scholarly journals Influence of Mg2+ on detection of somatogenic and lactogenic components of growth-hormone-binding protein in mammalian sera

1993 ◽  
Vol 293 (2) ◽  
pp. 345-349 ◽  
Author(s):  
T Amit ◽  
Z Hochberg ◽  
R J Barkey
Keyword(s):  
Growth Hormone ◽  
Binding Protein ◽  
Rabbit Serum ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Binding Studies ◽  
Type Iii ◽  
Wide Range ◽  
Human Sera ◽  
Serum Gh

We recently classified the growth-hormone (GH)-binding protein (GH-BP) in a wide range of mammalian [including human (h)] sera and reported the existence of a major lactogenic component in GH-BP of type-III sera (rabbit, horse, dog, pig and cat), based on the capacity of bovine (b) and ovine prolactin (PRL) to displace 125I-labelled human growth hormone (hGH) binding and on direct 125I-bPRL binding studies. In this study, we demonstrate the high degree of Mg2+ dependence of the binding of the classically lactogenic hGH and bPRL, but not that of the somatogenic bGH to various mammalian sera (types I-IV). Serum GH-BP was assayed using a previously described and validated charcoal-separation assay. 125I-hGH binding to rat, ovine, bovine, rabbit, horse, dog and human sera was enhanced 1.5-2.5-fold in the presence of 70 mM Mg2+. The Mg2+ effect was concentration-dependent between 3.7 mM and 70 mM, causing a significant and proportional increase in 125I-hGH binding to serum. Like 125I-hGH, 125I-bPRL binding to type-III sera was also Mg(2+)-dependent. In contrast, 125I-bGH binding to all types of serum GH-BP was not affected by Mg2+ concentrations of up to 35 mM, while 70 mM Mg2+ slightly, but significantly, reduced (by approx. 15%) bGH binding to rabbit serum. In keeping with the Mg(2+)-dependent stimulation of lactogenic hormone binding to GH-BP, 70 mM Mg2+ caused a shift to the left in the displacement curves of hGH and bPRL competing with 125I-hGH binding to rabbit, dog, horse and human sera, while the effects of the somatogens bGH and rabbit GH were shifted to the right. Scatchard analysis of hGH displacement curves with sera from various species yielded linear plots and revealed that Mg2+ significantly increased (2.3-3.0-fold) the affinity constants, but not the binding capacities. These results demonstrate the ability of changes in Mg2+ concentration to determine the degree of differential recognition of somatogens versus lactogens by serum GH-BP. It remains to be determined whether such bivalent cation effects may account, at least in part, for the growth retardation seen in Zn2+ or Mg2+ ion deficiencies.

Growth hormone binding protein in patients with renal failure

1992 ◽  
Vol 127 (6) ◽  
pp. 485-488 ◽  
Author(s):  
Hiralal G Maheshwari ◽  
Ian Rifkin ◽  
Joan Butler ◽  
Michael Norman
Keyword(s):  
Growth Hormone ◽  
Renal Failure ◽  
Renal Disease ◽  
Binding Protein ◽  
Scatchard Analysis ◽  
Gel Chromatography ◽  
Hormone Binding ◽  
Binding Studies ◽  
Growth Hormone Binding Protein ◽  
Hormone Binding Protein

To investigate changes in the growth hormone binding protein (GH-BP) in renal disease, gel chromatography was used to separate free and bound hormone after incubation with 125I-GH, the results being expressed as a percentage of radioactive GH eluting in a high molecular weight (70–80 kD) peak. In 26 normal individuals, binding was 39.3±8.0%, while in 11 patients with renal disease who were off dialysis binding was reduced to 16.8±5.6%. Similarly, in 9 patients undergoing hemodialysis binding was reduced to 24.6±6.8%, in 8 patients undergoing chronic ambulatory peritoneal dialysis binding was reduced to 25.7±7.6%, and in 9 patients within three months of a renal transplant binding was reduced to 25.1±8.6%. Scatchard analysis showed that these changes were not a result of decreased affinity of GH-BP for GH, and receptor binding studies showed that uremic serum was not inhibiting binding. The decreased concentration of GH-BP may indicate decreased expression of the GH receptor in target tissues, and hence diminished responsiveness to GH in renal failure.

