Specific Growth Hormone Binding Research Articles

Molecular cloning and characterization of growth hormone receptor and its homologue in the Japanese eel ( Anguilla japonica)

Two cDNAs encoding growth hormone receptor (GHR)-like genes, eGHR1 and eGHR2, were isolated from Japanese eel ( Anguilla japonica) liver tissue. The putative eel GHR proteins showed conserved structural features of vertebrate GHRs, including six cysteine residues and a YGEFS motif in the extracellular domain, a single transmembrane region, and proline-rich box 1 and box 2 domains. Northern blot analysis showed a single eGHR1 transcript in liver, while two sizes of eGHR2 transcripts, thought to be produced by alternative splicing, were present. RT–PCR revealed that eGHR1 and eGHR2 transcripts were widely distributed throughout the whole body of the Japanese eel. Moreover, the results of binding assays showed the specific binding of growth hormone to recombinant eGHR1. Since these putative eGHR proteins show all characteristics of the GHR family, we conclude that eGHR1 and eGHR2 cDNA encode two different GHRs in Japanese eel. We confirmed the ligand specificity of eGHR1 by binding assay, and further research is needed to allow characterization of the binding capability of eGHR2.

Inability of heterologous growth hormone (GH) to regulate GH binding protein in GH-transgenic swine.

Transgenic pigs expressing bovine, ovine, or human growth hormone (GH) structural genes fused to mouse metallothionein-I (mMT-bGH), ovine MT (oMT-oGH), or mouse transferrin (mTf-hGH) promoters were used to study the effects of GH on the regulation of serum GH-binding protein (GHBP). In the 14 transgenic pigs studied, circulating concentrations of heterologous GH ranged from 15 to 2,750 ng/mL. Using chromatographic methods, specific binding of GH was detected in serum from normal pigs but was undetectable in serum from all the transgenic pigs used, probably as a result of the high serum concentrations of heterologous GH present in these animals. Thus, to avoid interference of binding by high GH concentrations, serum samples were subjected to immunoblotting using a specific anti-GHBP antibody. A specific 54-kDa band was detected in normal pig serum as well as in sera from mMT-bGH, oMT-oGH, and mTf-hGH pigs. Additionally, sera from transgenic mMT-bGH pigs and their sibling controls were subjected to immunoprecipitation with an anti-GHBP antibody followed by immunoblotting with the same antibody. With this technique, we detected two specific bands of 53 and 45 kDa that could represent different degrees of glycosylation of GHBP. As determined by densitometric analysis the amount of GHBP in transgenic pig sera was similar to that detected in sera of the respective control animals. The amount of circulating GHBP remained unchanged even in oMT-oGH and mTf-hGH pigs that were exposed from birth to circulating concentrations of GH as high as 2,750 ng/mL. Thus, we conclude that heterologous GH do not act as modulators ofthe serum GHBP in pigs.

Growth Hormone Receptors and Insulin-like Growth Factor I Receptors on Human Peripheral Blood Cells

Growth hormone (GH) and insulin-like growth factor I (IGF-I) may be able to modulate some functions of the immune system. In this study, we examined the expression of the receptors for GH and IGF-I on human peripheral blood cells in physiological and pathological conditions.By using two-colour analysis on flow cytometry with a biotinavidin system, there was a specific GH binding site on human peripheral lymphocytes. GH-binding was more prominent on CD20+ (=B cells) than on CD2+ (=T cells), although GH receptor messenger ribonucleic acid (mRNA) could be detected in the T cells as well as in the B cells. There was no age-dependency of GH-binding on the CD20 cells, and there was no relationship between GH-binding and serum GHbinding protein activities, which reflect the tissue levels of GH receptors.We detected αIR3, anti IGF-I receptor antibody, bindings and IGF-I receptor mRNA expression in peripheral blood cells. Based on two-colour analysis, there were relatively higher bindings of αIR3 on CD4+ and CD16+ cells, intermediate bindings on CD8+ cells and weak bindings on CD20+ cells. In cord blood cells, αIR3 bindings and IGF-I receptor mRNA were higher than in adult cells. In GH-deficient patients, αIR3 bindings were higher than in control subjects, and reverted to normal after GH therapy.These results suggest that 1) GH and IGF-I may have hormonal effects on the immune system, 2) GH receptors on lymphocytes dose not correlate with the tissue levels of GH receptors, and 3) the cellular levels of IGF-I receptors may be inversely related to the ambient IGF-I concentrations.

