Characterization of high affinity receptors for insulin-like growth factors I and II on rat anterior pituitary cells

Primary cultures of rat anterior pituitary cells were assessed for the presence of specific receptors for insulin and for the somatomedin peptides, insulin-like growth factors I and II (IGF-I and IGF-II). Specific binding per 100,000 pituitary cells averaged 9.45 +/- 1.69% (mean +/- SD) for [125I]IGF-II, 0.83 +/- 0.06% for [125I]IGF-I, and only 0.11% for [125I]insulin, IGF-II was twice as potent as IGF-I in displacing [125I]IGF-II, while insulin was totally nonreactive, IGF-I was 5-fold more potent than IGF-II at displacing [125I]IGF-I and 1000-fold more potent than insulin. Scatchard analysis of [125I]IGF-II binding revealed a curvilinear plot, which could be resolved into a high affinity receptor with a Ka of 7.0 X 10(8) M-1 and 120,000 receptor sites/cell, and a low affinity receptor with a Ka of 1.1 X 10(8) M-1 and 720,000 receptor sites/cell. The existence of abundant high affinity somatomedin receptors (especially for IGF-II) on rat anterior pituitary cells is consistent with a potential role for these peptides in the regulation of GH secretion.     

Increased somatomedin receptor sites in newborn circulating mononuclear cells.

To evaluate the potential role of somatomedin as a growth factor in fetal development, we studied somatomedin receptors on circulating mononuclear cells from placental cord blood from 13 normal term newborns. Twelve healthy young adults served as controls. By radioreceptor assay of acid-chromatographed plasma, total somatomedin peptide content of cord blood was 0.30 +/- 0.02 U/ml (mean +/- SEM) compared to 1.12 +/- 0.08 U/ml for adults. Specific binding of [125I]somatomedin C to 50 X 10(6) mononuclear cells was 11.38 +/- 0.74% for newborns and 6.67 +/- 0.51% for adults. This increase in specific binding appears to be due to an increased number of receptor sites per cell. These data support a hypothesis of increased fetal sensitivity to somatomedin.     

The bovine placenta: a specific radioreceptor assay for both insulin-like growth factor I and insulin-like growth factor II

Binding of labelled IGF-I and IGF-II was studied to bovine, ovine and human placental cell membranes. The data show a preponderance of type I receptors in human placental membranes, and of type II receptors in ovine placental membranes, confirming reported data. In contrast, bovine placental membranes are rich in both type I and type II receptors. Therefore, the bovine placenta offers a good model for measuring specifically IGF-I (cross-reactivity with IGF-II 7%) and IGF-II (cross-reactivity with IGF-I 4%). By Scatchard analysis the apparent Kd (1-1.36 nmol/l) for the high affinity binding sites of the type I receptor is similar in all three preparations. Total binding capacity in ovine placental membranes is, however, 4 times lower. The affinity for the type II receptor is lower than for type I, whereas total binding capacity is higher. Affinity cross-linking confirms the competition experiments, showing binding of IGF-I to typical type I and of IGF-II to type II receptors.     

The effect of solubilization on the properties of the insulin receptor of human placental membranes.

The insulin receptor for human placental membranes has been solubilized in Triton X-100 and its properties have been examined in detail. Binding of [125 I]iodoinsulin to the soluble receptor is markedly inhibited by increas-ng concentrations of Triton X-100, due to a fall in receptor affinity. In 0.02--0.10% Triton X-100, the soluble receptor exhibits all the essential characteristics of the intact or particulate receptor. These include strict specificity for insulin and its analogues, increase in steady state binding with decrease in temperature, a pH optimum at 7.8--8.0, and negatively cooperative site-site interactions. The initial association rate of [125 I]iodoinsulin and the soluble receptor is a direct function of temperature, but the level of steady-state binding is lower at higher temperatures due to a marked increase in dissociation rate. Scatchard binding plots are curvilinear and show a large increase in affinity at 4 C with no change in total binding capacity (R0); increased binding to the particulate placental membrane at 4 C is due chiefly to an increase in R3. Negative cooperatively in the soluble receptor has been confirmed by kinetic experiments; thus, the dissociation of [125I]iodoinsulin from the receptor in the presence of "infinite" dilution is accelerated in the presence of 10(-8) M unlabeled insulin. The apparent molecular weight of the placental receptor, determined by gel filtration on 6% agarose, is approximately 300,000. These studies show that the basic properties of the insulin receptor do not depend on it being an integral conponent of the cell membrane.     