THE PRESENCE OF LACTOGEN BUT NOT GROWTH HORMONE BINDING SITES IN THE ISOLATED RAT HEPATOCYTE

1977 ◽  
Vol 74 (2) ◽  
pp. 323-334 ◽  
Author(s):  
A. C. HERINGTON ◽  
N. M. VEITH
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Binding Sites ◽  
Specific Binding ◽  
Female Rats ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Binding Studies ◽  
Membrane Function ◽  
Liver Membranes

The binding of 125I-labelled human growth hormone (hGH) and bovine growth hormone (bGH) has been studied in hepatocytes isolated from female rats by perfusion with collagenase in situ. The cells appeared to retain normal membrane function, in that amino acid ([14C]α-aminoisobutyric acid) transport was both saturable and temperature-dependent. Amino acid ([14C]leucine) incorporation into protein was also linear over 3 h and was inhibited by cycloheximide. Binding of 125I-labelled hGH was dependent on time, temperature, hepatocyte concentration and hGH concentration. At 22 °C, binding reached a steady-state after 2·5 h and had a half-life of dissociation of 2–3 h. Hormone specificity studies indicated that binding was specific for hormones with prolactin-like activity (hGH, prolactins) and not for growth hormones themselves (bGH). Scatchard analysis revealed a single class of binding site with a binding capacity of 26·74 ± 3·73 fmol/106 cells and a binding affinity of 1·24 × 109 ± 0·17 × 109 (s.e.m.) l/mol (n = 10). There was a significant sex difference in binding (female > male) and binding was subject to marked regulation by oestrogens (stimulation of binding) and by androgens (inhibition). The lactogen-binding sites, therefore, were comparable in many respects to those previously reported in rat liver membranes. No distinct GH binding sites were demonstrable as shown by the lack of specific binding by 125I-labelled bGH, purified either by Sephadex chromatography or by binding to and elution from GH receptors in rabbit liver membranes. The value of receptor purification of tracer for use in hormone binding studies was indicated by a substantial lowering of non-specific binding.

Ontogeny and nutritional regulation of the serum growth hormone-binding protein in the rat

1991 ◽  
Vol 125 (4) ◽  
pp. 409-415 ◽  
Author(s):  
Nkuadi Mulumba ◽  
Guy Massa ◽  
Jean-Marie Ketelslegers ◽  
Marc Maes
Keyword(s):  
Growth Hormone ◽  
Binding Protein ◽  
Female Rats ◽  
Male Rats ◽  
Nutritional Regulation ◽  
Hormone Binding ◽  
Serum Growth Hormone ◽  
Growth Hormone Binding Protein ◽  
Hormone Binding Protein ◽  
Serum Gh

Abstract. The ontogeny and the nutritional regulation of the serum growth hormone-binding protein in Wistar rats was studied in vitro using Ultrogel AcA34 filtration of serum incubated with 125I-bovine growth hormone. The level of the specific binding of GH to serum GH-binding protein was low in 1-week-old rats (female rats 2.3±0.9%; N=6, and male rats 2.1±8%; N=6 ) and increased with puberty, to reach 10-fold higher levels in 12-week-old adult female (26.1±2.3%; N=6) and 5-fold higher levels in adult male rats (11.2±0.9%; N=6). From 6 weeks of age and onwards, the level of serum GH-binding protein was significantly higher in female than in male rats, reflecting sexual dimorphism. The nutritional dependence of GH-binding protein was supported by the 46% decline of serum GH-binding protein levels in 6-week-old female rats fasted for 3 days (11.6±0.9 and 6.3±1.1% in control and fasted rats, respectively; N=8/group; p<0.001). After 4 days of refeeding, no difference was found between control and experimental animals. During development and after nutritional manipulation, Scatchard analysis revealed that the changes in GH-binding protein were due to changes in binding capacity and not affinity. The levels of serum GH-binding protein were positively correlated with the levels of hepatic GH binding sites, suggesting that the regulation of both proteins is closely related during development and in states of nutritional sufficiency and deprivation.

Inhibition of growth hormone bioactivity by recombinant human growth hormone-binding protein in the eluted stain assay system

1994 ◽  
Vol 140 (3) ◽  
pp. 445-453 ◽  
Author(s):  
M T Dattani ◽  
P C Hindmarsh ◽  
C G D Brook ◽  
I C A F Robinson ◽  
N J Marshall
Keyword(s):  
Growth Hormone ◽  
Radioligand Binding ◽  
Binding Protein ◽  
Recombinant Human Growth Hormone ◽  
Assay System ◽  
Tetrazolium Salt ◽  
Dependent Manner ◽  
Binding Studies ◽  
Nb2 Cells ◽  
Wide Range