Upregulation of GH receptor and GH binding protein during pregnancy in the GH deficient rat

During pregnancy there are dramatic changes in the endocrine and metabolic status of the mother: growth hormone (GH) is an important regulator of growth and development. A proportion of GH is bound by specific GH binding proteins (GHBP) that closely resemble the GH receptor (GHR). In the rodent both GHBP and the GHR are considered to be GH dependent, and consequently during pregnancy the increase in serum GH is associated with an increase in GHBP. To examine whether an increase in maternal GH is obligatory for elevation of maternal GHBP or GHR during pregnancy, we used a unique GH-deficient (GHD) strain of rats, to avoid the methodological complications of hypophysectomy and assessed serum GH, GHBP and hepatic GHR binding during the course of pregnancy. In GH normal rats, serum GH concentrations increased twofold and GHBP levels increased threefold; there was no change in hepatic GHR binding. In GHD rats, serum GH concentrations were low and did not increase during pregnancy. Nonetheless, levels of both serum GHBP and hepatic GHR binding increased to that measured in normal rats. Thus, an increase in maternal GH concentration is not required for the gestational upregulation of maternal GHBP or hepatic GHR binding, suggesting that other hormones may be essential in modulating the GH axis during pregnancy.

Pulsatility of plasma growth hormone and hepatic growth hormone receptor characteristics of broiler chickens divergently selected for abdominal fat content.

1. Plasma growth hormone (GH) pulsatility and hepatic GH receptor characteristics were compared in experimental lines of meat-type chickens selected for high (HF) or low (LF) abdominal fat content. 2. Mean GH concentration, baseline and amplitude of pulses were slightly, but not significantly, greater in LF chickens. Length and frequency of pulses were similar. 3. LF chickens exhibited higher plasma triiodothyronine (T3) concentrations. This difference between genotypes disappeared when the diet was supplemented with 1 mg/kg T3. 4. Specific binding of GH to liver membranes was higher for the fat line but was depressed by T3 supplementation to the same level in both lines. No difference was observed between lines for affinity constants. 5. It is concluded that direct selection for leanness has a less pronounced, if any, effect on GH pulsatility as compared with selection for food conversion efficiency; therefore, different physiological mechanisms are triggered to achieve leanness.

Immunoreactive growth hormone receptor/binding protein is present on fibroblasts and in serum of Laron-type dwarfs

Laron-type dwarfism is an autosomal recessive disorder characterised by extreme growth retardation and growth hormone (GH) resistance and has been shown in some cases to be associated with mutations in the GH receptor gene. Limited data suggest that in this condition specific liver GH binding is absent. In the majority of reported cases specific GH binding is also absent in serum. However it is not known whether the GH receptor and/or the serum GH binding protein are expressed in this condition. Using the techniques of immunohistochemistry and Northern blotting we have demonstrated that in cultured skin fibroblasts derived from four patients with Laron-type dwarfism the GH receptor gene is transcribed and the GH receptor protein is expressed on the cell surface. Further study of one of these patients, who has not previously been reported, has also revealed low but detectable levels of GH binding protein in serum using a two-site immunradiometric assay which does not depend on GH binding. These results indicate that the growth hormone receptor/binding protein is expressed in Laron-type dwarfism.

Subcellular distribution of the long and short isoforms of the growth hormone (GH) receptor in rat adipocytes: both isoforms participate in specific binding of GH

Subcellular distribution of the long and short isoforms of the growth hormone (GH) receptor in rat adipocytes: both isoforms participate in specific binding of GH

Study of the hepatic growth hormone (GH) receptor at different ages in chickens selected for a good feed conversion (FC) and a fast weight gain (GL)

The influence of genetic selection for improved feed conversion (FC) and fast growth (GL) in broiler chickens, on the hepatic growth hormone receptor was investigated as a function of age. Selection for improved feed conversion resulted in lean chickens, whereas selection for fast growth resulted in fat chickens. Growth hormone receptor characteristics were determined in liver microsomal fractions and plasma GH concentrations were measured by radioimmunoassay. Embryos from the FC line showed higher specific GH binding than GL embryos at day 16 of embryonic development only (P<0.05). On day 18 of embryonic development, the FC line had slightly more GH receptors than the GL line (P<0.05), whereas simultaneous plasma GH concentrations were greater in the GL line. The effect of selection on hepatic growth hormone binding was greatest in 4 week old growing chicks at which age the GL line showed a 2- to 3-fold higher % of specific binding than the FC line (P<0.001). The difference at this stage is probably due to down regulation of the receptor by the significantly higher GH concentrations in the FC line. The adult GL chickens also showed higher specific binding than FC, but the difference was less pronounced than in the growing stage (P<0.05). This was due to a higher number of GH receptors, while receptor affinity was unchanged. Because no line difference in plasma GH concentrations was found in adult hens, other unknown mechanisms probably play a role in determining differences in GH receptor binding between these selected lines at older ages.