Insulin binding to human ovaries

Human ovarian tissue samples were obtained at the time of laparotomy, and plasma membrane fractions were prepared and used in receptor assays. Incubations of the membrane fraction were performed with [125I]insulin (porcine), and Scatchard analysis of binding showed biphasic curves. The high affinity sites had an average concentration of 57.4 +/- 7.9 (+/- SEM) fmol/mg protein and a dissociation constant of 3.5 +/- 0.9 nM (n = 9). Neither affinity nor number of binding sites changed significantly during the menstrual cycle. We conclude that there is high affinity binding of [125I]insulin to human ovarian plasma membranes.     

Affinity-labeled somatomedin-C receptors and binding proteins from the human fetus

We investigated the ontogeny of the human placental membrane somatomedin-C (Sm-C)/insulin-like growth factor I (IGF-I) receptor by affinity labeling with the cross-linking agent disuccinimidyl suberate (DSS). Specific Sm-C receptors, identified from as early as 6 weeks gestation, demonstrated no apparent structural changes through the course of gestation. Second trimester human fetal brain membranes cross-linked to [125I]Sm-C exhibited an identical pattern of receptor binding. These findings in both placenta and fetal brain membrane are consistent with the proposed heterotetrameric structure of the Sm-C/IGF-I receptor. Using a similar DSS cross-linking technique, we identified Sm-C-binding proteins in second trimester amniotic fluid [apparent molecular mass (Mr), 38,000 and 35,000] and term cord plasma (Mr, 41,000, 38,000, and 35,000). Identically sized binding components (Mr, 35,000-45,000) were also found in membrane preparations of preterm placenta and brain after cross-linking. Evidence that these binding species represent contamination of preterm membrane preparations with soluble amniotic fluid and/or fetal plasma Sm-C-binding proteins and that they are not derived from membrane receptors is as follows: (1) these binding species, like amniotic fluid and cord plasma binding proteins, were insensitive to competition with insulin (in concentrations as high as 0.6 mg/ml), a characteristic not shared with membrane receptor binding components; (2) these binding species were largely removed from placental membranes by extensive washing and appeared in the supernate when membrane preparations were incubated at 4 C for 18 h, indicating that they were soluble and not an integral part of the particulate membrane; (3) limited proteolysis of placental membrane preparations did not result in the appearance of similar binding species; and (4) preparation of preterm placental membranes in the presence of protease inhibitors did not eliminate these binding species. Use of traditional methodology to study binding of somatomedin to receptors in membranes prepared from preterm human tissues may be misleading because of contamination by amniotic fluid and/or plasma-derived binding proteins.     

Evidence for the presence of type I insulin-like growth factor receptors on rat pancreatic A and B cells

Purified rat pancreatic islet cells express somatomedin receptors which are identified by their affinity for insulin-like growth factor (IGF)-I, IGF-II, and insulin. Binding of [125I]IGF-I to islet A cells was half-maximally inhibited by 7.10(-10) M IGF-I, while IGF-II, insulin, and proinsulin were respectively 10-, 500-, and 10,000-fold less potent displacers of IGF-I binding. Unrelated hormones such as glucagon or GH did not compete with [125I]IGF-I binding to A cells. The concentration of IGF-I receptors on A cells was estimated at 5000 IGF-I binding sites per cell with affinity constant (Ka) of 2 X 10(9) M-1. Islet B cells were found to exhibit a reversible time- and temperature-dependent binding with [125I]IGF-I. Specificity and affinity of IGF-I binding sites were identical for islet A and B cells. Linear Scatchard plots of competitive binding data on B cells suggest 1 single class of IGF-I receptors in a concentration of 12,000 sites per cell. The presence of high affinity receptors for IGF-I on adult islet A and B cells provides a molecular basis for this growth factor to influence growth, survival, and/or function of these endocrine cell types. Their low affinity for insulin should be considered as a potential mechanism for this hormone to influence, at high concentration, the function of islet A and B cells.     

