scholarly journals Evidence that non-covalent forces, thiol and disulphide groups affect the structure and binding properties of the prolactin receptor on hepatocytes from pregnant rats

1985 ◽  
Vol 228 (2) ◽  
pp. 383-390 ◽  
Author(s):  
K Yamada ◽  
D B Donner
Keyword(s):  
Growth Hormone ◽  
Binding Capacity ◽  
Prolactin Receptor ◽  
Sodium Dodecyl ◽  
Specific Effect ◽  
Scatchard Analysis ◽  
Pregnant Rats ◽  
Receptor Complexes ◽  
Affinity Labelling ◽  
Triton X

Incubation of hepatocytes from pregnant rats with dithiothreitol decreased specific 125I-prolactin (125I-prl) binding to such cells by about 20% relative to control. This was not due to a non-specific effect of dithiothreitol on the cell membrane, since reduction also altered the binding of prl to solubilized partially purified receptor. Exposure of hepatocytes to N-ethylmaleimide (6 mM) for periods as brief as 1 min decreased the subsequent specific binding of 125I-prl by more than 50%. N-Ethylmaleimide was less effective as an inhibitor of binding when applied after hepatocytes had been exposed to 125I-prl, binding being decreased by about 15%. Scatchard analysis demonstrated that the effect of N-ethylmaleimide resulted from loss of receptor-binding capacity without any substantial effect on the affinity of the prl receptor for hormone. Dithiothreitol diminished the affinity of lactogenic sites for prolactin without altering cellular binding capacity. These observations suggest that thiol and disulphide groups are present in the prl receptor and that these functional moieties regulate the formation and properties of prl receptor complexes. The species to which 125I-prl had bound were identified by affinity labelling. 125I-prl was covalently coupled into saturable complexes of Mr 65000 and 50000. 125I-human growth hormone (125I-hGH) was covalently incorporated into complexes of Mr 300 000, 220 000, 130 000, 65 000 and 50 000. Bovine growth hormone (bGH), but not prl, competed for 125I-hGH uptake into the 300 000-, 220 000- and 130 000-Mr complexes, indicating that these species were somatogenic. Prl, but not bGH, inhibited 125I-hGH uptake into 65 000- and 50 000-Mr complexes. This demonstrated that 125I-hGH in the presence of bGH could affinity-label lactogenic receptors. 125I-prl aggregates in Triton X-100, whereas 125I-hGH does not. Therefore lactogenic complexes to which 125I-hGH was bound in the presence of excess bGH were solubilized in Triton X-100 and characterized sequentially by gel filtration and affinity labelling. Prl receptors were eluted from columns of Sepharose 6B as a species of Mr380 000. Fractionation of the 380 000-Mr species on sodium dodecyl sulphate polyacrylamide gels resulted in the isolation of complexes of Mr 65 000 and 50 000. Thus non-covalent forces stabilize aggregates of the monomeric prolactin receptor.

Prolactin Receptor in Human Mammary Carcinoma

1979 ◽  
Vol 65 (6) ◽  
pp. 695-702 ◽  
Author(s):  
Raffaele Di Carlo ◽  
Giampiero Muccioli
Keyword(s):  
Protein Concentration ◽  
Binding Capacity ◽  
Specific Binding ◽  
Prolactin Receptor ◽  
Scatchard Analysis ◽  
Breast Carcinomas ◽  
Human Breast ◽  
Human Mammary Carcinoma ◽  
Human Breast Carcinomas ◽  
Lactogenic Hormones

The specific binding of labelled human prolactin was determined in 83 human breast carcinomas. Twenty-seven tumors (32.5 %) contained specific binding for prolactin of at least 1 % and were considered prolactin receptor positive. The binding was found linearly related to membrane protein concentration and specific only for lactogenic hormones. By Scatchard analysis the dissociation constant appeared similar to that observed in other target tissues, with a low binding capacity.

Partial purification and properties of the TSH receptors from human thyroid plasma membranes

1983 ◽  
Vol 103 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Yasuhiro Iida ◽  
Junji Konishi ◽  
Kanji Kasagi ◽  
Keigo Endo ◽  
Takashi Misaki ◽  
...  
Keyword(s):  
Sodium Acetate ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Human Thyroid ◽  
Partial Purification ◽  
Affinity Column ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Con A ◽  
Triton X