Abstract The effects of a recombinant human GH-binding protein (rhGHBP; amino acids 1–238) on GH stimulation of rat Nb2 lymphoma cells were examined with an eluted stain assay system (ESTA). This precise bioassay utilizes the colorimetric reduction by stimulated Nb2 cells of a yellow tetrazolium salt (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a purple formazan as its end-point. The use of a lactogenic bioassay allowed the investigation of hGHBP specificity for human GH (hGH) as opposed to prolactin. rhGHBP inhibited pituitary hGH bioactivity in a dose-dependent manner. No significant inhibition of prolactin or ACTH bioactivity occurred. It was confirmed that recombinant 20 kDa hGH also stimulated the Nb2 cells and that its relative potency was ∼ 10% of that of pituitary-derived hGH. Stimulation by 20 kDa hGH was also inhibited by rhGHBP. The highly quantitative ESTA system demonstrated that the binding protein inhibited in a competitive manner. hGH activation of the Nb2 cells did not appear to be governed by a Michaelian first-order reaction. As might then be anticipated, the concentration of rhGHBP required for 50% inhibition of GH bioactivity (IC50) changed with agonist concentrations for both 20 kDa and 22 kDa hGH. However, with equimolar concentrations of these two isohormones, the IC50 of the binding protein was virtually identical. Potentiation of hGH bioactivity in vivo by low concentrations of hGHBP has been reported but was not observed in our in vitro system when tested over a wide range of binding protein concentrations. In conclusion, the ESTA bioassay system permitted a detailed characterization of the inhibition of hGH bioactivity by rhGHBP. The hormonal specificity confirms earlier radioligand binding studies, except that we found that the 20 kDa hGH variant interacts with the rhGHBP. Journal of Endocrinology (1994) 140, 445–453

scholarly journals Serum and liver cytosolic growth-hormone-binding proteins are antigenically identical with liver membrane ‘receptor’ types 1 and 2

1986 ◽  
Vol 237 (3) ◽  
pp. 885-892 ◽  
Author(s):  
R Barnard ◽  
M J Waters
Keyword(s):  
Growth Hormone ◽  
Binding Site ◽  
Binding Proteins ◽  
Membrane Receptor ◽  
Rabbit Serum ◽  
Receptor Subtypes ◽  
Hormone Binding ◽  
Liver Growth ◽  
Serum Gh ◽  
Gh Receptors

Studies with a panel of monoclonal antibodies (MAbs) reactive towards the presumptive rabbit liver growth-hormone (GH) receptor show that the rabbit serum GH-binding proteins share seven antigenic determinants (three at the hormone-binding site and four located elsewhere) with the liver cytosolic GH-binding proteins and the putative GH ‘receptors’ associated with the hepatocyte membrane. The rabbit serum binding proteins have an affinity for GH similar to the membrane GH receptors [for human GH, Ka = 2.45 (+/- 0.15) X 10(9) M-1 (mean +/- S.E.M., n = 8)] and high capacity relative to membrane ‘GH receptors’. Analogues of the postulated membrane ‘receptor’ subtypes 1 and 2 exist in the serum, but not subtype 3, which is also absent from liver cytosol. The serum and cytosolic binding proteins have identical cation-dependence properties; hGH binding is Ca2+-dependent, whereas oGH binding is Ca2+-independent. Affinity labelling of hGH-affinity-purified serum binding proteins with 125I-hGH demonstrated a major GH-binding subunit, of Mr 55,000, identical with the major component purified from membranes. In view of their high affinity and capacity, the serum binding proteins could control availability of GH to membrane receptors. It is suggested that the cytosolic binding proteins may be newly synthesized serum binding proteins. The existence of a close relationship between subsets of membrane-associated GH-binding sites, the serum GH-binding proteins and cytosolic GH-binding proteins dictates a reappraisal of earlier ligand-binding studies, which did not distinguish between binding-site subsets in the liver.

Growth hormone-binding protein levels: Studies of children with short stature

Metabolism ◽  
1994 ◽  
Vol 43 (3) ◽  
pp. 357-359 ◽  
Author(s):  
Nelly Mauras ◽  
Lena M.S. Carlsson ◽  
Suzanne Murphy ◽  
Thomas J. Merimee
Keyword(s):  
Growth Hormone ◽  
Short Stature ◽  
Binding Protein ◽  
Hormone Binding ◽  
Protein Levels ◽  
Growth Hormone Binding Protein ◽  
Hormone Binding Protein
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