Establishment of a growth hormone responsive chondrogenic cell line from fetal rat tibia

Reproducible effects of growth hormone (GH) on primary isolated cells in monolayer are highly dependent on the culture conditions and/or the fraction of GH responsive cells. To study the effect of GH at the cellular level, a homogenous cell line with both GH responsiveness and chondrogenic properties was established. Primary isolated cells from 18-day-old fetal rat tibia were subcultured using a strict protocol for passages (every third day and a seeding density of 15,000/cm 2). Of six established cell lines, one fetal tibia cell line No. 5 (FTC 5) expressed adipogenic and chondrogenic properties at a low frequency. Cells from FTC 5 were subcultured in soft agar suspension with the addition of bovine GH (100 ng/ml). After 14 days in culture eight monoclonal cell lines were established from individual large colonies. Two subclones, FTC 5:3 and FTC 5:6, expressed a chondrogenic phenotype as demonstrated by chondrocyte foci, alcian blue staining and production of type II collagen. Further characterization of FTC 5:3 revealed specific binding of bovine GH with an affinity of 1.7 × 10 9 M −1, and approximately 7300 receptors/cell. Northern blot analysis of FTC 5:3 with a 32P-labeled RNA probe complementary to an extracellular part of the rat GH receptor, revealed two major labeled bands (4.0 and 1.2 kilobases). Both GH and insulin-like growth factor-I (IGF-I) stimulated 3H-thymidine uptake in FTC 5:3 (194 ± 28% and 405 ± 127% over control, respectively), while proteoglycan synthesis, as measured by [ 35S]sulphate uptake, was stimulated by IGF-I only (101 ± 18% over control). In conclusion, a chondrogenic cell line was established that expressed GH receptors and exhibited GH responsiveness.

Serum concentrations and peritoneal loss of growth hormone and growth-hormone-binding protein activity in older adults undergoing continuous ambulatory peritoneal dialysis: comparison with haemodialysis patients and normal subjects

Serum concentrations and peritoneal losses of growth hormone (GH) and of growth-hormone-binding protein (GH-BP) activity in 13 patients undergoing continuous ambulatory peritoneal dialysis (CAPD) were compared with those of 13 patients on haemodialysis and 13 normal subjects. The individuals in the three groups were matched by age (40-83 years), gender, and serum glucose concentration. In addition CAPD and haemodialysis patients were matched by haematocrit, serum creatinine and albumin concentrations, and period of time on dialysis (0.5-127 months). GH in the serum and in the peritoneal effluent were measured by radioimmunoassay (RIA) and NB2 bioassay. GH-BP activity was analysed by binding assay and expressed as a percentage of the specific binding of GH. In the haemodialysis patients, serum GH was significantly greater and serum GH-BP activity significantly less than in the CAPD patients and control subjects. Between the two latter groups no significant differences in GH or in GH-BP activity were observed. GH bioactive/immunoactive ratios in the three groups were similar. Both GH and GH-BP were detected in the peritoneal effluent of CAPD patients, in whom an overnight (8-h) peritoneal loss of GH (8.0 +/- 1.4 x 10(-3) micrograms/h/1.73 m2) was strongly correlated with serum GH (r = 0.840). Circulating GH and GH-BP activity were influenced by serum creatinine and haematocrit. In addition a positive relationship was observed between GH-BP activity and body mass index and between GH and time on dialysis. These data reaffirm that older adults undergoing haemodialysis, unlike CAPD patients, exhibit persistent abnormalities in GH-GH-BP axis.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective membrane expression of human growth hormone (GH) receptor causes Laron-type GH insensitivity syndrome.