Abstract. Human thyroid plasma membranes were solubilized with 0.5% Triton X-100 and TSH receptors were purified by using DEAE-Sephadex, Con A and TSH affinity chromatography. A TSH binding activity was bound to DEAE-Sephadex equilibrated with 0.05 m sodium acetate, pH 6.3, 0.2% Triton X-100 and was eluted by a linear gradient of 0.1 m to 1.0 m ammonium acetate, pH 6.3. Eighty-five per cent of the activity was absorbed to Con A Sepharose and was eluted with 0.5 m α-methyl-D-mannoside, 0.05 m sodium acetate, pH 6.0. Seventy-five per cent of the TSH binding capacity could be absorbed to TSH-affinity column and was eluted with 0.1 m glycine-HCl, pH 3.0. By sequential application of the above procedures, more than 100-fold purification of the receptor activity was attainable. [125I]TSH binding of this fraction was inhibited by addition of unlabelled TSH in a dose-dependent manner. Scatchard analysis gave a curvilinear plot with a high affinity association constant of 0.72 × 109 m−1. By using Ultrogel AcA 34 gel filtration, the molecular size of the hormonereceptor complex was estimated to be 180 000.

scholarly journals An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit

1981 ◽  
Vol 198 (3) ◽  
pp. 605-614 ◽  
Author(s):  
H F Cadman ◽  
M Wallis
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Specific Binding ◽  
Bovine Somatotropin ◽  
Scatchard Analysis ◽  
Single Class ◽  
Pregnant Rabbit ◽  
Membrane Bound ◽  
Ovine Prolactin ◽  
Triton X

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).

D-Glucose uptake and insulin binding by the human adipose cell plasma membrane as a function of its polypeptide composition

1977 ◽  
Vol 55 (2) ◽  
pp. 126-133 ◽  
Author(s):  
Bluma G. Brenner ◽  
Shiro Ozaki ◽  
Norman Kalant ◽  
Arthur Kahlenberg
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insulin Binding ◽  
Partial Purification ◽  
Scatchard Analysis ◽  
Molecular Weight Range

A preparation of plasma membranes isolated from human omental lipocytes is composed of about 15 major polypeptide components including three major glycoproteins with an apparent molecular weight range from 100 000 to 23 000, as determined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Extraction of this membrane preparation with sodium iodide or 2,3-dimethylmaleic anhydride solubilized 50 and 70% of the membrane protein, respectively, resulting from the extensive extraction of protein from all but two of the major membrane polypeptide components. This removal of protein did not affect the membrane's stereospecific D-glucose-uptake activity but did reduce its total specific [l25I]insulin-binding activity by 46–67%. The binding of [125I]insulin to its specific receptor on lipocyte plasma membranes was detected at physiologic concentrations of the hormone and could be competitively displaced by increasing concentrations of native insulin. The kinetic behaviour of this reaction was approximated by Scatchard analysis, and both the affinity and binding capacity of the plasma membrane for insulin were increased at lower temperatures.These results suggest that D-glucose transport in human adipose tissue is mediated by an intrinsic component of the hydrophobic structure of the lipocyte plasma membrane, and represent a partial purification of this component. In addition, these studies demonstrate and characterize the binding of insulin to the plasma membrane isolated from human lipocytes. A quantitative study of this binding reaction may provide further understanding of the mechanisms underlying the decreased insulin responsiveness characteristic of human diabetes.

scholarly journals Vasoactive intestinal peptide receptors in rat liver after partial hepatectomy

1992 ◽  
Vol 285 (2) ◽  
pp. 515-520 ◽  
Author(s):  
L G Guijarro ◽  
A Couvineau ◽  
M S Rodriguez-Pena ◽  
M G Juarranz ◽  
N Rodriguez-Henche ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Vasoactive Intestinal Peptide ◽  
Structural Changes ◽  
Binding Capacity ◽  
Regenerating Liver ◽  
Vip Receptors ◽  
Receptor Complexes ◽  
Binding Data ◽  
The Status ◽  
Affinity Labelling

We describe the status of vasoactive intestinal peptide (VIP) receptors in regenerating liver. VIP-stimulated adenylate cyclase activity was markedly decreased in proliferating liver 3 days after partial (70%) hepatectomy. This was associated with a reduced efficacy of VIP (53% compared with controls), with no change in the potency of the peptide (ED50 0.8 nM). In contrast, forskolin- and guanosine 5′-[beta gamma-imido]triphosphate (Gpp[NH]p)-stimulated enzyme activities were not decreased after hepatectomy. The expression of Gs protein subunits (alpha and beta) was studied by cholera toxin-catalysed ADP ribosylation of alpha s and by immunoblotting of alpha s and beta subunits. Both subunits were increased in regenerating liver, further suggesting that the decreased response to VIP was not related to a decreased expression of Gs proteins. In fact, the reduced adenylate cyclase response to VIP in regenerating liver was associated with quantitative and structural changes in VIP receptors. Equilibrium binding data obtained with 125I-VIP indicated the presence of two classes of binding sites, the Kds of which were not altered after hepatectomy. In contrast, changes in binding capacity (Bmax.) were as follows: 0.11 +/- 0.01 and 0.05 +/- 0.01 pmol/mg of protein for high-affinity sites in control and hepatectomized rats respectively; and 2.3 +/- 0.2 and 0.65 +/- 0.03 pmol/mg of protein for low-affinity sites in control and hepatectomized rats respectively. Moreover, affinity labelling experiments showed that the M(r) value of 125I-VIP-receptor complexes was higher in regenerating liver than in quiescent hepatocytes, e.g. 58,000 and 53,000 respectively. It is concluded that VIP receptors are altered in regenerating liver, resulting in a decreased response of adenylate cyclase to the neuropeptide.

