Evidence for specific binding and stimulatory effects of recombinant human erythropoietin on isolated adult rat Leydig cells

1992 ◽  
Vol 127 (5) ◽  
pp. 459-465 ◽  
Author(s):  
Roberto Mioni ◽  
Francesco Gottardello ◽  
Paola Bordon ◽  
Gianni Montini ◽  
Carlo Foresta
Keyword(s):  
Recombinant Human Erythropoietin ◽  
Leydig Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Maximal Effect ◽  
Adult Rat ◽  
Testosterone Production ◽  
Human Erythropoietin ◽  
Binding Data

The presence of specific binding of recombinant human erythropoietin and its effect on testosterone production were evaluated in isolated intact adult rat Leydig cells. Maximal specific binding was observed after 135 min incubation at 34°C. Scatchard analysis of the binding data revealed two distinct classes of binding sites for [125I]-recombinant human erythropoietin with dissociation constant of(Kd1) 1.9× 10−10mol/l and (Kd2) 1.37× 10−8 mol/l respectively and binding capacity of (Bmax1) 12.3fmol/l 106 cells and (Bmax2) 42.8 fmol/106 cells, respectively. GnRH, hCG, IGF-I and EGF did not induce any modification of recombinant human erythropoietin-specific binding. Recombinant human erythropoietin added to isolated adult rat Leydig cells exerted a stimulatory effect on testosterone production reaching its maximal effect at the dose of 10−10 mol/l (testosterone production from 14.9±1.7 to 45.1±6.2 pmol/106 cells/3 h). Addition of anti-recombinant human erythropoietin serum completely blocked the recombinant human erythropoietin-stimulated testosterone production. These results show that purified adult rat Leydig cells possess recombinant human erythropoietin specific binding, and suggest that this glycoprotein directly influences rat Leydig steroidogenesis.

BINDING OF HUMAN CHORIONIC GONADOTROPHIN BY RAT TESTIS: EFFECT OF SEXUAL MATURATION, CRYPTORCHIDISM AND HYPOPHYSECTOMY

1975 ◽  
Vol 64 (1) ◽  
pp. 59-66 ◽  
Author(s):  
JOACHIM FROWEIN ◽  
WOLFGANG ENGEL
Keyword(s):  
Dissociation Constant ◽  
Sexual Maturation ◽  
Leydig Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Scatchard Analysis ◽  
Rat Testis ◽  
Relative Binding

SUMMARY The specific binding of 125I-labelled human chorionic gonadotrophin (HCG) by rat testicular homogenate as compared with isolated Leydig cells differs with respect to total binding capacity but not to the dissociation constant (KD) as revealed by Scatchard analysis. The maximal binding capacity for [125I]HCG of crude testicular homogenate was 95 ng/g rat testis. Hypophysectomy causes a decline in binding capacity within the first three days but on the 20th and 30th day after hypophysectomy the relative binding capacity no longer differs from that of controls. Binding capacity is enhanced in cryptorchid testes relative to normal, and increases during sexual maturation to a peak shortly before puberty.

Calcium and the Fc Receptor on Human Platelets

1987 ◽  
Vol 57 (03) ◽  
pp. 298-301
Author(s):  
William F Clark ◽  
Gerald J M Tevaarwerk ◽  
Bruce D Reid ◽  
Suzanne Hall ◽  
Anita Caveney ◽  
...  
Keyword(s):  
Specific Binding ◽  
Normal Subjects ◽  
Human Platelets ◽  
Fc Receptor ◽  
Normal Male ◽  
Scatchard Analysis ◽  
Normal Female ◽  
Apparent Difference ◽  
Binding Data ◽  
Direct Binding

SummaryWe have described the calcium dependence of the IgG Fc receptor (Fc-R) on human platelets by analyzing the direct binding of radiolabelled Fc fragments, monomers and dimers of IgG. Specific binding to platelets was undetectable at 37° C in a calcium-free preparation but readily detected when calcium was restored. Scatchard analysis of the binding data for the calcium-restored platelets permitted calculation of the available Fc-R and the Ka of binding for the different IgG ligands. The mean Ka of binding for 12 normal subjects varied from 107 to 108 L/M, with an equal receptor number measured by Fc fragments and dimers of IgG, but a lesser amount for monomeric IgG. There was no apparent difference in Fc-R number for platelets from 6 normal male versus 6 normal female subjects.At 4° C binding was detectable for dimers and polymers of IgG in a calcium-free preparation and this was markedly increased with recalcification. Thus, our data are consistent with an Fc receptor population on human platelets whose avidity for binding is significantly enhanced in a calcium-restored medium.

scholarly journals Hepoxilin A3-specific binding in human neutrophils

1996 ◽  
Vol 313 (2) ◽  
pp. 537-541 ◽  
Author(s):  
Denis REYNAUD ◽  
Peter DEMIN ◽  
Cecil R. PACE-ASCIAK
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Leukotriene B4 ◽  
Proteinase K ◽  
Human Neutrophils ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Specific Binding Sites ◽  
Binding Data ◽  
Hepoxilin A3

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 °C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 °C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3±9.1 nM and 8.86±1.4 pmol/ml per 2×106 cells (±S.E.M.) respectively reflecting approx. 2.67×106 sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.

