Structural characteristics of growth hormone receptors on mouse 3T3 cells having different biological responses to growth hormone

Cultured 3T3-F442A preadipocytes are able to undergo GH-promoted differentiation into adipocytes. The relationship between the structure and function of GH receptors on 3T3 cells (3T3-F442A preadipocytes, differentiated adipocytes and 3T3-C2 cells, which vary in susceptibility to adipose conversion or with respect to carbohydrate and lipid metabolism) was studied by the covalent cross-linking of 125I-labelled human (h) GH to intact cells with the bifunctional reagent disuccinimidyl suberate. When preadipocytes were cross-linked and analysed using sodium dodecylsulphate-polyacrylamide gel electrophoresis, a prominent 125I-labelled hGH-receptor complex of Mr 130,000 was observed along with minor complexes (Mr 300,000, 230,000 and 60,000) on autoradiography. Non-reducing-reducing two-dimensional gel electrophoresis revealed that the higher molecular weight complexes also contained the Mr 130,000 complex. Neuraminidase and tunicamycin treatment demonstrated that the GH receptor on F442A preadipocytes is a sialo-glycoprotein with N-linked carbohydrate chains. When the differentiated 3T3-F442A adipocytes and 3T3-C2 cells (a sub-line with no susceptibility to adipose conversion with GH) were examined in the same way as 3T3-F442A preadipocytes, no differences were observed in the specificity of GH binding and in the molecular size of the 125I-labelled hGH-receptor complexes and their glycosylation characteristics. This suggests that the structural characteristics of the GH receptor are closely related in each cell type, but that the hormonal signals produced after GH binding to the receptor may cause different effects according to the cell type.     

Glycosylation characteristics of the mouse liver lactogenic receptor

The structural characteristics and glycosylation properties of the lactogenic receptor were examined in 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized plasma membranes from female mouse liver. The specific binding of the radioiodinated human growth hormone [( 125I]hGH) was displaced with an equivalent potency by both hGH and prolactin. After a mild neuraminidase treatment, this binding was increased by 40%, as a result of an increase in receptor affinity. Affinity chromatography on immobilized lectins revealed that the [125I]hGH-receptor complexes were specifically retained and eluted from ricin lectin-agarose, concanavalin A and lentil lectin, indicating the presence of N-linked glycans. Covalent cross-linking of solubilized [125I]hGH-receptor complexes with disuccinimidyl suberate, followed by analysis by sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE) under reducing conditions, and autoradiography resulted in the appearance of two bands with apparent Mr approximately 62,000 and approximately 100,000. The labelling of these bands was prevented by unlabelled hGH or ovine prolactin (oPrl) but not by bovine growth hormone (bGH). Neuraminidase treatment of the two receptor forms resulted in increased electrophoretic mobility which was inhibited by simultaneous addition of sialyl-lactose, a neuraminidase substrate. The both cross-linked forms were unaffected by endoglycosidase H, while endoglycosidase F decreased the molecular weight of each of the forms by about 8000 Da, yielding bands at Mr approximately 54,000 and approximately 92,000. In conclusion, taking into account that hGH is a Mr 22,000 polypeptide, the two forms of the receptor correspond to glycoproteins of Mr approximately 40,000 and approximately 78,000, respectively. They contain polypeptide backbones of Mr approximately 32,000 and approximately 70,000, and complex N-linked oligosaccharide chains with terminal sialic acid residues which could be involved in receptor binding affinity.     

Purification and biological activity of a single charge isomer of pituitary-derived chicken growth hormone