Crystallization of ovine placental lactogen in a 1:2 complex with the extracellular domain of the rat prolactin receptor

1998 ◽  
Vol 54 (6) ◽  
pp. 1408-1411 ◽  
Author(s):  
Hans W. Christinger ◽  
Patricia A. Elkins ◽  
Yael Sandowski ◽  
Edna Sakal ◽  
Arieh Gertler ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Growth Hormone ◽  
Prolactin Receptor ◽  
Human Growth ◽  
Cross Reactivity ◽  
Placental Lactogen ◽  
Asymmetric Unit ◽  
Receptor Complexes ◽  
Endocrine Hormones ◽  
Shed Light

Growth hormone and prolactin control somato-lactogenic biology. While high-resolution crystal structures have been determined for receptor complexes of human growth hormone, no such information exists for prolactin. A stable 1:2 complex was formed between ovine placental lactogen, a close prolactin homologue, and two copies of the extracellular portion of the rat prolactin receptor. Using synchrotron radiation, native data have been collected to 2.3 Å. Crystals contain one complex per asymmetric unit. The crystal structure of this complex will shed light on the structural reasons for cross-reactivity and specificity among the endocrine hormones, placental lactogen, prolactin and growth hormone.

scholarly journals Different characteristics of solubilized lactogen receptors from livers of pregnant and non-pregnant female rats

1982 ◽  
Vol 203 (3) ◽  
pp. 653-662 ◽  
Author(s):  
Norio Sasaki ◽  
Yuko Tanaka ◽  
Yasuo Imai ◽  
Toshio Tsushima ◽  
Fukashi Matsuzaki
Keyword(s):  
Binding Capacity ◽  
Pregnant Female ◽  
Female Rats ◽  
Scatchard Analysis ◽  
Female Rat ◽  
Binding Studies ◽  
Pregnant Rats ◽  
Pregnant Rat ◽  
Stokes Radius ◽  
Rat Livers

Receptors specific for lactogenic hormones were solubilized by 1% (v/v) Triton X-100 from the crude particulate membrane fraction of livers of pregnant and non-pregnant female rats and the characteristics of both preparations were compared. Human 125I-labelled somatotropin was used for binding studies of lactogenic hormone. The solubilized receptor retained most of the characteristics noted in the particulate fraction. The binding of human 125I-labelled somatotropin to the solubilized receptor is a saturable process, depending on temperature and time. Scatchard analysis of displacement curves revealed similar affinity constants ranging from 1.02 × 109 to 1.20 × 109 1/mol, while the binding capacity was 4.5 times greater in the pregnant rat livers than in the non-pregnant female rat livers. The receptors for human 125I-labelled somatotropin from livers of non-pregnant and pregnant female rats were equally adsorbed onto a concanavalin-A-Sepharose column and were dissociated from the column with α-methyl-d-glucoside or α-methyl-d-mannoside in the same manner. By gel filtration on Sepharose 6B, however, the molecular sizes of the hepatic receptors were found to be different. The apparent Mr value was approx. 270000 with a Stokes‘ radius of 5.5nm in the non-pregnant female rats and approx. 330000 with a Stokes’ radius of 5.5nm in the pregnant rats. Furthermore, isoelectric-focusing experiments showed that a major part of the receptor from the non-pregnant female rat livers had a neutral pI (7.0—8.5), whereas that from pregnant-rat livers had an acidic pI (4.2—4.7). These data suggest that the increase in the lactogenic binding capacity in rat liver membranes during pregnancy may be associated with marked changes of the physicochemical properties of the receptors.

scholarly journals Structure of covalent insulin-receptor complexes (I-S-S-R) in isolated rat adipocytes and human placental membranes

1985 ◽  
Vol 229 (2) ◽  
pp. 513-519 ◽  
Author(s):  
S Clark ◽  
L C Harrison
Keyword(s):  
Insulin Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Species ◽  
Buffer System ◽  
Rat Adipocytes ◽  
Receptor Complexes ◽  
Placental Membranes ◽  
Triton X ◽  
Binding Subunit

