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1.
A highly sensitive radioreceptor assay (RRA) for human prolactin (hPRL) based on membrane preparations obtained from chemically induced rat mammary tumour is described. The binding of 125I-labelled, highly purified pituitary human prolactin was specific for lactogenic hormones and depending on time, temperature, and concentration of receptor protein. Optimal specific receptor binding (18-20%) was obtained by incubation at 21 degrees C for 18 h. The prolactin receptor was shown to have a single "class" of binding sites with an affinity constant (Ka) of 6.0 X 10(10) mol-1. The binding capacity was 8-33 fmol/mg membrane protein. The sensitivity of the radioreceptor assay was 0.5 ng/ml ovine prolactin (NIH-PS-10) or 0.84 ng/ml human prolactin (NIH-VLS-4). The receptor binding activity of various purified prolactin preparations from different species was comparable to the biological hormone activities, indicating that this in vitro assay system measures values which are biologically relevant. 相似文献
2.
T B Ng T Y Hui C H Cheng 《Comparative biochemistry and physiology. A, Comparative physiology》1991,99(3):387-390
1. 125I-labelled ovine prolactin and bovine growth hormone were used to test for the presence of prolactin and growth hormone receptors in membrane prepared from tissues of the white eel Anguilla japonica, the carp Ctenopharynogodon idellus and the ricefield eel Monopterus albus. 2. High levels of specific 125I-labelled ovine prolactin binding were found in white eel liver membranes and carp kidney membranes. 3. High levels of specific 125I-labelled bovine growth hormone binding were detected in white eel liver membranes. 4. Tissues of the ricefield eel did not bind 125I-labelled ovine prolactin or bovine growth hormone. 5. The results suggest the presence of prolactin receptors in white eel liver and carp kidney membranes and growth hormone receptors in white eel liver membranes. 相似文献
3.
Prolactin binding in the developing rat fetal liver 总被引:1,自引:0,他引:1
The binding of prolactin by fetal rat liver cell membrane fractions from 17 to 21 days gestation was studied. Particulate liver membranes were prepared in Dulbecco's Phosphate Buffered Saline (PBS) by ultracentrifugation and incubated at 22 degrees C for 16 hours with [125I] iodo-human growth hormone (hGH). Non-specific binding was assessed by parallel incubations in the presence of a 2000-fold excess ovine prolactin. Specific prolactin binding sites were detected only at 21 days gestation (2932 +/- 401 cpm/mg protein) in freshly prepared membranes. On freezing at -20 degrees C for 24 to 48 hours, the membranes of 20 days gestation animals were able to specifically bind prolactin (1295 +/- 239 cpm/mg protein). Freezing led to a 45 +/- 7% increase (4270 +/- 701 cpm/mg protein) in prolactin binding at 21 days gestation. No hormonal binding was detected from 17 through 19 days gestation in either fresh or freeze-thawed membranes. Scatchard analysis revealed a high affinity binding site with a Ka of approximately 1.4 X 10(8)M-1 in both fresh and freeze-thawed membrane preparations. The data show that 1) prolactin receptors appear in liver only during late fetal life and that 2) freezing of membranes may unmask binding sites that are initially unavailable to specifically bind prolactin. 相似文献
4.
An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit. 总被引:8,自引:5,他引:3
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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). 相似文献
5.
L C Teh C J Ormandy A S Surus R L Sutherland G E Chapman 《Hormones et métabolisme》1988,20(5):278-281
The distribution of 125I radioactivity in the liver, kidneys, adrenals and serum of male rats was measured 10 minutes after an intravenous bolus of 125I-labelled human growth hormone (hGH) was administered in the presence or absence of a large excess of ovine growth hormone or ovine prolactin. The hGH binding sites in the adrenals had displacement properties characteristic of lactogenic receptors, whereas those in the liver had displacement properties characteristic of somatogenic receptors. Bovine and ovine adrenal microsomal membrane fractions contained high affinity (Ka = 1.4-3.3 nM-1) binding sites for hGH which showed ligand specificity typical of lactogenic receptors. It is concluded that the hGH binding site in the adrenal gland is a classical lactogenic receptor and that this tissue is a convenient and rich (42.6 +/- 6.4 fmol hGH specifically bound/mg protein) source of receptor suitable for further characterization. 相似文献
6.