The chicken pituitary gland contains a number of naturally occurring, developmentally regulated forms of GH which have identical molecular weights but differ in their isoelectric points. In order to characterize their biological properties, each must be separated from non-GH proteins and other forms of GH. Chickens GH (cGH) was separated from other pituitary proteins by immunoaffinity chromatography using an anti-GH monoclonal antibody covalently linked to Sepharose 4B. The cGH eluted from this column as a single peak and migrated as a single band during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), but showed multiple bands on isoelectric focussing. This material was chromatographed on a high-performance cation exchange column, and separation of charge isomers was monitored by a combination of isoelectric focussing and immunoblotting. Chicken GH eluted from this column in two distinct peaks. The minor peak (cGH P1) contained an isomer with an isoelectric point of 6.86 and the major peak (cGH P2) an isomer with an isoelectric point of 7.52. Each isomer migrated as a single band during isoelectric focussing and SDS-PAGE (Mr = 23,500), and as a single peak during high-performance gel permeation chromatography and reverse-phase high-performance liquid chromatography. Analysis of cGH P2 through 30 cycles in a gas-phase microsequencer gave an amino acid sequence identical to that predicted by translation of the GH complementary DNA nucleotide sequence. This single charge isomer increased the rate of lipolysis in chicken adipose tissue explants by about fourfold and was able to displace 125I-labelled cGH from binding sites in liver membranes with a dissociation constant of about 4 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins.

The present study constitutes the characterization of a specific, high-affinity GH-binding protein (GHBP) in the serum of a teleost, the goldfish (Carassius auratus). GH-binding assay and ligand blotting techniques were employed to identify GHBPs in goldfish serum and hepatocyte culture medium. The binding characteristics and apparent molecular weights (Mr) of goldfish GHBPs were also compared with those of rabbit and rat. LIGAND analysis identified a single class of high-affinity and low-capacity binding sites for iodinated recombinant carp GH (rcGH) in the goldfish serum, with an association constant (Ka) of 20.1x10(9) M-1 and a maximum binding capacity (Bmax) of 161 fmol ml-1 serum. A single class of binding sites for iodinated recombinant sea bream GH and bovine GH (bGH) was also found in goldfish serum, but with a much lower affinity than that of rcGH. The binding affinity for iodinated bGH in rabbit and rat sera was found to be similar to that reported previously. Ligand blotting revealed multiple forms of GHBPs in sera of goldfish, rabbit and rat with Mr ranging from 70 kDa to 400 kDa and 27 kDa to 240 kDa under non-reducing and reducing conditions respectively. A prominent band with Mr of 66 kDa and a minor band with Mr of 27 kDa were observed to occur in sera from all three species under reducing conditions. Iodoacetamide promoted the shedding of three GHBPs with Mr of 25, 40 and 45 kDa from the cultured goldfish hepatocytes. The appearance of all bands was completely inhibited by the presence of excess unlabeled rcGH. Our results provide clear evidence that a GHBP exists in the goldfish and indicate that more information on teleost GHBPs is needed if the physiology of growth in teleosts is to be fully understood.     

Production of anti-idiotypic antisera to rat GH antibodies capable of binding to GH receptors and increasing body weight gain in hypophysectomized rats

Anti-idiotypic antibodies to rat GH antibodies were produced in both sheep and mice and shown to be capable of mimicking GH by inhibiting 125I-labelled ovine GH (oGH) binding to sheep liver membranes. The sheep anti-idiotypes were characterized further and shown to (1) inhibit 125I-labelled oGH binding to oGH antibodies, (2) inhibit 125I-labelled oGH binding to rat adipocytes and (3) be incapable of inhibiting the binding of either 125I-labelled ovine prolactin or 125I-labelled bovine insulin to sheep liver membranes. This indicated that the antibodies were not limited to certain species or tissues, but were hormone specific. Finally, these anti-idiotypic antibodies were also capable of stimulating an increase in body weight gain in hypophysectomized rats, suggesting that they may be functional as well as structural mimics of GH, although the increased body weight gain was not accompanied by any increase in circulating concentrations of insulin-like growth factor-I.     

Growth hormone-binding sites in chicken hypothalamus

Specific binding of 125I-labelled recombinant DNA-derived chicken GH (rcGH; 2.1 +/- 0.41 (S.E.M.) % of total counts) and of 125I-labelled bovine GH (1.80 +/- 0.27% of total counts) to crude plasma membranes of the chicken hypothalamus was demonstrated. Binding of 125I-labelled rcGH was related to the amount of tissue incubated and was significant over the range 250-950 micrograms membrane protein per tube. Binding of 125I-labelled rcGH was saturable over the range 0.14-0.40 pmol and was to a single class of high-affinity (33.5 pM) low-capacity (2.14 fmol/mg protein) binding site. Binding of 125I-labelled rcGH was displaced by ovine GH and by ovine prolactin. These results demonstrate, for the first time, central GH-binding sites in a vertebrate species.     

