of refed animals was comparable to controls. A significant correlation between the plasma bioassayable somatomedin and the hepatic level of somatotropic binding sites was found. It is proposed that, in fasting, the loss of somatotropic binding sites in the liver is one of the possible causes of the decreased plasma somatomedin bioactivity.

Structures of the somatotropin receptor and prolactin receptor on rat hepatocytes characterized by affinity labelling.

Human somatotropin competed for 125I-human somatotropin binding to hepatocytes from female or male rats. Bovine somatotropin and prolactin each inhibited part, but not all, of the uptake of 125I-human somatotropin. The binding of 125I-prolactin was inhibited by human somatotropin and prolactin, but not by bovine somatotropin. Bovine somatotropin and human somatotropin, but not prolactin, competed for 125I-bovine somatotropin binding sites. 125I-labelled hormones were covalently coupled to membrane receptors with higher efficiency on hepatocytes from female than from male rats, allowing structural descriptions of lactogenic and somatogenic binding sites that had not been possible previously. Disuccinimidyl suberate covalently coupled 125I-human somatotropin into saturable complexes of Mr 300 000, 220 000, 130 000, 65 000 and 50 000. Bovine somatotropin inhibited the incorporation of 125I-human somatotropin into complexes of Mr 300 000, 220 000 and 130 000, whereas low concentrations of prolactin competed for incorporation into the 65 000- and 50 000-Mr species. 125I-bovine somatotropin was incorporated into complexes of Mr 300 000, 220 000 and 130 000. Human somatotropin and bovine somatotropin, but not prolactin, inhibited the production of these complexes. 125I-prolactin binding produced complexes of Mr 65 000 and 50 000. Native prolactin and human somatotropin, but not bovine somatotropin, inhibited uptake of 125I-prolactin into these species. Thus direct affinity labelling, as well as competition for covalent coupling, suggests that the 300 000-, 220 000- and 130 000-Mr species are components of the somatotropin receptor and that the 65 000- and 50 000-Mr complexes result from hormone binding to the prolactin receptor. By subtracting the Mr of prolactin, it was calculated that the hormone was bound to species of Mr 43 000 and 28 000. These Mr values were not affected by reduction of solubilized membranes, suggesting that the structure of the prolactin receptor is not stabilized by interchain disulphide bonds between subunits. Subtracting the Mr of somatotropin from somatogenic complexes indicated that the hormone had bound to species of Mr 280 000, 200 000 and 100 000. The 300 000- and 220 000-Mr complexes were not isolated from reduced membranes, whereas the amount of the 130 000-Mr species was augmented. These observations could suggest that a major component of the somatotropin receptor is a trimeric aggregate in which some subunits are retained in a larger complex by interchain disulphide bonds.

Characterization of a monoclonal antibody to human sex hormone binding globulin

We have produced a monoclonal hybridoma cell line (S 1B 5) that secretes an IgG 2α immunoglobulin with a high affinity ( K d 0.38 × 10 −11 M) for 125I-labelled sex hormone binding globulin (SHBG), and which will specifically immunoprecipitate SHBG from serum. The antibody is produced in high titre in culture medium and ascites fluid, and can be purified to apparent homogeneity by protein A affinity chromatography. When examined by isoelectrofocussing, a characteristic series of bands, which bind 125I-SHBG, are observed at pH 8.5–9. Competitive, superimposable displacement of 125I-SHBG from the antibody is achieved with dilutions of human, chimpanzee and gorilla sera at comparable SHBG concentrations. Partial cross-reaction is observed with sera from other Old World primates, but not with sera from New World monkeys or any other vertebrate species studied.

Peripheral metabolism of parathyroid hormone in patients with primary hyperparathyroidism as judged by immunological and biological studies.