Structural determinants of ligand recognition by type I insulin-like growth factor receptors: use of semisynthetic insulin analog probes

We undertook a systematic analysis of the structural determinants necessary for ligand recognition by the type I insulin-like growth factor (IGF) receptor by investigating the binding of semisynthetic insulin analogs to IGF receptors from human placental cell membrane fragments. Analogs were prepared by synthetic and semisynthetic methods. Three groups of insulin analogs were synthesized: the first group contained insulin analogs modified at the amino-terminal position of the insulin A chain and included acetyl-insulin and human proinsulin; the second group included analogs in which B chain residues B26-B30 [despentapeptide insulin (DPI)], B25-B30 (deshexapeptide insulin), and B24-B30 (desheptapeptide insulin) were removed; the third group contained insulin analogs in which B chain residues B26-B30 were removed (DPI) and phenylalanine(B25) substituted with other amino acids, including alanine, serine, leucine, and tyrosine. Half-maximal inhibition of binding of radiolabeled IGF-I to placental cell membrane fragments was used as an index of relative binding affinity (K1/2). To determine further if semisynthetic insulin analogs bound to the type I IGF receptor, placental membrane fragments were affinity labeled with radiolabeled IGF-I in the presence and absence of submaximal concentrations of unlabeled hormone, insulin, or semisynthetic analogs, and the labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Insulin had a 500 times lower affinity for the type I IGF receptor than IGF-I [K1/2 = 140 +/- 69 nM (mean +/- SD)] whereas proinsulin and acetyl insulin had a more than 100 times lower affinity than insulin for this receptor type. Removal of insulin B chain amino acid residues 26-30 (DPI) did not negatively affect the binding of the insulin-derived peptide and actually increased the apparent affinity of ligand-receptor association approximately 2-fold. However, further removal of phenylalanine(B25) (deshexapeptide insulin) and phenylalanine(B24) (desheptapeptide insulin) decreased the binding of ligand to the type I IGF receptor progressively by several orders of magnitude. Substitution of phenylalanine(B25) of DPI with tyrosine, a substitution that actually increased the homology of this analog to IGF-I, resulted in a 4- to 5-fold increase in the relative apparent affinity of the analog for the type I IGF receptor (K1/2 = 31 +/- 4 nM). On the other hand, substitution of phenylalanine(B25) with alanine, serine, and leucine decreased the relative apparent binding affinity approximately 2- to 8-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of prolactin binding sites in the brain of the ring dove (Streptopelia risoria)

Radiolabeled ovine prolactin ([125I]oPRL) was found to bind specifically to crude membrane fractions prepared from ring dove brain homogenates. Specific binding changed with incubation time and temperature but did not fluctuate systematically with pH. Specific binding levels were highest in hypothalamus and forebrain and were lowest in cerebellum and brain stem. A linear increase in specific binding was observed in pooled midbrain and forebrain membrane fractions as membrane protein concentrations increased from 0.2 to 6.0 mg/ml. Scatchard analysis of binding saturation and competitive binding data revealed that dove midbrain/forebrain membranes bound oPRL with high affinity (Kd = 2.1-2.6 X 10(-10) M) but had low binding capacity (Bmax = 2.9-4.8 fmol/mg protein). Unlabeled oPRL and human growth hormone competed most effectively with radiolabeled oPRL for occupation of midbrain/forebrain binding sites. Turkey PRL and human placental lactogen were less effective as competitors but were 8-15 times more effective than ovine or turkey growth hormone, porcine insulin, or ovine luteinizing hormone. Subcutaneous injection of unlabeled oPRL lowered specific binding in liver and midbrain/forebrain by 76 and 39%, respectively, over that observed after saline injection. Prior MgCl2-induced desaturation of membrane binding sites greatly reduced or eliminated these treatment differences. These results suggest the existence of specific, saturable binding sites for PRL in dove brain which conceivably could mediate the reported effects of PRL on behavior and gonadal function in this species.     

Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes

In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 A and 38 A. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 A) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 A to 72 A. Similar results are observed for a glycoprotein fraction obtained from rat adipocyte membranes but are not observed for an identical fraction isolated from human erythrocyte membranes. We conclude that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin.     