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 less than 0.01 vs. controls) and serum SM-C by 53% (P less than 0.01), while GH and PRL binding capacities decreased respectively by 63% (P less than 0.01) and 62% (P less than 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 less than 0.01). No significant changes in serum growth hormone concentrations occurred with fasting or refeeding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of glycosylated growth hormone-binding proteins in rat serum

Affinity cross-linking technique revealed the presence of three growth hormone-binding proteins (GHBP) in dealbuminized rat serum. The apparent molecular weights, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 52,000, 44,000 and 39,000. By use of carbohydrate chain cleaving enzymes it was found that the binding proteins contain N-linked complex carbohydrate chains, representing 8000, 4000 and 5000 in apparent molecular weight, respectively. Gel permeation chromatography and sucrose density gradient centrifugation revealed a growth hormone-binding entity with Stoke's radius 94.5 A (+/- 2.8, n = 5) and an s-value of 10.8 S (+/- 0.31, n = 5). The molecular weight could be calculated to 413,000. Both for gel chromatography and sucrose density gradient experiments unlabelled growth hormone reduced the radioactive peaks from 0 to 50%. The relation of this binding entity to the GHBP's described with affinity cross-linking technique and the nature of this binding entity is at present unclear. No serum binding protein(s) for prolactin was detected by affinity cross-linking technique, gel permeation chromatography or sucrose density gradient analysis.     

Autoradiographic localization of the binding of 125I-labelled prolactin to rat tissues in vitro.

The binding of 125I-labelled prolactin to normal rat tissues was examined with autoradiographic techniques. Tissue slices were incubated with 125I-labelled ovine prolactin in the presence or absence of excess unlabelled hormone to distinguish between the specific and the non-specific localization of grains. Specific binding was found in the liver, adrenal gland and kidney from female rats, and in the mammary gland, ovary, testis and prostate gland. Conversely, fat, muscle, heart, lung, and spleen from female rats and uterine tissue did not bind 125I-labelled prolactin appreciably, or show competition for binding in the presence of unlabelled hormone. In the testis, prolactin bound exclusively to Leydig cells; in the prostate gland, binding was localized in the secretory epithelium. Kidney tubules in the cortex displayed specific prolactin localization whereas the medulla did not bind prolactin. Adrenal medulla showed no hormone binding; however, the zona reticularis and to a lesser extent the zona fasciculata bound prolactin. In the ovary, grains were limited to the theca and to a lesser extent the corpus luteum and follicles. In all cases, binding was essentially abolished when unlabelled prolactin was included in the incubation medium. These results confirm reports of the presence of prolactin receptors in these tissues and serve to identify the cells within a particular organ that respond to prolactin.     

Insulin-like growth factor carrier proteins in neonatal and adult rat serum are immunologically different: demonstration using a new radioimmunoassay for the carrier protein from BRL-3A rat liver cells

A carrier protein for insulin-like growth factors (IGFs) has been purified from serum-free medium conditioned by the Buffalo rat liver (BRL)-3A cell line and used to immunize rabbits. Purified carrier protein was 125I labeled and affinity purified on IGF-Sepharose. The major labeled protein had a mol wt of about 33,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (appropriate for the IGF carrier protein subunit) and gave a single predominant peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and acid-urea gel electrophoresis that was immunoprecipitated by immune serum and comigrated with unlabeled proteins that bind [125I] IGF. A RIA was developed using affinity purified [125I]carrier protein and immune serum. Tracer binding was inhibited only by preparations containing IGF carrier proteins, but not by unrelated proteins or by the IGFs themselves. Carrier proteins from BRL-3A cells gave equivalent strong reactivity either after dissociation of endogenous IGF or as an IGF-carrier protein complex. The antiserum effectively recognized the approximately 40,000 mol wt (Mr approximately 40,000) carrier protein from neonatal rat serum, both as a native complex and after acid stripping. It did not effectively recognize the Mr approximately 150,000 carrier protein from adult rat serum either as endogenous complex or after acid stripping. These results suggest that the Mr approximately 40,000 carrier protein of neonatal rat serum and the Mr approximately 40,000 binding subunit of the Mr approximately 150,000 carrier protein in adult rat serum are immunologically distinct. These antisera to the BRL-3A carrier protein should be useful tools with which to dissect the relationships between different carrier protein species and to study the regulation of IGF carrier protein gene expression.     