The present study characterizes the immunological and biological activity of circulating forms of parathyroid hormone (PTH) in patients with primary hyperparathyroidism. In addition, the rate of elimination of intravenously injected 125I-labelled bovine parathyroid hormone (125I-bPTH) was studied in patients with this disease before and after operation. The different molecular forms of serum PTH were characterized by gel chromatography followed by radioimmunoassay employing two antisera with specificities directed against the N-terminal and mid-region part of the peptide, respectively. The major part of immunoreactive PTH (iPTH; on the average above 50%) eluted corresponding to fragments with a molecular size about 7,500 daltons in both radioimmunoassays. Specific immunoreactivity coeluting with the intact hormone represented 9-15%. The biological activity of hyperparathyroid serum after gel chromatography was tested in a hormone-sensitive rat kidney adenylyl cyclase assay system. The basal and PTH-stimulated adenylyl cyclase activity (half-maximal) stimulation at 5 micrograms/l or 0.6 nM) was dependent on Mg2+ and ATP. Maximal responses to PTH, calcitonin, and prostaglandin E2 were 50-200% above basal activity and were obtained in the presence of both GTP and Gpp(NH)p (5 X 10(-4) M). Serum from patients with hyperparathyroidism and PTH extracted from parathyroid tissue stimulated the adenylyl cyclase in a dose-dependent manner, as did the chromatographic fraction representing the intact hormone. Elimination of 125I-bPTH from circulation after intravenous injection to patients with this disease suggested that the hormone, but not its degradation products, were removed more rapidly before than after successful surgery. We conclude that the major part of circulating iPTH in patients with primary hyperparathyroidism is unable to stimulate the rat kidney adenylyl cyclase, and that the biological PTH activity is represented by the intact hormone (15% or less of total iPTH). These patients degrade more rapidly the injected 125I-bPTH and this mechanism introduces a new concept to protect target cells against excessive hormone action.

Filter-binding assay procedure for thyroid hormone receptors

An assay procedure for thyroid hormone receptor activity which used nitrocellulose membrane filters was developed. Receptor proteins, extracted from washed rat liver nuclei with a 0.4 m NaCl solution, were incubated with 125I-labeled thyroid hormone (T 3), and filtered on the cellulose ester membranes under suction at 2°C. The filters were subsequently washed with cold buffer and counted for 125I radioactivity. The method allowed an accurate estimation of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentrations. The usefulness of this filter-binding method became evident when it was compared with the conventional procedure that employs Sephadex G-25 columns. For practical application to routine assays, various filtration conditions were examined, and a standard procedure was established. Using this technique, the isolated receptors were determined to possess an apparent K d of 1.38 × 10 −10 m and a pH optimum of T 3 binding at 8.2–8.4.

Discrimination between intact and mid-C-regional PTH using selective radioimmunoassay systems

Abstract. Two 125I-labelled hormone preparations (synthetic 44–68(Tyr)hPTH bearing a tyrosin at position No. 44 and 1–84 bPTH) were used to characterize antiserum G III for PTH radioimmunoassay. PTH fragments were used for displacement of either tracer and included 1–34 hPTH, 28–48 bPTH, 25–48 bPTH, 28–54 hPTH, 32–43 hPTH, 42–55(Tyr)hPTH (tyrosin at position No. 42), 1–65 bPTH fragments and intact 1–84 hPTH. Standard curves of the 44–68 hPTH, 1–65 bPTH and the 1–84 hPTH run in parallel to each other independent of the tracer used for the assay. When using the 44–68(Tyr)hPTH tracer, half-maximal displacement (1/2 max) was achieved at 24 pg/tube for 44–68 hPTH, 450 pg/tube for 1–65 bPTH and at 1300 pg/tube for 1–84 hPTH. When carrying out the assay with the 1–84 bPTH tracer, the values for 1/2 max were 11.5 pg/tube for 44–68 hPTH, 135 pg/tube for 1–65 bPTH and 370 pg/tube for 1–84 hPTH. Gel filtration of plasma and peritoneal dialysate from one patient with terminal renal insufficiency and secondary hyperparathyroidism indicate that due to the high affinity of the antibody for the mid-C-regional hPTH, this assay system is appropriate for the discrimination of intact PTH and large mid-C-regional fragments, that include most of the 44–68 hPTH sequence.

Receptors for insulin and insulin-like growth factor in cultured arterial smooth muscle cells depend on their growth state.

The binding of 125I-labelled insulin and 125I-labelled insulin-like growth factor (IGF) to cultured arterial smooth muscle cells from rats was studied during various growth states of the cells. The level of binding of 125I-labelled insulin to the cells was low in growing cells and high in stationary cells. The level of 125I-labelled IGF binding to the cells was high in growing cells and low in stationary cells. In addition, the effect of unlabelled IGF and insulin on the binding of both 125I-labelled hormones to the cells was examined during various growth states. In growing cells insulin displaced 125I-labelled insulin from its binding sites; IGF competed weakly with 125I-labelled insulin for the binding sites. In parallel, IGF displaced 125I-labelled IGF binding whereas insulin competed weakly with 125I-labelled IGF for the binding sites. In stationary cells both hormones displaced 125I-labelled IGF binding. Insulin-like growth factor also displaced 125I-labelled insulin binding whereas insulin could not significantly displace 125I-labelled insulin from the binding sites. Insulin only competed with 125I-labelled insulin for the binding sites after removal of the fetal calf serum from the culture medium.