Somatomedin-C receptor ontogeny and levels in porcine fetal and human cord serum.

The ontogeny of somatomedin receptors in tissues of fetal pigs and levels of somatomedin-C in fetal pig serum at various gestational ages and in human cord serum was investigated. Specific binding of 125I somatomedin-C by particulate membranes prepared from fetal organs from a variety of gestational ages almost always exceeds specific 125I insulin binding. In liver, kidney, heart and the maternal portion of the placenta, apparent binding affinity for somatomedin is relatively constant throughout gestation and is the same for membranes from fetal and adult animals. In contrast, in the fetal portion of the placenta, specific somatomedin-C binding and apparent binding affinity increases as gestation progresses. The changes in this tissue correlate temporally with the acceleration of growth of the pig fetus. Membranes prepared from fetal lungs exhibit higher specific binding of somatomedin and higher affinity constants than adult lung membranes. Somatomedin levels in fetal pig serum are about 25% of those observed in the sow and are constant throughout fetal life. Somatomedin in human cord serum is likewise low compared to adult levels. Small-for-gestational age infants and large, postmature infants have lower mean somatomedin levels than normal weight, full-term infants. The identification of specific somatomedin receptors in fetal tissues opens the possibility that somatomedin-C stimulates growth of the fetus. Although not resolved, the relatively low levels of somatomedin in fetal serum may reflect low levels of the somatomedin binding protein rather than an absolute deficiency of biologically active somatomedin.     

Partial purification, characterization, and assay of a slightly acidic insulin-like peptide (ILAs) from human plasma.

An insulin radioreceptor assay (RRA) using human placental microsomal membranes was used to measure insulin-like activity (ILA) extracted from human plasma concentrates (Cohn fraction IV-4) by acid ethanol. The soluble activity (ILAs), chromatographed on Sephadex G-75 in 1 M acetic acid, migrated as a small molecule (fractional elution volume, 0.56) ahead of insulin (fractional elution volume, 0.70), whereas at neutral pH, ILAs migrated as a large molecular weight species. The ILAs peak from acid gel filtration on Sephadex was further purified by chromatography on carboxymethyl cellulose (CMC). The ILAs peak from both Sephadex and CMC diluted parallel to the porcine insulin standard in the insulin RRA and was totally unreactive in an insulin RIA. The CMC-purified material was iodinated and purified by binding to and elution from human placental membranes. The binding of [125I]ILAs to human placental membranes was inhibited only minimally by insulin and proinsulin and not at all by epidermal growth factor, nerve growth factor, glucagon, or lactogenic hormones, including human growth hormone. Multiplication-stimulating activity (MSA) inhibited in a manner parallel to ILAs. A Scatchard plot of the binding data was nonlinear. Sephadex ILAs was subjected to isoelectric focusing. The fractions assayed in both insulin and ILAs RRAs yielded comparable results. Peaks of ILA were observed at pHs 5.3, 6.6, and 8.4. When CMC-ILA was subjected to isoelectric focusing in polyacrylamide, a single peak of activity migrating between pH 6.2-6.8 was seen. [125I]ILAs focused at exactly the same pH. Electrophoresis of CMC-ILAs in acid-urea revealed a sharp peak of activity migrating with one of the five protein bands seen after staining. Again, [125I]ILAs comigrated with unlabeled ILAs. The molecular weight of ILAs, as determined on a calibrated Sephadex G-150 column at neutral pH, was 9,000-10,000 daltons. CMC-ILAs stimulated [14C]glucose incorporation into triglycerides of rat adipose tissue and augmented [3H]thymidine incorporation into human fibroblasts, chicken embryo fibroblasts, and BALB 3T3 cells as well as [35S]sulfate incorporation into macromolecules of rabbit chondrocyte culture medium. In summary, ILAs isolated on the basis of a RRA for insulin is a slightly acidic peptide with some of the biological activities expected of a somatomedin.     

Isolation of a somatomedin from plasma of rats bearing growth hormone secreting tumors.