Mutations in the growth hormone receptor (GHR) gene can cause growth hormone (GH) resistance. Given the sequence homology between the extracellular domain of the GHR and a soluble GH-binding protein (GH-BP), it is remarkable that GH-BP binding activity is absent from the serum of patients with Laron-type GH insensitivity, a hereditary form of severe dwarfism. We have previously identified a mutation within the extracellular domain of this receptor, replacing phenylalanine by serine at position 96 of the mature protein, in a patient with Laron syndrome. We have now investigated the effect of this Phe----Ser substitution on hormone binding activity by expressing the total human GHR cDNA and mutant form in eukaryotic cells. The wild-type protein expressed was able to bind GH but no plasma membrane binding was detectable on cells transfected with the mutant cDNA; this was also the case of cells transfected with a Phe96----Ala mutant cDNA, suggesting that the lack of binding activity is not due to a posttranslational modification of serine. Examination of the variant proteins in subcellular fractions revealed the presence of specific GH binding activity in the lysosomal fraction, whereas immunofluorescence studies located mutant proteins in the cytosol. Our findings suggest that these mutant GHRs fail to follow the correct intracellular transport pathway and underline the potential importance of this phenylalanine residue, which is conserved among the GH, prolactin, and erythropoietin receptors that belong to the same cytokine receptor superfamily.

Effects of hypo or hyper-thyroidism on growth hormone-binding protein

Growth hormone (GH) receptors are influenced by the thyroidal state of experimental animals. It has been suggested that GH-binding protein (BP) might serve as an indirect measure of the GH receptors. The present study was undertaken to investigate the growth hormone binding protein in patients with hypo or hyperthyroidism. Patients included 42 adults with untreated hyperthyroidism (FT4 greater than 25 pmol/l; TSH less than 0.15 mU/l) and 29 adults and three children with untreated hypothyroidism (FT4 less than 10 pmol/l; TSH greater than 15 mU/l). Growth hormone binding protein was measured by a binding assay with dextran-coated charcoal separation. The specific binding of 125I-human GH (1 ng) obtained with 50 microliters serum was expressed as a percentage of the total c.p.m. Growth hormone binding protein specific binding in hypothyroid adults and children was significantly lower than in their respective controls in both adults and children (P less than 0.001). In patients with FT4 levels greater than 40 pmol/l, the mean (+/- SEM) growth hormone specific binding (12.89 +/- 0.59%) was higher than in controls (P less than 0.05). However, in 15 hyperthyroid patients with levels of FT4 of 25-40 pmol/l, the mean growth hormone binding protein specific binding (11.22 +/- 0.76%) was not different from that in normal human subjects. The affinity constants (Ka) obtained by Scatchard analysis for the hypothyroid and hyperthyroid patients' sera were not significantly different from that for normal human sera. Binding capacity for the hypothyroid sera was significantly lower (P less than 0.02), while that of hyperthyroid sera was increased (P less than 0.001). Growth hormone binding protein correlates positively with the thyroid status. It can be indirectly deduced that this reflects a similar relationship with the human GH receptor.

Human Plasma Growth Hormone (GH)-Binding Proteins Are Regulated by GH and Testosterone*

Possible regulation of GH-binding proteins (GH-BPs) in human plasma was examined. Eight children with isolated GH deficiency had a very low level of plasma GH-binding activity (10.2 +/- 1.1% of radioactivity). Under GH treatment the hormone binding to the high affinity BP (peak II-BP) increased in every patient to reach the mean value of 18.5 +/- 1.4%. In one patient, Scatchard plot analysis indicated that this increase was related to a higher binding capacity without any significant change in the binding affinity. A positive correlation existed between the GH-binding activity and insulin-like growth factor-I plasma levels. In nine boys with pubertal delay, the GH-specific binding to peak II-BP was normal (30.6 +/- 3.7% of radioactivity); it decreased significantly after testosterone treatment. In four boys with precocious puberty, the specific GH binding to peak II-BP was low (16.6 +/- 1.1%). It increased significantly to 21.6 +/- 1.1% of radioactivity after treatment with a LHRH analog. A negative correlation existed between plasma testosterone levels and GH binding to peak II-BP in boys presenting pubertal delay or precocious puberty. The high affinity GH-BP is regulated, and among the factors that play a role in this regulation, GH and testosterone have opposite effects.