Growth hormone receptors in rat liver membranes: Effects of fasting and refeeding, and correlation with plasma somatomedin activity

The effects of fasting and refeeding on hepatic growth hormone receptors, on insulin receptors and on plasma somatomedin activity were studied. Female rats were either subjected to fasting for 4 days, refed for 3 days after a 4-day fasting, or allowed free access to food (controls). The specific binding of 125I-labelled bovine growth hormone was low in liver microsomal membranes (45% that of controls) and in plasma membranes (52% that of controls) of fasted rats. The number of somatotropic sites rather than the affinity of the binding was decreased. Lactogenic sites as judged by the binding of 125I-labelled human growth hormone were not significantly reduced in liver membranes of fasted rats. 125I-labelled insulin specific binding was enhanced in microsomal (184% that of controls) and plasma membranes (136% that of controls) of fasted rats; these modifications were associated with a decreased insulinaemia. But immunoreactive rat growth hormone levels were not different in plasma of fasted, refed and control animals. Decreased plasma bioassayable somatomedin was associated with the low number of somatotropic binding sites in liver membranes of fasted rats. Somatomedin activity of refed animals was comparable to controls. A significant correlation between the plasma bioassayable somatomedin and the hepatic level of somatotropic binding sites was found. It is proposed that, in fasting, the loss of somatotropic binding sites in the liver is one of the possible causes of the decreased plasma somatomedin bioactivity.

Insulin degradation by human skeletal muscle.

Although previous studies from this and other laboratories have extensively characterized insulin degrading activity in animal tissues, little information has been available on insulin responsive human tissues. The present study describes the insulin degrading activity in skeletal muscle from normal human subjects. Fractionation of a sucrose homogenate of skeletal muscle demonstrated that 97% of the total neutral insulin degrading activity was in the 100 000 x g supernatant with no detectable glutathione-insulin transhydrogenase activity. The 100000 x g pellet contained 85% of the total acid protease activity and all the glutathione-insulin transhydrogenase activity. The soluble insulin degrading activity was purified 1400-fold by ammonium sulfate fractionation, molecular exclusion, ion-exchange and affinity chromatography. Enzymatic activity was determined by measuring an increase in trichloroacetic acid-soluble products of the 125I-labeled hormone substrates. The purified enzyme showed marked proteolytic specificity for insulin with a Km of 1.63 X 10(-7)M (+/-0.32) and was competitively inhibited by proinsulin and glucagon with Ki values of 2.1 X 10(-6)M and 4.0 X 10(-6)M, respectively. This insulin protease exhibited a pH optimum between 7 and 8, a molecular weight of 120000 and was capable of degrading glucagon. Inhibition studies demonstrated that a sulfhydryl group is essential for activity. Molecular exclusion chromatography of [125I]insulin degraded products revealed a time-dependent increase in degradation products with molecular weights intermediate between intact insulin and iodotyrosine. These studies demonstrate that the major enzymatic system responsible for insulin degrading activity is a soluble cysteine protease capable of rapidly metabolizing insulin under physiologic conditions.

Effect of various drugs on the binding of thyrotrophin to thyroid plasma membranes.

Effects of enzyme inhibitors and membrane-active drugs on the binding of 125I-labelled thyroid-stimulating hormone (TSH) to human thyroid membranes and membrane adenylate cyclase (AC) activity were studied. FOY, a synthetic polyvalent proteolytic enzyme inhibitor, Trasylol, alpha- and beta-adrenergic blocking agents, tranquilizers, anti-histamines and polyene antibiotics enchanced TSH binding in a dose-dependent manner, whereas selective enzyme inhibitors and adrenergic stimulating agents had no effect. Both propranolol and FOY inhibited basal and TSH stimulated AC activity of thyroid membranes. FOY, as well as propranolol was found to have protective effects on hypotonic erythrocyte lysis. These results suggest that propranolol and FOY increased TSH binding by the same mechanism, probably the so-called membrane-stabilizing effects. Although the detailed mechanisms underlying the increased TSH binding by these drugs remain unknown, they may change the membrane structure, thereby enhancing the TSH receptor affinity.