We have purified a protein which has somatomedin-like properties from the serum of Wistar-Furth rats bearing a growth hormone producing pituitary tumor (MStT/W15). Activity was measured by a placental insulin and/or somatomedin C radioreceptor assay (SmC-RRA). The serum was initially filtered through Sephadex G-150 equilibrated with 0.1 M NH4HCO3 and 0.02% NaN3. On the G-150 column, radioreceptor insulin (RRI) and radioreceptor somatomedin C (RRSm-C) activities coincided and appeared predominantly in the 160,000 mol wt range with a minor proportion in the 50,000 mol wt range. The pooled active fractions were boiled for 30 min at pH 5.5. After removing denatured protein by centrifugation, the extract was passed through G-50 Sephadex equilibrated with 1% formic acid and 0.15 M NaCl. Sixty to 90% of the SmC-RRA activity in the effluent appeared in the 9000 mol wt range. This material has an isoelectric focusing range of 8.4--9.6, similar to that described for human somatomedin C. On SDS-urea polyacrylamide gel electrophoresis only one protein band was seen. The isolated peptide (rSm) stimulated sulfate uptake in hypophysectomized rat cartilage. The potency of two preparations was variously assayed from 14.0 to 54.7 units/mg. Rat somatomedin was iodinated and purified by absorption on and elution from placental membranes. Eight to 12% of rat [125I]Sm was specifically bound by human placental membranes. Rat [125I]Sm was displaced by hSmC and rSm and human NSILA-S, partially displaced by procine proinsulin and poorly displaced by rat insulin. In preliminary studies, rat [125I]Sm was displaced from receptors on human placental membranes by sera from pituitary tumor bearing rats greater than normal rat sera greater than hypophysectomized rat sera.     

Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes.

High-affinity [3H]serotonin binding activity has been solubilized from bovine cerebral cortical membranes by using Triton X-100, Tween-80, and octyl-beta-D-glucopyranoside. This mixture of detergents solubilizes the high-affinity [3H]serotonin binding activity present in crude membrane preparations with retention of 75-90% specific binding. The detergent mixture was chosen because it can easily be removed from the solubilized fraction by dialysis and polystyrene bead adsorption, thus permitting further purification and isolation of the binding sites. Saturation analysis reveals multiple components of high-affinity [3H]serotonin binding. In crude bovine cortical membranes, at least two binding components are present. A higher-affinity binding component, as defined from curvilinear Scatchard plots, has a Kd for [3H]serotonin of 1-3 nM, whereas a lower-affinity component has a Kd of 10-20 nM. In the solubilized preparation, only a single class of binding sites is apparent, with a Kd of 50-100 nM. Removal of detergents by dialysis and polystyrene bead adsorption results in restoration of the curvilinear Scatchard plot with apparent Kds similar to those observed in crude membrane preparations and with increased Bmax values for each component. [3H]Serotonin binding activity in the solubilized preparation is stable to Sephacryl S-300 column chromatography and to glycerol gradient sedimentation. Saturation analysis of the peak binding fractions from both these procedures once again yields curvilinear Scatchard plots, indicating that the multiple high-affinity binding components are preserved and migrate together. The molecular weight, Stokes radius, and frictional coefficient of the binding site(s) have been calculated. After detergent removal the solubilized material shows many of the characteristics usually attributed to S1 receptors, such as high affinity for [3H]serotonin and its analogs and low affinity for serotonin antagonists.     

Growth hormone receptors in isolated rat adipocytes

Specific GH binding sites in isolated rat adipocytes have been partially characterized. Binding of [125I]iodohuman(h)GH was rapid, reversible, and was time and temperature dependent. Maximum specific binding occurred at 37 C in approximately 40 min at pH 7.4. Bound labeled hGH was rapidly dissociable, with the addition of excess unlabeled hormone. Specific binding is inhibited by as little as 1.0-1.5 ng/ml hGH, and 50% inhibition was obtained with 15-20 ng/ml. No inhibition was observed with insulin, glucagon, hPRL, or hTSH at concentrations up to 1 micrograms/ml. This receptor does not discriminate between monkey GH, rat GH, bovine GH, and porcine GH. Specific binding varied linearly with cell concentration. Scatchard analysis revealed linear plots with a Ka of approximately 10(9) M-1 and 15,000 sites per cell. There was less than 15% degradation of [125I]iodo-hGH over 90 min at 37 C. There was a striking increase in [125I]iodo-hGH binding to adipocytes at pH 4.85. Scatchard analysis of binding at pH 4.85 revealed a curvilinear plot with an apparent increase of sites per cell from 15,000 to 60,000, and a modest increase in the apparent affinity constant of the high affinity, low capacity sites using the two-compartment model for curvilinear plots. The GH receptors in rat fat cells displayed no ability to bind labeled hPRL or human placental lactogen, consistent with minimal recognition of lactogenic peptides by these receptors. Thus, the rat adipocyte contains specific binding sites for GH that fulfill the major criteria for receptor binding. The presence of such receptors in these cells may facilitate the study of GH receptors in relation to the biological effects of the hormone on adipose tissue in various metabolic settings.     