Effect of hypophysectomy and acute administration of growth hormone (GH) on GH-receptor binding in chick liver membranes

The effects of hypophysectomy on GH binding to liver membranes of young chicks were studied 3 days and 1 week after surgery. Specific binding of 125I-labelled chicken GH (cGH) to MgCl2-treated liver microsomal fractions of hypophysectomized animals was two- to fivefold greater than to those of sham-operated or control (non-operated) birds. This effect was due to a rise in binding capacity rather than a change in binding affinity of the GH receptor. Two daily injections of cGH (20 micrograms/animal) returned the number of hepatic GH receptors from hypophysectomized chicks to the level of the sham-operated ones. Administration of GH to the latter group did not cause a significant lowering of specific binding or number of receptors. No positive correlation between GH binding and plasma concentrations of insulin-like growth factor-I (IGF-I) was observed; although GH binding increased, IGF-I levels were lower for the hypophysectomized group. Since the number of hepatic GH receptors and the plasma GH levels were inversely correlated, it was concluded that the GH receptors in the liver of the chicken can be down-regulated by GH. This possibly explains why GH binding is low in posthatch and young chicks, because circulating GH concentrations are high during this period.

Influence of nutrition and bovine growth hormone (GH) on hepatic GH binding, insulin-like growth factor-I and growth of lambs

The effect on young lambs of 0.25 mg recombinant bovine GH (bGH)/kg per day on plasma concentrations of insulin-like growth factor-I (IGF-I), glucose, specific hepatic GH binding and body composition changes was examined at two levels of nutrition (lucerne pellets; 3 and 1.7% of body weight/day). Lambs on low levels of nutrition had low plasma IGF-I (P less than 0.001). Plasma concentrations of IGF-I were increased by bGH treatment at both levels of nutrition, with the high nutrition group showing the greatest IGF-I response after 3 and 40 days of bGH treatment. Plasma glucose, after 40 days, was higher overall (P less than 0.05) in lambs on high nutrition. bGH treatment increased plasma glucose, with the response being greater in the well-fed lambs. Specific binding of GH to liver membranes was highest in lambs on high nutrition and on bGH treatment; no significant interaction between nutrition and bGH treatment was detected, indicating that specific binding of GH was increased proportionally by bGH at both nutritional levels. The major change in body composition was the reduced level of fatness in lambs treated with bGH. There was no significant effect of bGH on body weight although bGH treatment tended to increase weight gain of well-fed lambs and decreased weight loss of poorly nourished lambs. The results show that, although there was a significant (P less than 0.05) bGH/nutrition interaction for IGF-I there was no such interaction for body weight/components or specific GH binding to the liver. The results indicate that an increase in plasma IGF-I does not necessarily result in increases in growth or changes in carcass composition.

Differences in hepatic growth hormone receptor binding during development of normal and dwarf chickens

The aim of this study was to compare the growth hormone (GH) receptor in liver microsomal fractions of normal chickens (Dw) and chickens carrying the dwarf gene (dw). Specific binding of GH to its hepatic receptor was significantly higher for Dw embryos from d 14 till d 20 of incubation than for dw embryos. The difference in binding was due to a decreased binding capacity but not affinity in the livers of the dwarf embryos. The same binding pattern was found in livers of adult chickens: lower binding was again caused by a lower number of GH receptors and at this stage the difference was even clearer than during embryonic development. Binding studies on livers of growing chicks demonstrated that binding was low for both genotypes, but a small though significant difference between them remained. The cause of this decrease in number of GH receptors in dwarf birds has yet to be determined but may be due to the primary action of the dwarf gene.

Growth Hormone (GH) Stimulates Protein Synthesis in Cells Transfected with GH Receptor Complementary DNA

An expression vector containing a rat GH receptor cDNA was transfected into Chinese hamster ovary (CHO) cells, and stable cell lines expressing GH receptors were established. In contrast to nontransfected CHO cells, expression of GH receptors in transfected cells resulted in the appearance of high affinity (Kd = 1.53 nM) specific binding of GH. Cross-linking of [125I]hGH to the receptors and subsequent sodium dodecyl sulfate (SDS)-electrophoresis gave an estimated receptor mol wt of 84,000. GH treatment stimulated protein synthesis 60% over basal levels in GH receptor-expressing CHO cells, but not in the receptor-negative parental cells. The effect was observed only under serum-free conditions and was time and dose dependent. These results show that heterologous expression of the rat GH receptor results in the appearance of specific binding of GH and the acquisition of a functional GH response.