Purification, partial characterization, and heterologous radioimmunoassay of growth hormone (cGH) in red deer.

Red deer growth hormone (cGH; 3.3 mg) was purified from an aqueous extract of seven pituitary glands (4.01 g wet weight) by preparative gel filtration on Sephadex G-100, gel filtration on Sephadex G-100 SF, and anion exchange chromatography on DEAE-Sepharose CL-6B. Purified cGH gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight under reducing conditions of 20,000 Da and gave a single peak on reverse-phase high-performance liquid chromatography. N-Terminal amino acid determination of 42 residues gave a sequence identical with those published for bovine and ovine GH. In a radioreceptor assay based on binding of iodinated recombinant bovine GH (rbGH) to liver microsomes prepared from a pregnant ewe, cGH was equipotent with an ovine GH (oGH) standard. In an oGH radioimmunoassay, cGH diluted in parallel with oGH and rbGH. Using this assay plasma GH concentrations were determined in adult nonpregnant red deer hinds over a 12-month period. There was a significant seasonality in plasma GH concentrations with concentrations consistently low between mid-May and mid-September. This is the period when voluntary food intake and liveweight gain are greatest. It is suggested that in the presence of low plasma GH concentrations nutrients may be diverted toward lipogenesis and hence promote fat deposition.     

Receptors for lactogenic hormones in the ovine corpus luteum. I: A major discrepancy in the specific binding of radiolabelled ovine prolactin and human growth hormone

The characteristics of the binding of 125I-labelled human GH (hGH) and ovine prolactin (oPRL) were studied in the ovine corpus luteum. Although oPRL is the homologous ligand for sheep lactogenic receptors, its binding was significantly and consistently lower than that of 125I-labelled hGH. This was not due to iodination damage of oPRL since: (1) 125I-labelled oPRL tracers which bound poorly relative to 125I-labelled hGH in the ovine corpus luteum were equipotent in the pig and rat corpus luteum, (2) the differences between 125I-labelled hGH and oPRL binding persisted with tracers of equivalent biopotency and (3) the iodination procedure affected neither oPRL bioactivity in the Nb2 tumour assay nor its binding activity with ovine corpus luteum receptors. Ovine luteal receptors were specific for lactogenic hormones. The specific binding of 125I-labelled hGH or oPRL could be inhibited completely by incubation with either unlabelled hormone, with similar potencies. However, oGH inhibited binding only at much higher concentrations, consistent with its known contamination with oPRL. Moreover, 125I-labelled oGH was not bound specifically to sheep luteal tissue. Fractionation of sheep luteal homogenates on sucrose density gradients (with or without cell-surface membrane perturbation by digitonin) demonstrated that binding of 125I-labelled hGH and 125I-labelled oPRL peaked in the same regions of the gradients, coincident with a number of luteal cell-surface membrane markers. We conclude that the marked discrepancy between the binding of hGH and oPRL tracers by sheep luteal tissue was not due to iodination damage of oPRL, binding of 125I-labelled hGH to somatogenic receptors or differential binding to luteal cell-surface versus intracellular receptors.     

