scholarly journals Use of calcium dependence as a means to study the interaction between growth hormones and their binding proteins in rabbit liver

1988 ◽  
Vol 250 (2) ◽  
pp. 533-538 ◽  
Author(s):  
R Barnard ◽  
M J Waters
Keyword(s):  
Growth Hormone ◽  
Binding Proteins ◽  
Specific Binding ◽  
Binding Protein ◽  
Liver Microsomes ◽  
Human Growth ◽  
Rabbit Liver ◽  
Scatchard Analysis ◽  
Calcium Dependence ◽  
High Affinity

The affinity of 22,000-Mr human growth hormone (22 K-hGH) for GH binding proteins in rabbit liver is increased approx. 19-fold by 25 mM-Ca2+. In contrast, ovine growth hormone (oGH) binding is Ca2+-independent up to 10 mM, and decreased by greater Ca2+ concentrations. The 20,000-Mr hGH variant (20K-hGH), lacking residues 32-46, exhibits intermediate behaviour. Without Ca2+ there is a residual 40% of maximum specific binding to liver microsomes, and this increases to 65% with liver cytosolic GH binding proteins. In contrast with 22K-hGH, Scatchard analysis of 20K-hGH binding to liver microsomes produces curvilinear plots in the presence of 25 mM-Ca2+. From these results and inhibition studies with monoclonal antibodies to the GH binding proteins, it is concluded that deletion of the region 32-46 from 22K-hGH has eliminated one component of high-affinity Ca2+-potentiable binding. The Ca2+-mediated increase in Ka for the 22K-hGH-binding protein interaction is consistent with convergence of unit negative charges on the hormone and binding protein towards an intercalated Ca2+ ion. A positive charge in the critical region of nonprimate GHs would render their interactions Ca2+-independent and of lower Ka compared with 22K-hGH. A likely candidate for the negatively charged interactive residue is glutamate-33, since it is unique to human GH and is replaced by a positively charged arginine in non-primate GHs. Its absence in 20K-hGH could explain the altered calcium-dependence of 20K-hGH binding to what is probably the type 2 binding protein [Barnard & Waters (1986) Biochem. J. 237, 885-892]. The Ca2+-dependence of 20K-hGH binding to a subset of GH binding proteins provides both a verification and a mechanistic basis for the proposal [Hughes, Tokuhiro, Simpson & Friesen (1983) Endocrinology (Baltimore) 113, 1904-1906] that 20K-hGH binds with high affinity to only a subset of binding proteins in rabbit liver membranes.

Comparison of growth hormone binding and metabolic response in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin

1985 ◽  
Vol 110 (1) ◽  
pp. 50-55 ◽  
Author(s):  
Stephen LaFranchi ◽  
Cheryl E. Hanna ◽  
Toni Torresani ◽  
Eugen Schoenle ◽  
Ruth Illig
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Metabolic Response ◽  
Specific Binding ◽  
Human Growth ◽  
Scatchard Analysis ◽  
Rat Adipocytes ◽  
Significant Difference ◽  
Glucose Incorporation ◽  
Number Of Binding Sites

Abstract. We undertook a comparison of human growth hormone (hGH) binding and metabolic responses in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin to determine whether the site of fat depot biopsy might affect the response to hGH stimulation. The results showed highest specific binding in epididymal (3.6%), followed by subcutaneous (2.3%) and retroperitoneal adipocytes (1.5%); half-maximal binding was achieved at 14–18 ng/ml hGH for the three sites. Scatchard analysis of the binding data from each site was linear; there was no significant difference in binding affinities (2.1 to 3.3 × 109, m−1), but the number of binding sites was statiticially higher in epididymal (9.8 × 103) as compared to subcutaneous (7.5 × 103, P < 0.05) and retroperitoneal cells (3.3 × 103, P < 0.01). Stimulation with 5 to 2500 ng pituitary hGH produced a dose-related increase in glucose incorporation, with the largest increase in epididymal fat cells (31%, P <0.05) followed by subcutaneous cells (18%, P < 0.05); no significant increase was seen with retroperitoneal cells. Biosynthetic hGH produced a similar pattern of glucose incorporation in the three sites. Addition of hGH antibodies blocked the glucose incorporation in epididymal adipocytes using both pituitary-derived and biosynthetic hGH. It seems clear that this insulin-like effect is caused by hGH, not an insulin-like impurity. We conclude that the number of binding sites, perhaps related to adipose cell size, differs in adipose tissue from different locations and this influences the metabolic response to hGH stimulation.