Effect of insulin and nutrition on serum levels of somatomedin A in the rat

The serum levels of somatomedin A, as measured by radioreceptor assay, were significantly reduced in rats 2 days after the administration of streptozotocin. The mean decrease was 45.4 +/- 2.9% of the initial values. In rats treated with insulin, blood glucose levels and glycosuria decreased, and serum somatomedin A returned to 108.3% +/- 11.7% of the initial values by the sixth day of treatment. In untreated diabetic rats, serum somatomedin A decreased progressively to 23.4 +/- 4.4% 8 days after streptozotocin administration. The total caloric intakes in the treated and nontreated diabetic rats were similar, suggesting that the low levels of somatomedin A in diabetic rats may be due to lack of insulin. A significant correlation was observed between serum somatomedin A values and body weight (r = 0.90) or the urinary glucose (r = -0.84) or blood glucose levels (r = -0.67). When the diabetic insulin-treated rats were fed a low protein diet, there was no increase in serum somatomedin A. Inhibitory factors in serum which interfere in the bioassay for somatomedin had no effect in our radioreceptor assay.     

In vivo modulation of somatomedin receptor sites: effects of growth hormone treatment of hypopituitary children

We have previously demonstrated that somatomedin (SM) can induce the loss of specific SMC receptors on cultured IM-9 lymphocytes in a time- and concentration-dependent manner. To investigate the acute regulation of SM binding under in vivo conditions, we have evaluated SM receptors on circulating mononuclear cells obtained from 12 hypopituitary dwarfs before and after 4 days of human GH administration (0.1 U/kg . day). Plasma SM levels, measured by radioreceptor assay, rose from 0.37 +/- 0.08 U/ml (mean +/- SEM) to 1.00 +/- 0.10 (P less than 0.001). Concomitantly, specific binding of [125I]SMC to 50 x 10(6) mononuclear cells/ml fell from 13.61 +/- 0.97% to 10.40 +/- 0.85% (P less than 0.02). The decrease in specific binding was predominantly secondary to a reduced number of SMC receptor sites per cell, with no alteration in receptor affinity. Overall, a significant inverse correlation was observed between plasma SM levels and mononuclear cellular binding of [125I]SMC (P less than 0.001). The data demonstrate that treatment of hypopituitary dwarfs with conventional therapeutic doses of human GH results in significant acute increases in plasma SM levels in the majority of subjects, with a reciprocal decline in the specific binding of [125I]SMC. We conclude that SM, like insulin, is capable of regulating homologous receptor concentrations under both in vitro and in vivo conditions.     

Annual variations in the binding of insulin to hepatic membranes of the frog Rana esculenta.

Amphibia undergo regular annual cycles of metabolic activity that are influenced by both exogenous factors and hormones. Insulin binding to crude frog hepatic membranes was studied throughout the year. The general character of insulin binding was similar to that in other vertebrates; the maximum specific binding was achieved after 4 hr at 4 degrees, the optimum pH was 7.8, half-maximal displacement of bound insulin was from 9 x 10(-10) to 1 x 10(-9) M, and insulin analogs competed for the insulin receptor in line with their relative biological potencies. A biphasic Scatchard plot and negative cooperativity of the receptor were also observed in frog liver membranes. Affinity constants from Scatchard plots revealed high and low affinity binding sites which were unchanged during the year. The seasonal cycle, however, markedly affected the binding capacity for both sites. Maximum binding occurred in May-June and the minimum in November-December for both classes of receptors. Binding capacities ranged from 1.71 to 11.33 fmol/mg protein for the high affinity sites and from 432 to 3171 fmol/mg protein for the low affinity sites. It is concluded that annual cycles of insulin binding reflect modulation of receptor number rather than receptor affinity.     