Receptors for lactogenic hormones in the porcine corpus luteum: properties and luteal phase concentrations

Homogenates of pig corpora lutea contained specific, high-affinity receptors for ovine prolactin (oPRL) and human GH (hGH). Specific hormone binding was enhanced by divalent metal ions, but only when included in the binding reaction. Divalent metal ions did not act by increasing the recovery of bound hormone by low-speed centrifugation, but appeared to promote the formation of a more stable hormone-receptor complex. Both oPRL and hGH tracers were bound in similar amounts and with similar affinities by pig luteal homogenates and the concentrations of either unlabelled hormone required to displace specific binding of either tracer by 50% were identical. In contrast, 125I-labelled oGH failed to bind to pig luteal homogenates and oGH competed poorly for hGH or oPRL binding. Only hormones with prolactin-like activity competed for 125I-labelled oPRL binding. Specific prolactin binding was low in recently ovulated and early luteal phase corpora lutea, increased significantly in the mid-luteal phase and declined once more in the late luteal phase. Receptor concentrations increased with increasing gestational age.     

Partial biochemical and biological characterization of purified chicken growth hormone (cGH). Isolation of cGH charge variants and evidence that cGH is phosphorylated

Chicken growth hormone (cGH) was purified from frozen pituitary glands obtained from recently sacrificed broilers. Glands were homogenized in a protease inhibitor solution (0.5 mM PMSF, 50 KIU/ml aprotinin, pH 7.2); extract was taken to pH 9.0 with calcium hydroxide and the supernatant was differentially precipitated with 20% (fraction A) and 50% (fraction B) ammonium sulfate. cGH (fraction B-DE-1) was obtained in pure form from fraction B after DEAE-cellulose chromatography at pH 8.6, with a yield of 2.9 mg/g tissue. Three charge variants of cGH (Rf = 0.23, 0.30, and 0.35) could be isolated by electroelution after semipreparative nondenaturing polyacrylamide gel electrophoresis of fraction B-DE-1. These charge variants showed the same apparent molecular weight (26,300 Da) by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing of fraction B-DE-1 revealed two major components (pI = 7.2 and 7.4) and four minor bands (pI = 6.2, 6.7, 7.1, and 7.5). It was found that fraction B-DE-1 contained a significant amount of esterified phosphate (1 nmol PO4/3.5 nmol protein) similar to that reported previously for ovine GH. The functional integrity of the cGH obtained here was characterized by two heterologous and one homologous bioassays. High activity was shown by fraction B-DE-1 in the tibia assay (1.76 UI/mg) and in the liver ornithine decarboxylase assay (sixfold over control), both made in hypophysectomized rats; and it also stimulated lipolysis (138 and 215% at 10 and 100 ng/ml, respectively) on chicken abdominal adipose tissue explants.     

Specific binding sites for LH/chorionic gonadotrophin, low-density lipoprotein, prolactin and FSH in homogenates of human corpus luteum. I: Validation of methods

The specific binding of 125I-labelled human chorionic gonadotrophin (hCG), human low-density lipoprotein (hLDL), human FSH (hFSH) and human prolactin (hPRL) to homogenates of human corpus luteum tissue was measured. Specific binding of 125I-labelled hCG was dependent on the temperature and duration of incubation, was inhibited by divalent metal ions or chelating agents, and increased linearly with homogenate concentration. Recovery of bound hormone was more effective using Millipore filtration or polyethylene glycol precipitation compared with centrifugation alone. Binding of 125I-labelled hCG was inhibited specifically by low levels of hCG and human LH (hLH) but not by ovine LH or bovine LH. Incubation of human luteal tissue with ice-cold citrate buffer (pH 3) released more than 90% of specifically bound 125I-labelled hCG within 5 min. This treatment inactivated LH receptors, but did not affect the immunoactivity of hLH released, enabling the measurement of released hormone by radioimmunoassay. Scatchard plots of binding of 125I-labelled LDL to human corpus luteum demonstrated a single class of binding sites. Binding was saturable, increased linearly with increasing concentration of homogenate, and was displaceable by low concentrations of unlabelled LDL. Binding of 125I-labelled hPRL to human luteal homogenates was increased by Mg2+ and was specific for lactogenic hormones (human prolactin, human growth hormone and ovine prolactin). Binding of 125I-labelled hFSH was not dependent on divalent metal ion concentration (in marked contrast to hFSH binding to immature pig granulosa cell receptors) and was displaced by hFSH preparations but not by hPRL, ovine LH or hCG at 1 microgram/ml. These results establish optimal conditions and hormone specificities for the measurement of human luteal gonadotrophin and LDL receptors, and methods for the estimation of hLH/hCG endogenously bound to human corpus luteum tissue.     