scholarly journals Monoclonal antibodies to the pituitary growth-hormone receptor by the anti-idiotypic approach. Production and initial characterization

1990 ◽  
Vol 266 (2) ◽  
pp. 467-474 ◽  
Author(s):  
M I Elbashir ◽  
T Brodin ◽  
B Åkerström ◽  
J Donnér
Keyword(s):  
Growth Hormone ◽  
Monoclonal Antibodies ◽  
Human Growth Hormone ◽  
Binding Sites ◽  
Binding Protein ◽  
Liver Microsomes ◽  
Human Growth ◽  
Hepatoma Cells ◽  
Number Of Binding Sites ◽  
Serum Binding Protein

We obtained 10/192 and 3/384 antibody-secreting hybrids after immunization of Balb/c mice with either human growth hormone or affinity-purified rabbit anti-(human growth hormone) respectively. Radiolabelled rabbit anti-(human growth hormone) antibodies, but not human growth hormone, were specifically bound by supernatants from the 13 hybrids. The binding was completely inhibited by human-growth-hormone serum binding protein. However, anti-(human growth hormone antibodies) were detected in the sera of all the mice immunized with human growth hormone. In an independent fusion, which was carried out after immunization with fewer doses of human growth hormone, anti-(human growth hormone) antibodies were also obtained. Five hybrids, where the starting antigen was human growth hormone, were selected for ascites production, and the corresponding monoclonal antibodies were partially purified and characterized with respect to their immunoglobulin isotype and their interaction with human-growth-hormone receptors. These antibodies were found to enhance the binding of radioiodinated human growth hormone to human-growth-hormone serum binding protein from human and rabbit plasma by 40%. Scatchard analysis of the effect of one of the monoclonal antibodies showed that this enhancement was due to an increased number of binding sites. All of the partially purified antibodies but one (F12) inhibited the binding of human growth hormone to rat but not rabbit, liver microsomes to various extents, as well as to H-4-II-E rat hepatoma cells. Monoclonal antibody F12 enhanced the binding of radiolabelled human growth hormone to rat liver microsomes and H-4-II-E hepatoma cells. This enhancement was found to be due to an increase in the number of binding sites.

scholarly journals Structural studies on membrane-bound and soluble growth-hormone-binding proteins of rabbit liver

1987 ◽  
Vol 242 (3) ◽  
pp. 713-720 ◽  
Author(s):  
S I Ymer ◽  
A C Herington
Keyword(s):  
Growth Hormone ◽  
Liver Cell ◽  
Binding Proteins ◽  
Binding Protein ◽  
Rabbit Liver ◽  
Cross Linking ◽  
Subunit Structure ◽  
Gh Receptor ◽  
Disulphide Bonds ◽  
Binding Subunit

Covalent cross-linking techniques have been used to investigate the structural characteristics of the growth-hormone (GH) receptor in a variety of rabbit liver cell membrane preparations (particulate and soluble). Two classes of GH-binding protein have been identified which differ in their Mr by gel filtration and susceptibility to precipitation with poly(ethylene glycol) (PEG). The first, a PEG-precipitable (Mr approximately 300,000) protein, contained Mr-65,000 and Mr-40,000 binding proteins linked by disulphide bonds. It was present in aqueous extracts derived from microsomal membranes but was not present in cytosol preparations. The second, a PEG-non-precipitable protein (Mr approximately 100,000) was composed of a non-disulphide-linked primary GH-binding subunit of Mr 60,000-66,000. This binding protein was present in all rabbit liver cell fractions and/or preparations. Both binding-protein classes contained intramolecular disulphide bonds. It is not clear whether the Mr-approximately 100,000 form, or perhaps higher-Mr species which have not been identified by cross-linking studies, represents the native, endogenous, form of the GH receptor present in particulate microsomal or plasma membranes. Accordingly, although these data have identified two classes of GH-binding protein, especially a primary GH-binding subunit of Mr 60,000-66,000, they indicate that, unlike studies on the insulin receptor, covalent cross-linking techniques alone are not sufficient to delineate the complete subunit structure of the native and endogenous form of the GH receptor.

