DESIGN OF A PHOTOAFFINITY LABEL FOR THE HORMONE BINDING SITE OF NEUROPHYSIN

The characteristics of the human serum growth hormone-binding protein (GHBP) were compared with those of a water-soluble GH-binding site prepared by incubating cultured IM-9 lymphocytes in assay buffer with 25 mmol/l iodoacetamide. High-performance liquid chromatography gel filtration of the water-soluble GH-binding site incubated with 125I-labeled human GH ([125I]hGH) revealed a large peak of bound [125I]hGH eluting at the same position as the peak of [125I]hGH bound to the GHBP in serum. The estimated Mr of the peak was 120 000, presumably representing one [125I]hGH bound to two binding sites. The binding specificities of the serum GHBP, the water-soluble GH-binding site and the GH receptor on IM-9 lymphocytes were identical. The binding affinities for 22 000 hGH and for 20 000 hGH of the serum GHBP were similar to the binding affinity of the water-soluble GH-binding site but lower than those of the cellular GH receptor. These findings show that the characteristics of the serum GHBP are comparable to those of the water-soluble GH-binding site released from IM-9 cells and support the hypothesis that in man the serum GHBP is produced by proteolytic cleavage of the cellular GH receptor.

Download Full-text

Binding of Testosterone and Oestradiol to Sex Hormone Binding Globulin, Human Serum Albumin and other Plasma Proteins: Evidence for Non-Specific Binding of Oestradiol to Sex Hormone Binding Globulin

Clinical Science ◽

10.1042/cs0640307 ◽

1983 ◽

Vol 64 (3) ◽

pp. 307-314 ◽

Cited By ~ 29

Author(s):

M. Jawed Iqbal ◽

Maureen Dalton ◽

Robert S. Sawers

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Binding Site ◽

Plasma Proteins ◽

Specific Binding ◽

Sex Hormone ◽

Sex Hormone Binding Globulin ◽

Normal Female ◽

Hormone Binding

1. The percentage binding of testosterone (T) and oestradiol (E2) to sex hormone binding globulin (SHBG) and human serum albumin (HSA) was determined over a range of SHBG concentrations of 16–250 nmol of dihydrotestosterone (DHT) bound/l. It was found that the binding of both T and E2 to HSA was a function of their binding to SHBG and bore an inverse relationship to it. After removal of both SHBG and HSA from plasma by affinity chromatography a ‘residual’ binding of about 11% for T and 12% for E2 was still apparent. in addition to the specific high-affinity, low capacity binding of E2 to SHBG, non-specific low-affinity binding of 7–12% was demonstrated after selective denaturation of the specific binding site of the latter. 2. Competition studies indicated that although at the relatively higher levels of SHBG found in the normal female the physiological concentrations of E2, T and DHT need not be taken into account in estimating the unbound fractions of steroids, at the relatively lower levels of SHBG found in normal men and hirsute women, the physiological concentrations of T and DHT are effective in causing statistically significant displacement of E2 from the common, specific binding site on SHBG. 3. A simple computerized technique is described for the determination of fractions of E2 and T respectively, that are unbound to SHBG, unbound to SHBG and HSA, and unbound to all plasma proteins, when the total plasma levels of E2, T, DHT and SHBG are known.

Download Full-text

Linkage between the Hormone Binding Site and the Reactive Center Loop of Thyroxine Binding Globulin

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.2000.2110 ◽

2000 ◽

Vol 384 (1) ◽

pp. 31-36 ◽

Cited By ~ 12

Author(s):

Scott A. Suda ◽

Peter G.W. Gettins ◽

Philip A. Patston

Keyword(s):

Binding Site ◽

Hormone Binding ◽

Thyroxine Binding Globulin ◽

Reactive Center Loop ◽

Reactive Center

Download Full-text

Influence of imprinting with A and B chains of insulin on binding and functional changes in tetrahymena

Bioscience Reports ◽

10.1007/bf01152289 ◽

1990 ◽

Vol 10 (5) ◽

pp. 431-436 ◽

Cited By ~ 4

Author(s):

G. Csaba ◽

P. Kovács

Keyword(s):

Binding Site ◽

Binding Capacity ◽

Combined Treatment ◽

The Other ◽

Hormone Binding ◽

Functional Effect ◽

Functional Changes ◽

Positive Material ◽

A Chain ◽

The One

Insulin and its A and B chain increased the quantity of intracellular PAS-positive material (glycogen) in tetrahymena, whereas the combined A+B chains decreased it. Imprinting—previous interaction—with insulin, its A and B chains in themselves and with the A+B chain increased the hormone binding capacity of tetrahymena, but the functional effect of imprinting (storage or breakdown of glycogen) showed a different tendency with insulin and A+B chain on the one hand, and A chain and B chain on the other. Since the imprinting potential of a molecule promotes the induction of receptor formation, the fact remains that both component chains of insulin were able to act as potential imprinters, although the A chain was superior to the B chain in this respect throughout, and combined treatment with the A+B chain ultimately induced the formation of a similar binding site as insulin itself.

Download Full-text

Osteocalcin and Sex Hormone Binding Globulin Compete on a Specific Binding Site of GPRC6A

Endocrinology ◽

10.1210/en.2016-1312 ◽

2016 ◽

Vol 157 (11) ◽

pp. 4473-4486 ◽

Cited By ~ 31

Author(s):

Luca De Toni ◽

Diego Guidolin ◽

Vincenzo De Filippis ◽

Simone Tescari ◽

Giacomo Strapazzon ◽

...

Keyword(s):

Energy Metabolism ◽

Binding Site ◽

Computational Modelling ◽

Sex Hormone ◽

Sex Hormone Binding Globulin ◽

Dependent Manner ◽

Hormone Binding ◽

Human Embryonic Kidney ◽

Molar Excess ◽

293 Cells

The undercarboxylated form of osteocalcin (ucOC) regulates male fertility and energy metabolism, acting through the G protein-coupled receptor (GPRC)6A, thus forming a new pancreas-bone-testis axis. Recently, GPRC6A has also been suggested to mediate the nongenomic responses of free testosterone (T). However, these data did not consider the physiological scenario, where circulating T is mainly bound to sex hormone-binding globulin (SHBG) and only a small percentage circulates freely in the blood. Here, by the use of computational modelling, we document the existence of similar structural moieties between ucOC and SHBG that are predicted to bind to GPRC6A at docking analysis. This hypothesis of competition was assessed by binding experiments on human embryonic kidney-293 cells transfected with human GPRC6A gene. Unliganded SHBG specifically bound the membrane of human embryonic kidney-293 cells transfected with GPRC6A and was displaced by ucOC when coincubated at 100-fold molar excess. Furthermore, specific downstream Erk1/2 phosphorylation after stimulation of GPRC6A with ucOC was significantly blunted by 100-fold molar excess of unliganded SHBG. Intriguingly previous incubation with unliganded SHBG, followed by incubation with T, induced Erk1/2 phosphorylation in a dose-dependent manner. Neither binding nor stimulating activities were shown for SHBG saturated with T. Experiments on mutation constructs of GPRC6A strengthened the hypothesis of a common binding site of ucOC and SHBG. Given the role of GPRC6A on energy metabolism, these data agree with epidemiological association between SHBG levels and insulin sensitivity, suggest GPRC6A as a likely SHBG receptor, and add bases for the possible regulation of androgen activity in a nonsteroidal manner.

Download Full-text