Somatotrophic receptors in hepatic tissue of the developing male pig

The development of hepatic somatotrophic receptors and plasma concentrations of insulin-like growth factor-I (IGF-I) were investigated at five different ages (2, 20, 35, 105 and 165 days) in four male pigs per group. The specific binding of 125I-labelled porcine GH (pGH) to hepatic somatotrophic membranes was very low at 2 days of age (0.53 +/- 0.12%), and increased progressively (P less than 0.01) with advancing age to 3.60 +/- 0.95% at 165 days of age. Specific binding of 125I-labelled bovine GH (bGH) to the same membrane preparations was markedly higher than binding of 125I-labelled pGH; it also showed a distinct developmental increase (P less than 0.01) with age from 4.4 +/- 0.55% at 2 days of age to 24.0 +/- 1.90% at 165 days of age. Plasma concentrations of IGF-I increased significantly (P less than 0.01) from 79 +/- 14.0 micrograms/l at 2 days of age to 610 +/- 64.0 micrograms/l at 165 days of age. Non-linear regression analysis of the competitive binding data using bGH as labelled and unlabelled ligands showed linear Scatchard plots in the three youngest age groups, with an association constant (Ka) of approximately 3.5 litres/nmol. Curvilinear Scatchard plots were observed in the two oldest age groups. The Ka for the higher affinity binding site (approximately 5.0 litres/nmol) was very similar to that for the sole site observed in the younger animals. The Ka of the lower affinity binding site was approximately 0.35 litres/nmol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific interactions of growth hormone (GH) with GH-receptors and GH-binding proteins in vivo in genetically GH-deficient Ames dwarf mice.

The fate of exogenous radiolabeled growth hormone (125I-hGH) was studied in Ames dwarf mice, which do not express growth hormone (GH) or prolactin (PRL) genes. Labeled GH was injected in low amounts that did not exceed the normal physiological GH concentration in mice. Binding of most of the injected 125I-hGH by the GH-binding proteins (GHBPs) present in plasma represents the first step in the handling of this material in vivo. The decay curve followed a two-compartment model and gave the equation: Conc = 2.807e-0067t + 15301e-0.0647t (coefficient of determination 0.9986+/-0.0019), while in normal mice, GH decay followed a three-compartment model as we have previously reported. The fast compartment with t1/2 of 1-2 min was virtually absent in dwarf mice, and chromatographic studies revealed the disappearance of free GH in these mice. We also present evidence of the labeled GH-forming complexes, presumably with GHBPs under in vivo conditions. The second step of processing labeled GH in vivo is the uptake by the liver, which was slower in dwarf than in normal mice (30-45 vs 15 min). Moreover, a lower GH uptake was found in dwarf than in normal mice (UB ratio of 1.75+/-0.29 [30 min] vs L/B ratio of 3.68+/-0.33 [15 min], respectively) due to lower concentration of free GH in plasma and to the reduced number of GH-receptors (GHRs). The radioactive material present in the liver was compatible with 125I-hGH-GHR complexes with Stokes radius of 59A. In summary, we provide evidence that plasma of dwarf mice contains proteins capable of binding GH in vivo and probably representing GHBPs not complexed with GH. The presence of these proteins modified the pharmacokinetics of 125I-hGH in plasma and its subsequent uptake by the liver. The presence of these binding proteins in the absence of endogenous GH suggests that a fraction of total GHBPs (one class?) is independent of GH concentration.     

Identification of growth hormone molecular variants in chicken serum.