Expression of insulin-like growth factor receptors in primary human thyroid neoplasms

1989 ◽  
Vol 121 (1) ◽  
pp. 112-120 ◽  
Author(s):  
Tohru Yashiro ◽  
Yoshito Ohba ◽  
Hitomi Murakami ◽  
Takao Obara ◽  
Toshio Tsushima ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Thyroid ◽  
Scatchard Analysis ◽  
Type I ◽  
Single Class ◽  
Normal Tissues ◽  
Igf I ◽  
Cancer Tissues

Abstract. The presence of IGF-I receptors was demonstrated in normal and neoplastic tissues of human thyroid. Binding of [125I]IGF-I to thyroid membranes was dependent on time and temperature of incubation, and maximal binding was achieved at 4°C and 18 h of incubation. [125I] IGF-I binding was dose-dependently displaced by unlabelled IGF-I; half-maximal inhibition occurred at concentrations of 10–20 μg/l. IGF-II and insulin had relative potencies of 5 and 1% compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with high affinity (Ka: 1.2–8.6 × 109 1/mol) in normal thyroid tissues. Affinity cross-linking and autoradiography demonstrated the type I IGF receptors. Specific binding of [125I] IGF-I in thyroid cancer tissues (9.69 ± 2.07% per 200 μg protein; mean ± sem, N = 8) was significantly (p <0.05) higher than that in the surrounding normal tissues (3.03 ± 0.35%, N = 8). In contrast, there was no difference in the binding between adenoma tissues (4.19 ± 0.53%, N = 5) and the adjacent normal tissues (2.94 ± 0.24%, N = 5). The higher IGF-I binding in cancer tissues was due to an increase in the binding capacity without any change in the affinity. The presence of IGF-I receptors suggests a possible role of IGF-I and its receptors in the growth of thyroid cancer cells.

Characterization of [3H]nifedipine binding to intact vascular smooth muscle cells

1988 ◽  
Vol 254 (1) ◽  
pp. C45-C52 ◽  
Author(s):  
K. Sumimoto ◽  
M. Hirata ◽  
H. Kuriyama
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Smooth Muscles ◽  
Muscle Cells ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Porcine Coronary Artery ◽  
Control Value

Specific binding of the dihydropyridine Ca2+ antagonist [3H]nifedipine to dispersed smooth muscle cells of the porcine coronary artery was investigated and the findings were compared with the binding to microsomes of smooth muscles. Specific binding to intact cells was saturable and reversible. The dissociation constant was 1.93 +/- 0.42 nM and the maximal binding capacity was 59.6 +/- 12.4 fmol/10(6) cells, as assessed by Scatchard analysis of the equilibrium binding at 25 degrees C. The Kd value with intact cells was slightly higher than that observed with microsomes. Specific binding of [3H]nifedipine to intact cells was completely displaced by unlabeled dihydropyridine derivatives. Among other Ca2+ antagonists, verapamil and d-cis-diltiazem partially and flunarizine completely inhibited the binding. In the case of microsomes, d-cis-diltiazem stimulated the binding of [3H]nifedipine. These results suggest that there may be multiple binding sites for different subclasses of Ca2+ antagonists. Polyvalent cations had no effect on the binding to intact cells. In the case of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated microsomes, the addition of CaCl2 and BaCl2 increased the Bmax, but the Kd value remained unchanged. MnCl2 and CdCl2 had stimulatory or inhibitory effects, depending on the concentrations, whereas LaCl3 had no effect. The effect of membrane depolarization on the binding was also examined. When the intact cells were incubated in high [K+]o solution for 60 min, the Kd was lowered to 1.4 nM from the control value of 2.0 nM, thereby indicating that [3H]nifedipine binds to Ca2+ channels, with a higher affinity, at depolarized states.

The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin

1981 ◽  
Author(s):  
P Silber ◽  
T H Finlay
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Binding Constant ◽  
Specific Binding ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Binding Data ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.

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.

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

1981 ◽  
Vol 241 (6) ◽  
pp. F605-F611 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Collecting Tubule ◽  
The Difference ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

SPECIFIC BINDING OF PROLACTIN TO SEMINAL VESICLE, PROSTATE AND TESTICULAR HOMOGENATES OF IMMATURE, MATURE AND AGED RATS

1977 ◽  
Vol 74 (2) ◽  
pp. 163-173 ◽  
Author(s):  
R. J. BARKEY ◽  
J. SHANI ◽  
T. AMIT ◽  
D. BARZILAI
Keyword(s):  
Seminal Vesicle ◽  
Binding Capacity ◽  
Specific Binding ◽  
Age Groups ◽  
Liver Homogenate ◽  
Binding Specificity ◽  
Scatchard Analysis ◽  
Total Binding ◽  
Ovine Prolactin ◽  
Rat Prostate

Ovine prolactin was iodinated by the lactoperoxidase method and purified by gel filtration on Sephadex G-100. The binding ability of the labelled hormone was determined, by incubation with liver homogenate from rabbits in late pregnancy, to be 8·8% total binding/ mg protein, of which 86% was specific. The fraction of 125I-labelled ovine prolactin which bound most strongly was subsequently used to study its binding to rat seminal vesicle, prostate and testicular homogenates. The total binding to the seminal vesicle homogenate taken from mature (80-day-old) rats was the highest (11·69%/mg protein), but the greatest degree of binding specificity (82·6%) was to immature (30-day-old) rat prostate. Both total and specific binding to rat testicular homogenate were consistently very low. The binding specificity was demonstrated by displacement studies: while ovine prolactin caused displacement of specific binding, human chorionic gonadotrophin, rat thyrotrophin and human follicle-stimulating hormone did not cause any significant displacement of bound 125I-labelled ovine prolactin. Affinity constants (Ka) and binding capacities for the seminal vesicle and prostate homogenates were determined by Scatchard analysis and the effect of age on these parameters was studied. There was no difference in Ka between the aged (220-day-old), immature and mature rat tissue homogenates; however, a significant fall in binding capacity was observed in the mature rat prostate, and a further fall in the aged rat prostate. No such change was observed in the binding capacity of the seminal vesicle, as estimated by Scatchard analysis, although total and specific binding to the mature homogenates was higher than that of the other age groups.

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