Modulation of prolactin binding sites in vitro by membrane fluidizers. II. Age-dependent effects on rat ventral prostatic membranes 总被引:1,自引:0,他引:1
The objectives of this study were to determine (i) if the age-related changes in 125I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24-25 days old), young-adult (80-90 days old) and aged (550-610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding. 相似文献
7.
Radioimmunoassay for ovine, caprine and bovine prolactin in plasma and tissue extracts 总被引:1,自引:0,他引:1
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1. A radioimmunoassay for ovine prolactin is described based on the inhibition of the reaction between (131)I-labelled ovine prolactin and guinea-pig or rabbit antiserum to ovine prolactin. The extent of the reaction after a 4-day incubation period is determined by chromatoelectrophoresis or by adsorption of unchanged (131)I-labelled ovine prolactin on charcoal. The sensitivity is equal to 5.9ng. of prolactin/ml. of plasma with chromatoelectrophoresis, or 0.2ng. of prolactin/ml. of tissue extracts with the charcoal separation. 2. A complete cross-reaction demonstrated between ovine prolactin and caprine pituitary extracts allows the assay to be used to measure caprine prolactin. The partial cross-reactions between ovine prolactin and bovine prolactin and between ovine prolactin and bovine pituitary extract differ, and an alteration in the immunological activity of bovine prolactin during its isolation is suggested. Bovine prolactin in plasma may be measured against a bovine pituitary extract as standard. No cross-reactions were demonstrated with pituitary extracts from a number of other species. The extent of the contamination of ovine and bovine growth hormone preparations by their respective prolactins is shown. 3. Dilutions of ovine and caprine plasma inhibit the reaction between (131)I-labelled ovine prolactin and antiserum with the same characteristics as ovine prolactin. 4. The immunoreactive material in plasma fractionates on Sephadex G-200 and in sucrose density gradients as a single peak similar to that shown by freshly dissolved ovine prolactin. There is no evidence that ovine prolactin is bound to a plasma protein. 5. By suppressing prolactin secretion and assaying serial samples of plasma thereafter it is shown that the immunological activity of the surviving hormone becomes progressively altered with time. It is suggested that this alteration is usually not detected but introduces an element of uncertainty into the quantitative but not the qualitative value of the measurements obtained by reference to standard ovine prolactin. 相似文献
8.
We have recently demonstrated specific binding for 3H-arginine8-vasopressin (3H-AVP) to high affinity sites on membranes of rat mesenteric arteries. We have now measured the biological activity of this peptide (AVP) and analogues on the perfused rat mesenteric artery. There was a close relationship between the ED50 of agonists or the pA2 of antagonists on the perfused tissue and the relative potency (IC50) of analogues for displacing 3H-AVP from the membrane preparation. The ED50 measured was 67 +/- 7 ng for AVP and 7.2 +/- 1.1 microgram for oxytocin. In sodium-depleted rats we have observed an increase (27%) of the maximal response to AVP with no significant change in ED50 (from 2.8 +/- 1.0 X 10(-8) M to 1.3 +/- 0.2 X 10(-8) M). On the membrane preparation, the number of binding sites for 3H-AVP was increased from 71 +/- 17 fmole/mg protein (Kd 3.5 +/- 0.5 nM) to 115 +/- 10 fmole/mg protein (Kd 4.8 +/- 0.3 nM) in the sodium-depleted rat by comparison to control animals. These results suggest that AVP and its analogues interact in a similar manner in the in vitro perfused rat mesenteric artery and with the membrane receptors isolated from the same tissue. Receptors for AVP are increased in the mesenteric vascular bed by sodium depletion. 相似文献
9.