It has been described that pituitary growth hormone shows molecular and functional heterogeneity. In birds, size and charge variants of chicken growth hormone (cGH) have been shown in the chicken pituitary gland and in purified preparations of the hormone. Here we demonstrate the existence of cGH molecular isoforms in chicken serum, thus suggesting that they are secreted from the gland. The isolation of total cGH present in sera was performed by immunoaffinity chromatography employing a specific monoclonal antibody against cGH. Different analytical electrophoretic methods (SDS-polyacrylamide gel electrophoresis, isoelectric focusing, bidimensional polyacrylamide gel electrophoresis) followed by Western blot and immunostaining were employed to characterize the serum cGH isoforms, and compared to those present in a fresh pituitary extract. Several identical immunoreactive bands comigrated in both serum and the gland extract in the different systems (SDS-PAGE, MW 16, 22, 26, 29, 52, 62, 66 kDa; IEF, pIs 8.1, 7.5, 7.1, 6.8, 6.2), thus revealing a high correspondence of molecular isoforms of the hormone in the two tissues. Additionally, a glycosylated variant of chicken growth hormone (G-cGH) was also revealed in the serum after concanavalin A-Sepharose chromatography.     

A radioreceptor assay for purified teleost growth hormone.

Highly purified ¹²⁵I-labelled tilapia (Sarotherodon mossambicus) growth hormone (GH) binds to membrane fractions prepared from tilapia liver. Specific (displaceable) binding occurred with the 600, 15,000, and 90,000g fractions with the greatest binding observed with the 90,000g microsomal membrane fraction. Binding was dependent on time and membrane concentration. Specificity studies showed that up to 60% of the ¹²⁵I-labelled tilapia GH bound to the liver microsomal membrane fraction could be displaced by 500 ng of unlabelled hormone. Scatchard analysis of the binding of ¹²⁵I-labelled tilapia GH revealed a single class of binding site with a binding capacity of 125 ± 7.7 fmol/mg protein and a binding affinity of 1.5 × 10¹⁰ ± 0.4 × 10¹⁰ (SEM) l/mol. GH preparations from several vertebrate classes and tilapia prolactin and ovine prolactin at high concentrations displayed competition for the tilapia GH-binding site.¹²⁵I-Labelled tilapia GH demonstrated specific binding to liver microsomal membrane fractions of the teleosts Gillichthys mirabilis, Salmo gairdneri, and Oncorhynchus tschawytscha but not to those of an amphibian Necturus maculosus or a bird Coturnix japonica. Slight, but significant, specific binding was observed with the microsomal membrane fraction of tilapia kidney and gill and of rat liver. These data suggest that this tilapia GH radioreceptor assay may have an application in the detection of teleost GH-like activity.     

Characterization of a specific insulin-like growth factor-I/somatomedin-C receptor on high density, primary monolayer cultures of bovine articular chondrocytes: regulation of receptor concentration by somatomedin, insulin and growth hormone

In order to obtain a phenotypically stable cell population of chondrocytes, high density primary monolayer cultures of bovine articular chondrocytes were established. Using these cultures, a specific insulin-like growth factor-I/somatomedin-C (IGF-I/SM-C) receptor was demonstrated and characterized. At 15 degrees C steady-state binding was attained by 5 h, and averaged 25% per 2.2 X 10(6) cells. Fifty per cent displacement of 125I-labelled IGF-I/SM-C by unlabelled IGF-I/SM-C occurred at concentrations of only 2.3 ng/ml, whereas IGF-II and porcine insulin were approximately 15- and 1000-fold less potent respectively. Scatchard analysis gave a linear plot, with a calculated association constant of 2.26 X 10(9) l/mol and a receptor number of 15 400 sites per cell. Preincubation of chondrocyte monolayers with either IGF-I SM-C or porcine insulin at 37 degrees C for 20 h resulted in reduction of 125I-labelled IGF-I/SM-C binding in a dose-dependent manner, although higher concentrations were required with insulin. More than 40% down-regulation of the receptor occurred with IGF-I/SM-C at concentrations of 10 nmol/l and nearly 70% reduction at 50 nmol/l. Interestingly, after preincubation with either human (h) or bovine (b) GH, 40% down-regulation of 125I-labelled IGF-I/SM-C binding was observed at concentrations of 10 mumol/l. Local production of IGF-I/SM-C by chondrocytes in response to GH stimulation may have occurred, but, because only 120 pmol IGF-I/SM-C and less than 30 pmol IGF-I/SM-C per litre were recovered from serum-free conditioned media preincubated with bGH and hGH respectively, this was not established.(ABSTRACT TRUNCATED AT 250 WORDS)