The insulin receptor of embryonic chicken cartilage.

Highly purified plasma membranes have been obtained from embryonic chicken cartilage by physical means rather than enzymatic digestion. Rapid and reversible binding of [125I]iodoinsulin to these membranes is demonstrated. Similar to the insulin-binding properties of rat liver and adipocytes and human mononuclear cells, optimal specific binding of insulin to chondrocyte plasma membranes has a sharp pH optimum at 8.0, and maximal binding occurs at 2--4 C. Analysis of equilibrium binding reveals a curvilinear Scatchard plot, whose high affinity segment generates a maximum affinity of 1.0 X 10(9) M-1, and a receptor concentration of 0.4 pmol/mg membrane protein. This affinity constant is similar to those generated for insulin binding to membranes prepared from embryonic chicken liver (2.5 X 10(9) M-1), rat liver (1.4 X 10(9) M-1), and mouse liver (0.6 X 10(9) M-1), whereas the receptor concentration is less than that of embryonic chicken liver membranes (1.1 pmol/mg), which in turn was less than those of rat liver membranes (2.8 pmol/mg) and mouse liver membranes (3.5 pmol/mg). Kinetic studies show augmentation of insulin-receptor dissociation by excess insulin when initial receptor occupancy, is low, suggesting that negative cooperativity is present. There is little or no interaction of other hormones with the chondrocyte insulin receptor, with the exception of proinsulin and the insulin-like growth factors. Porcine proinsulin, bovine proinsulin, somatomedin C, and nonsuppressible insulin-like protein prevent [125I]iodoinsulin binding to chondrocyte plasma membranes with dose-response curves which are parallel to that of unlabeled porcine insulin itself, but with molar potencies relative to porcine insulin of 15%, 9%, 2.5%, and 1.4%, respectively. Porcine insulin and proinsulin both prevent binding of [125I]iodosomatomedin C to chondrocyte plasma membranes but with molar potencies less than 1% that of unlabeled somatomedin C. These observations are consistent with the presence of a specific independent insulin receptor in embryonic chicken cartilage which is similar in its characteristics to the insulin receptor in previously described tissues. Insulin has a weak interaction with the chondrocyte receptor for somatomedin C. Interaction with the somatomedin receptor may be the mechanism by which insulin exerts anabolic effects on cartilage when used in pharmacological amounts.     

Separation of two thyrotropin binding components from porcine thyroid tissue by affinity chromatography: characterization of high and low affinity sites.

Two distinct thyrotropin (TSH) binding species have been separated from solubilized porcine thyroid membranes. Membranes was solubilized with 1% Triton X-100, and the supernatant was recovered by centrifugation at 105,000 X g. Scatchard analysis of thyrotropin binding to solubilized membranes (SM) yielded a nonlinear plot with Kd values for the high and low affinity components similar to those of intact membranes. Chromatography of the SM preparation on concanavalin A-Sepharose 4B resulted in the retention of 10-20% of the binding activity. Upon elution of the column, a peak of binding material (5-7% of total activity) was eluted at 0.3 M alpha-methyl-D-mannoside. This concanavalin A (Con A) bound fraction exhibited a linear Scatchard plot with a Kd value similar to that of the high affinity component of the SM. The protein fraction that did not bind to Con A (Con A unbound) also exhibited a linear Scatchard plot, but with affinity similar to that of the low affinity component of SM. Discontinuous sucrose density gradient ultracentrifugation revealed the presence of two major binding peaks in the solubilized membrane preparation. The slowly sedimenting peak corresponded to that seen in the Con A bound fraction, whereas the rapidly sedimenting peak corresponded to that of the Con A unbound fraction. Sepharose 6B chromatography indicated that in the case of the Con A unbound fraction, a single peak of specific binding activity was eluted in the void volume, and in the case of the Con A bound fraction, one major peak with an approximate Stokes radius of 67 A and several other minor peaks were eluted. These results demonstrate the physical separation of two distinct TSH binding species from thyroid membranes and provide further support for the model of multiple classes of binding sites.