The study was designed to compare the direct effect of three prolactin-like hormones on steroidogenesis of ovine luteal cells collected at day 40-45 of pregnancy. 100 ng/ml of ovine placental lactogen or 100 ng/ml of ovine growth hormone or 100 ng/ml of ovine prolactin were added to the media of luteal cell cultures. After 48 h incubation, all cultures were terminated and the media were frozen until further steroid analysis. To determine to what extent growth hormone (GH), prolactin (PRL) and lactogen (PL) regulate the activity of 3 beta-HSD, an enzyme involved in progesterone synthesis, the classical steroidal competitive inhibitor of 3 beta-HSD trilostane, was investigated for its effects on basal and GH-, PRL-, and PL-stimulated progesterone biosynthesis since there is a possibility that the luteotropic effect of these hormones are mediated via 3 beta-HSD. oPL resulted in an increase of progesterone secretion in a statistically significant manner, while GH or PRL had no effect on progesterone secretion. A decrease in progesterone secretion as an effect of 100 mM trilostane was observed in all culture types. An explanation for the luteotropic effect of PL and the lack of this effect for GH is that the GH receptor associates with a different molecule within the ovarian tissue and forms a heterodimeric receptor for PL, and the possibility that physiological effects of native oPL may be mediated through its binding to specific PL receptors, which have low affinities for oGH and oPRL. 相似文献
10.
Binding of ovine LH to uterine tissue preparation from intact and ovariectomised rat clearly indicates that uterus possesses specific binding sites for LH. Binding characteristics of LH to uterine tissue preparation from intact rat showed saturability with high affinity and low capacity. Scatchard plot analysis showed dissociation constant of the specific binding site to be 0.12 x 10(-9) mol/l and the number of binding sites was 2.31 +/- 0.05 f mol/mg protein. Ovariectomy did not change the binding affinity but effected a decrease in the number of binding sites (1.7 +/- 0.08 f mol/mg protein). LH treatment of ovariectomized (ovx) rat had no effect on binding affinity but significantly increased the number of binding sites (3.23 +/- 0.1 f mol/mg protein). Reduction of uterine weight due to ovariectomy and marked increase of ovx rat uterine weight by LH administration indicate a source of estrogen in ovx rat. An in vitro uterine tissue slice (from intact and ovx rat) incubation showed depletion of 17 beta-estradiol (E2) content in ovx rat which significantly elevated on LH addition. Data suggest that LH binding to rat uterine tissue has biological relevance. 相似文献
11.
Specific binding of 125I-labelled human somatotropin was demonstrated in isolated hepatocytes from male mice. In the presence of divalent cations (Ca2+ and Mg2+) the binding of 125I-labelled human somatotropin was competitive with ovine prolactin. Scatchard analysis of competition data indicated a KD of 1.4 +/- 0.2 nM and a binding capacity of 13 000 +/- 2000 sites/cell. In the absence of divalent cations and in the presence of EDTA, human and bovine somatotropins were found to be equally effective to displace bound 125I-labelled human somatotropin, while ovine prolactin showed a weak competition. In this case, the binding capacity was 8400 +/- 1500 sites/cell and the KD was 1.1 +/- 0.1 nM. 相似文献
12.
《Journal of receptor and signal transduction research》2013,33(4):407-417
AbstractThe specific binding of lactoperoxidase-labelled 125I-labelled ovine prolactin was determined in a membrane particulate of the pigeon crop-sac mucosal epithelium. Binding was found to be dependent upon the particulate preparation used, its protein concentration and the length of the incubation at 5°C. Scatchard analysis of the binding to crop-sacs from saline or prolactin-injected (1.9 μg per pigeon) revealed that prolactin stimulated 7-fold its own receptors by increasing the number of binding sites per mg protein: saline - 392±75 fmol/mg protein and prolactin 2736±602 fmol/mg protein (p<0.01). This increase did not affect the affinity constant (Ka): saline - 5.28±0.75x108 l/mol and prolactin-3.28±0.40x108 l/mol (N.S.), in keeping with the stimulatory effect of prolactin in the rat liver and mammary gland. This study further demonstrates the physiological role of endogenous prolactin in maintaining its own binding-sites in the pigeon crop-sac, since the administration of 0.8 ml anti-serum to prolactin resulted in a 63% reduction in the specific binding of the labelled hormone in vitro. These results confirm the prolactin binding to the pigeon crop-sac mucosa, quantify the stimulation of this binding by prolactin itself, and demonstrate the role of the endogenous hormone in the maintenance of these receptors. 相似文献
13.