Ontogeny of growth hormone (GH) binding in the domestic turkey: evidence of sexual dimorphism and developmental changes in relationship to plasma GH

The post-hatch ontogeny of hepatic GH binding and its relationship to GH plasma profile characteristics in male and female turkeys of slow- (RBC-2) and fast-growing (F; selected from RBC-2) genetic lines were determined. Specific binding of 125I-labelled recombinant chicken GH to crude hepatic membrane preparations (100,000 g pellet) was determined at 2, 4, 8, 14 and 24 weeks of age for both total (occupied plus free; 4 mol MgCl2/l pretreatment) and free (without MgCl2 pretreatment) binding sites. Characteristics of the plasma GH profile were measured at each age by serial blood sampling through indwelling jugular vein catheters. When specific binding to either free or total sites was expressed on a whole organ basis (i.e. hepatic GH-binding capacity/bird), binding increased dramatically (P less than 0.0001) with increasing age over both lines and sexes. Total binding capacity (free plus occupied sites) per bird was greater for females than for males at 24 weeks of age (P less than 0.04), as birds reached sexual maturity, but did not differ between fast- and slow-growing lines at any age. Available binding capacity (free sites) per bird was greater for the faster growing F than RBC-2 line at the older ages when body size was most divergent (14 and 24 weeks of age; P less than 0.01, P less than 0.06 respectively), but did not differ between sexes. Correlation analysis at individual ages revealed a progressive change in the nature of the relationship between hepatic GH binding, plasma GH and somatic growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of growth hormone receptors in cultured rat epiphyseal chondrocytes by specific binding of growth hormone and immunohistochemistry.

Growth hormone has been reported to exert direct effects on rat and rabbit epiphyseal chondrocytes in vitro, indicating that GH interacts with specific receptors on these cells. To investigate this possibility, binding of GH to cultured rat epiphyseal chondrocytes was studied under various experimental conditions. Chondrocytes were isolated enzymatically from epiphyseal growth plates of the proximal tibia of 20-day-old male rats and were cultured in monolayer in Ham's F-12 medium supplemented with 10% calf serum and 1% of a serum substitute. The cells were seeded at various densities (25,000-200,000 cells/well) and cultured for 5-16 days. Twenty-four hours before binding experiments, the medium containing calf serum was changed for one containing serum obtained from hypophysectomized rats, in order to avoid binding of GH present in the calf serum. Binding was studied by incubating 125I-labelled human GH (hGH) with the cells in the presence or absence of various concentrations of unlabelled hGH, bovine GH (bGH), rat GH (rGH) and ovine prolactin (oPRL). Specific binding could be demonstrated in cells cultured for 5-16 days. Binding was dependent upon time and temperature, and maximal binding was obtained by incubating the labelled hormone for 4-6 h at 24 degrees C. An increase in binding was noted between 7 and 12 days in culture. In cells cultured for 12 days, addition of unlabelled hGH, bGH or rGH caused a dose-dependent displacement of 125I-labelled hGH, whereas oPRL was ineffective. Scatchard analysis resulted in a linear plot, and the number of binding sites/cell was approximately 5700, with a dissociation constant of 0.46 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of GH binding to specific cellular receptors in vitro--a new model of growth regulation in vivo.

A new theory on the complex growth hormone (GH) mediated promotion of tissue growth has been developed on the basis of in vitro studies of growth hormone receptors on human peripheral mononuclear cells (PMC). It is hypothesized that GH acts through its tissue receptors and regulates its own receptors through two different pathways: first GH directly downregulates GH binding, second a partially GH-dependent serum factor (the so-called SM-B) enhances GH binding. Some experimental evidence for this hypothesis is presented using a new method to investigate GH receptors in circulating human blood cells: The effect of trypsin, antitrypsin, growth hormone, somatomedin-B (SM-B) and anti-SM-B-antiserum on GH binding to PMC was studied. Trypsinization of cells leads to a decrease both of specific binding and of binding affinity (affinity constant after 60 minutes of trypsinization 0.5 X 10(6) M-1 versus 1.5 X 10(6) M-1 in untreated control cells). Exposure of PMC to antitrypsin activities was followed by an increase of binding affinity and specific binding (affinity constants with 10 KIU 1.9 X 10(6) M-1, with 100 KIU 2.4 X 10(6) M-1, with 1000 KIU 3.6 X 10(6) M-1). This antitrypsin effect exceeds the binding values expected after blocking trypsin activities possibly being present in the incubation medium. In a subset of experiments the partially GH-dependent serum factor SM-B was used as the antitrypsin moiety and was shown to increase specific GH binding to PMC in a similar manner as did antitrypsin (with 1000 ng SM-B affinity constant 12.0 X 10(6) M-1, specific binding 9.7%).(ABSTRACT TRUNCATED AT 250 WORDS)