The structure of naturally-formed covalent disulphide-linked complexes between insulin and its receptor was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. To prevent destabilization of disulphide bonds at alkaline pH the standard discontinuous electrophoresis conditions were changed to a continuous buffer system at pH 7.0. 125I-insulin was first bound to either rat adipocytes or human placental membranes for 10 min at 37 degrees C. After washing, non-dissociable radioactivity was extracted from cells or membranes in Triton X-100 and immunoprecipitated with an antiserum (B-2) to the insulin receptor. Electrophoresis of the immune precipitate revealed the two smaller of the three reported species of native insulin receptor (Mr values approx. 350 000, 290 000 and 260 000); in addition, a species of Mr 200 000 was also frequently observed in adipocytes. When non-dissociable 125I-insulin was chemically crosslinked to adipocytes or placental membranes, prior to solubilization and immunoprecipitation, all three species of the native receptor were labelled; after reduction, only a single species of Mr 130000 was observed. These findings indicate that disulphide exchange of insulin occurs with the Mr 130000 (alpha) binding subunit within partially reduced species of the native, oligomeric receptor. The degree of disulphide binding of insulin could therefore depend on the relative abundance of partially reduced receptor species and on the redox state of the cell membrane.

scholarly journals Characterization of human somatotropin binding to detergent-solubilized lactogenic receptors from rat liver

1981 ◽  
Vol 194 (2) ◽  
pp. 385-394 ◽  
Author(s):  
J S Bonifacino ◽  
S H Sánchez ◽  
A C Paladini
Keyword(s):  
Rat Liver ◽  
Rate Constant ◽  
Binding Capacity ◽  
Inhibitory Effect ◽  
Binding Activity ◽  
Dissociation Rate ◽  
Scatchard Analysis ◽  
Single Class ◽  
Solubilized Form ◽  
Triton X

Lactogenic receptors from rat liver microsomal fraction (‘microsomes’) were extracted by treatment with 1% (w/v) Triton X-100. Triton X-100 exerts an inhibitory effect on both the binding reaction and the separation of the free hormone from the complex. The association and dissociation of 125I-labelled human somatotropin are time- and temperature-dependent processes. The association rate constant, k1, is 6.7 × 10(6) mol . litre-1 . min-1 at 25 decrees C, and the dissociation rate constant, k-1, is 1.1 × 10(-3) min-1 at 25 degrees C. Scatchard analysis of saturation data reveals the existence of a single class of receptors and that solubilization leads to a slight decrease in affinity and a sharp increase in binding capacity. The dissociation constant, Kd, of the solubilized preparation is 0.22 nM and the binding capacity 2900 fmol/mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the solubilized receptors is specifically inhibited by hormones with lactogenic activity. Incubation of the solubilized preparation with trypsin resulted in an 80% decrease in binding activity. The solubilized form of the receptor has a slightly increased sensitivity to the inactivation by trypsin, heat and extremes of pH, with respect to the membrane-bound form.

SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF HUMAN AND PORCINE THYROTROPHIN RECEPTORS

1978 ◽  
Vol 78 (1) ◽  
pp. 89-102 ◽  
Author(s):  
P. J. D. DAWES ◽  
V. B. PETERSEN ◽  
B. REES SMITH ◽  
R. HALL
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Binding Activity ◽  
Human Thyroid ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
Approximate Molecular Weight ◽  
Membrane Bound ◽  
Triton X

Thyrotrophin (TSH) receptors have been extracted from human and porcine thyroid membranes by treatment with Triton X-100.125I-Labelled bovine TSH was used to monitor receptor activity. Analysis by gel filtration and electrophoresis on acrylamide gels containing sodium dodecyl sulphate suggested that Triton extracts of human thyroid membranes contained TSH receptors with a molecular weight in the region of 50 000 closely associated with Triton micelles of approximate molecular weight 300 000. Isoelectric focusing studies indicated that the Triton-solubilized TSH binding activity had an isoelectric point of pH 4–4·5. The soluble TSH receptors were heat-labile, showed optimum TSH binding at pH 7·4 and reduced hormone binding at high ionic strength. The TSH binding characteristics of membrane-bound and solubilized human TSH receptors were similar and both preparations gave curved Scatchard plots. Solubilized porcine TSH receptors appeared to have a similar molecular weight to the human receptors and were also closely associated with Triton micelles of approximate molecular weight 300 000. Scatchard analysis of TSH binding to membrane-bound or solubilized porcine TSH receptors gave approximately linear plots with association constants of 2·8 ± 0·95 (s.e.m.) × 109 and 1·7 ± 0·27 × 1091/mol respectively. Comparison of the binding capacities of the solubilized and membrane-bound porcine receptors indicated that the 0·5% Triton extracts contained 40% of the original TSH binding activity and that this was present at a concentration of 25 ng/ml.

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