The objectives of this study were to determine (i) if the age-related changes in 125I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24–25 days old), young-adult (80–90 days old) and aged (550–610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding. 相似文献
14.
Lipophorin binding to the midgut of Manduca sexta larvae was characterized in a midgut membrane preparation, using iodinated larval high-density lipophorin ((125)I-HDLp-L). The iodination procedure did not change the affinity of the preparation for lipophorin. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The time-course of lipophorin binding to the membranes was affected by the lipophorin concentration in the medium, and at a low lipoprotein concentration, a longer time was required for equilibrium to be reached. The specific binding of lipophorin to the midgut membrane was a saturable process with a K(d) = 1.5+/-0.2x10(-7) M and a maximal binding capacity = 127+/-17 ng lipophorin/microg of membrane protein. Binding did not depend on calcium, was maximal around pH 5.5, was strongly inhibited by an increase in the ionic strength, and abolished by suramin. However, suramin did not completely displace lipophorin that was previously bound to the membrane preparation. The lipid content of the lipophorin did not significantly affect the affinity of the membrane preparation for lipoprotein. 相似文献
15.
The binding of cyclosporin A and ovine prolactin to rabbit mammary gland membranes was determined. CsA bound with a Kd of 2.2 X 10(-6)M whereas prolactin bound with a Kd of 2 X 10(-10)M. The binding of each ligand was an independent event and neither ligand influenced the binding of the other ligand showing that CsA does not inhibit the binding of prolactin to its specific receptor in this system. 相似文献
16.
Several classes of lipids are transported in insect hemolymph by lipophorin, a major hemolymphatic lipoprotein. The binding of lipophorin to the midgut of the hematophagous insect Rhodnius prolixus was characterized in a midgut membrane preparation, using purified lipophorin radiolabelled in protein moiety ((125)I-HDLp). Lipophorin specific binding to membranes achieved equilibrium after 30-40 min, was sensitive to pH, and was maximal at pH 7.0. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The specific binding of lipophorin to the membrane preparation was a saturable process, with K(d)=0.9+/-0.06 x 10(-7) M and a maximal binding capacity of 70+/-11 ng lipophorin/microg of membrane protein. Lipophorin binding did not depend on calcium, but it was affected by ionic strength and was inhibited in the presence of increasing salt concentrations. Suramin interfered with lipophorin binding to the midgut receptor, and it was abolished in the presence of 2 mM suramin, but at concentrations between 0.05 and 0.2 mM it was slightly increased. Condroitin 4-sulfate also affected lipophorin binding, which was reduced to 56% of control. Pre-incubation of the midgut membrane preparation with trypsin or at high temperature inhibited binding. Midgut capacity to bind lipophorin varied at different days after blood meal. It was highest at second day after feeding, and then gradually decreased. 相似文献
17.