Effect of human growth hormone (GH)-binding protein in human serum on GH binding to rabbit liver membranes

Metabolism ◽  
1992 ◽  
Vol 41 (7) ◽  
pp. 732-737 ◽  
Author(s):  
Tamar Amit ◽  
Ronnie J. Barkey ◽  
Moussa B.H. Youdim ◽  
Zeev Hochberg
Keyword(s):  
Growth Hormone ◽  
Human Serum ◽  
Human Growth Hormone ◽  
Binding Protein ◽  
Human Growth ◽  
Rabbit Liver ◽  
Liver Membranes

Chronic treatment with bovine growth hormone upregulates high-affinity hepatic somatotropic receptors in sheep

1991 ◽  
Vol 124 (3) ◽  
pp. 307-313 ◽  
Author(s):  
Helga Sauerwein ◽  
Bernhard H. Breier ◽  
John J. Bass ◽  
Peter D. Gluckman
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Hormone Treatment ◽  
Bovine Growth Hormone ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Gh Therapy ◽  
High Affinity

Abstract. We evaluated the effect of chronic bovine growth hormone treatment on the hepatic somatotropic receptor. Growing lambs were treated with bGH at 0, 0.05, 0.15, 0.25 or 0.55 mg · kg−1 · day−1 daily (N=5/group) for 56 days. The binding of ovine GH to hepatic membranes washed with 4 mol/l MgCl2 and prepared in the presence of aprotinin was examined. The specific binding of oGH was increased (p<0.01) from 7.1±1.2% in saline-treated controls to 17.4±1.5% in the 0.55 mg · kg−1 · day−1 group. Scatchard analysis showed curvilinear plots that best fitted a two-site model in 22/25 livers. The two sites had estimated dissociation constants (Kd) of 3 to 13 nmol/l for the low-affinity site and a Kd ranging from 0.17 to 0.31 nmol/l for the high-affinity site. Treatment with bGH had no consistent effect on the affinity of either binding site. However, bGH therapy was associated with a dose-dependent increase (p<0.01) in the number of high-affinity somatotropic receptors. There was no effect of bGH therapy on the concentration of low-affinity binding sites. The concentration of high-affinity receptors correlated with weight gain (r=0.54, p<0.01), fat content (r=−0.54, p<0.01), protein content (r=0.40, p<0.05), and plasma IGF-I (r=0.57, p<0.005). The concentrations of low-affinity binding sites showed no such correlations. These observations demonstrate that an important effect of chronic GH therapy in animals with an intact somatotropic axis is to increase significantly the concentration of high-affinity somatotropic receptors. It is suggested that enhancement of the number of high-affinity somatotropic receptors is central to the determination of the efficacy of GH therapy.

Specific binding sites for ovine prolactin in three amphibian cell lines

1985 ◽  
Vol 248 (1) ◽  
pp. C80-C87 ◽  
Author(s):  
M. Dunand ◽  
M. L. Aubert ◽  
J. P. Kraehenbuhl ◽  
B. C. Rossier
Keyword(s):  
Growth Hormone ◽  
Cell Lines ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Growth ◽  
Functional Differentiation ◽  
Water Metabolism ◽  
Ovine Prolactin ◽  
Follicle Stimulating Hormones

Established cell lines (TB-6c and TB-M) obtained by continuous culture of epithelial cells from toad Bufo marinus urinary bladder, which, in culture, maintained a high degree of functional differentiation, exhibited a significant number of high-affinity (KA = 1-2 X 10(10) M-1) binding sites detected both with radioiodinated (125I) ovine prolactin (oPRL) and human growth hormone (hGH). Binding capacity was higher in the case of TB-6c cells (7,573 +/- 581 sites/cell) than with the TB-M cells (1,160 +/- 87). Similarly, binding sites for oPRL were characterized on Xenopus laevis kidney-derived cell line A6. With oPRL used both as tracer and standard, significant cross-reaction was observed with hGH, less with human or rat prolactin (PRL), and none with human chorionic somatomammotropin, bovine growth hormone, and rat luteinizing hormone or follicle-stimulating hormones. B. marinus pituitary extracts completely displaced the binding of 125I-oPRL to toad bladder binding sites. This finding of specific sites for PRL on amphibian bladder and kidney cells confirms that PRL exerts specific biological actions for the control of electrolyte and water metabolism in the amphibians.

Sign in / Sign up

Export Citation Format

Share Document