Binding sites of a novel neuropeptide pituitary-adenylate-cyclase-activating polypeptide in the rat brain and lung 总被引:2,自引:0,他引:2
H C Lam K Takahashi M A Ghatei S M Kanse J M Polak S R Bloom 《European journal of biochemistry》1990,193(3):725-729
Pituitary-adenylate-cyclase-activating polypeptide (PACAP) is a novel 38-amino-acid neuropeptide isolated from ovine hypothalamic tissues based on its activity of stimulating adenylate cyclase of rat pituitary cells. Binding sites for PACAP were studied in rat tissue membranes using a 27-amino-acid N-terminal derivative of PACAP [PACAP(1-27)] labelled with 125I. Particularly high specific binding sites of 125I-PACAP(1-27) were noted in the hypothalamus, brain stem, cerebellum and lung. Specific binding sites are also present in the pituitary gland, but at a lower concentration, and mainly in the anterior lobe. Very low concentration of 125I-PACAP(1-27)-binding sites were found in the colon, aorta and kidney membranes and no binding sites were detected in the pancreas and testis. Maximal binding of 125I-PACAP(1-27) was observed at pH 7.4. Interaction of 125I-PACAP(1-27) with its binding site was rapid, specific and saturable as well as time, pH and temperature dependent. PACAP(1-27) is more potent than PACAP in displacing the binding of 125I-PACAP(1-27) with brain membranes [concentration that inhibits 50% of the binding (IC50) = 7.45 +/- 1.52 nM and 11.45 +/- 3.65 nM, respectively; mean +/- SEM, n = 4] and lung membranes (IC50 = 4.41 +/- 0.87 nM and 10.68 +/- 3.09 nM, respectively). Vasoactive intestinal peptide displaced the binding of 125I-PACAP(1-27) in lung membrane (IC50 = 16.88 +/- 5.14 nM) but not in brain membranes. The equilibrium binding of 125I-PACAP(1-27) at 4 degrees C was characterized by a single class of binding site for the brain membrane with a dissociation constant (Kd) of 2.46 +/- 0.53 nM and a maximal binding capacity (Bmax) of 8.44 +/- 3.13 pmol/mg protein, but there were two classes of binding site for lung membranes with Kd of 1.02 +/- 0.51 nM and 5.19 +/- 0.99 nM, and Bmax of 2.84 +/- 0.72 pmol/mg protein and 9.13 +/- 1.89 pmol/mg protein, respectively. These findings suggest that subtypes of PACAP-binding sites exist and PACAP may have a physiological role in the hypothalamus/pituitary axis as well as in other regions of the brain and lung. 相似文献
18.
Prolactin secreted by mouse anterior pituitaries was purified by gel filtration on Sephadex G-100. Electrophoretic homogeneity of the purified hormone was demonstrated in several gel systems, and electrophoresis in the presence of sodium dodecyl sulfate indicated an apparent molecular weight of 21 000 +/- 2000. Mouse and ovine prolactin displayed parallel dose vs. response curves in radio-receptor binding studies, indicating that these two hormones compete for identical receptor sites on rabbit mammary membranes. Comparative peptide mapping studies carried out on tryptic digests of mouse and ovine prolactin suggested only partial homology between the two hormones. Internally labeled monomeric mouse prolactin was observed to undergo aggregation following storage at --20 degrees C for 2 months. 相似文献
19.
Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we described the optimal conditions for the measurement of 125I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (Kd approx. 2.36 - 10(-9) M), hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lactation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7--8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in a prompt 3--6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eighth and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro. 相似文献
20.
Summary The binding of intravenously administered prolactin to choroid plexus and brain tissue was determined radioautographically in the ring dove, a species that exhibits prolactin-induced alterations in brain function. An intense autoradiographic reaction was detected over the epithelial cells of the choroid plexus 5 min after the intravenous injection of 125I-ovine prolactin. A significant reaction was also observed over the infundibulum but no significant uptake of prolactin occurred in other brain areas. The binding of radiolabelled prolactin to infundibulum appeared to be non-specific, since excess unlabelled hormone did not reduce silver grain density. In contrast, 125I-ovine prolactin binding in choroid plexus was significantly reduced by excess unlabelled ovine prolactin or human growth hormone, but not by ovine luteinizing hormone. Specific binding to choroid plexus was also detected in vitro. The lack of significant brain uptake of prolactin in vivo is discussed in relation to recent in vitro evidence for specific binding sites for prolactin in several dove brain regions. Similarities between the binding results obtained in this avian species and those reported previously in mammals suggest that the two vertebrate groups exhibit similar patterns of prolactin interaction with neural target tissues. 相似文献