scholarly journals Influence of divalent cations on the detection of somatogenic and lactogenic binding sites in mouse liver cells

1985 ◽  
Vol 228 (3) ◽  
pp. 761-764 ◽  
Author(s):  
G N Ciccia-Torres ◽  
J M Dellacha
Keyword(s):  
Binding Sites ◽  
Mouse Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Divalent Cations ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Scatchard Analysis ◽  
Male Mice ◽  
Ovine Prolactin

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.

Specific binding sites for ovine prolactin in three amphibian cell lines

1985 ◽  
Vol 248 (1) ◽  
pp. C80-C87 ◽  
Author(s):  
M. Dunand ◽  
M. L. Aubert ◽  
J. P. Kraehenbuhl ◽  
B. C. Rossier
Keyword(s):  
Growth Hormone ◽  
Cell Lines ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Growth ◽  
Functional Differentiation ◽  
Water Metabolism ◽  
Ovine Prolactin ◽  
Follicle Stimulating Hormones

Established cell lines (TB-6c and TB-M) obtained by continuous culture of epithelial cells from toad Bufo marinus urinary bladder, which, in culture, maintained a high degree of functional differentiation, exhibited a significant number of high-affinity (KA = 1-2 X 10(10) M-1) binding sites detected both with radioiodinated (125I) ovine prolactin (oPRL) and human growth hormone (hGH). Binding capacity was higher in the case of TB-6c cells (7,573 +/- 581 sites/cell) than with the TB-M cells (1,160 +/- 87). Similarly, binding sites for oPRL were characterized on Xenopus laevis kidney-derived cell line A6. With oPRL used both as tracer and standard, significant cross-reaction was observed with hGH, less with human or rat prolactin (PRL), and none with human chorionic somatomammotropin, bovine growth hormone, and rat luteinizing hormone or follicle-stimulating hormones. B. marinus pituitary extracts completely displaced the binding of 125I-oPRL to toad bladder binding sites. This finding of specific sites for PRL on amphibian bladder and kidney cells confirms that PRL exerts specific biological actions for the control of electrolyte and water metabolism in the amphibians.

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.

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).

PGF2 alpha and PGE2 binding to rat myometrium during gestation, parturition, and postpartum

1990 ◽  
Vol 258 (5) ◽  
pp. E740-E747
Author(s):  
M. Molnar ◽  
F. Hertelendy
Keyword(s):  
Placebo Group ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Time Dependent ◽  
Scatchard Analysis ◽  
Plasma Progesterone ◽  
High Affinity ◽  
The Third ◽  
Venous Plasma

The specific binding of prostaglandins (PG) F2 alpha and E2 was studied in a rat myometrial membrane-enriched fraction during the latter part of gestation and parturition, as well as in the postpartal period. Tritiated PGE2 and PGF2 alpha binding was specific, saturable, time dependent, and directly proportional to the amount of membrane protein. Scatchard analysis indicated the presence of high-affinity (Kd2) and low-affinity (Kd2) binding sites for both PGs. The affinity of both binding sites for PGF2 alpha and the apparent Kd2 for PGE2 remained essentially the same throughout gestation and post-partially and were similar to nonpregnant rats. The apparent Kd1 of PGE2, however, increased by 10-fold from day 21 of gestation to 1 day postpartum. Although the maximal binding capacity of the high-affinity (Bmax1) and low-affinity (Bmax2) binding sites of PGF2 alpha showed a nonsignificant increase compared with prepartum values, reaching maximal values 12-24 h postpartum, those of PGE2 showed a significant increase on the third day after delivery. The concentration of prostanoids in uterine venous plasma and amniotic fluid increased significantly with approaching parturition, whereas plasma progesterone decreased, raising the estradiol-progesterone ratio 25-fold. After unilateral fetectomy, the binding sites for PGF2 alpha and PGE2 increased significantly compared with the contralateral pregnant horns. Administration of the PG synthetase inhibitor, indomethacin, also increased two- to threefold both PGF2 alpha and PGE2 binding compared with the placebo group, whereas intrauterine administration of PGF2 alpha and PGE2 significantly reduced it.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

1984 ◽  
Vol 219 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
Y A Lefebvre ◽  
J T Venkatraman
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Intact Animal ◽  
Binding Capacity ◽  
Specific Binding ◽  
Male Rat ◽  
Scatchard Analysis ◽  
Specific Binding Sites ◽  
Number Of Binding Sites

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3′,5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3′,5′-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.

Binding sites for interferons on ovine and human endometrial membranes

1993 ◽  
Vol 5 (2) ◽  
pp. 219 ◽  
Author(s):  
DL Russell ◽  
GG Manalo ◽  
JK Findlay ◽  
LA Salamonsen
Keyword(s):  
Binding Sites ◽  
Luteal Phase ◽  
Binding Capacity ◽  
Specific Binding ◽  
Major Product ◽  
Human Endometrium ◽  
Scatchard Analysis ◽  
Ovarian Steroids ◽  
Steroid Dependence ◽  
Proliferative Phase

In the ewe, the major product of the preimplantation blastocyst is ovine trophoblast protein-1 (oTP-1), which is now classified as an omega-interferon (IFN). Receptors for IFN are present on sheep endometrium and vary cyclically, presumably modified by the actions of ovarian steroids. This study examined whether or not IFN receptors were present on human endometrium at any stage during the menstrual cycle. In addition, the steroid dependence of ovine endometrial IFN receptors was determined. Specific binding of 125I-labelled IFN (125I-IFN) to ovine endometrial membranes was substantially higher than binding to membranes derived from bovine spleen, human placenta or pooled human endometrium (relative specific binding 100:33:36:20). Human endometrial membrane preparations from proliferative-phase tissue showed very little specific binding (mean 0.8 +/- 0.3%, n = 4) in contrast to luteal-phase endometrium (2.1 +/- 0.3%, n = 8). Treatment of ovariectomized ewes with oestradiol-17 beta (E) resulted in significantly increased binding (117 +/- 7%) of 125I-IFN to endometrial tissues compared with tissue from ovariectomized (OvX, 75 +/- 7%), progesterone (P)-treated (69 +/- 7%), or (E + P)-treated (81 +/- 8%) groups (P < 0.05); all were compared with binding to pooled ovine luteal-phase tissue, 100%. There were no differences between the other three groups. Scatchard analysis showed binding affinity of the same order for the sheep and human receptors (Kd = 10(-10) mol L-1) but binding capacity was considerably lower for human (6.0 fmol mg-1) than for sheep (47-123 fmol mg-1) endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormone receptors in the epiphysial cartilage

1984 ◽  
Vol 103 (2) ◽  
pp. 125-131 ◽  
Author(s):  
K. W. Kan ◽  
R. L. Cruess ◽  
B. I. Posner ◽  
H. J. Guyda ◽  
S. Solomon
Keyword(s):  
Growth Plate ◽  
Binding Sites ◽  
Hormone Receptors ◽  
Binding Capacity ◽  
Specific Binding ◽  
Serum Insulin ◽  
Skeletal Growth ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Epiphysial Cartilage

ABSTRACT In order to assess which hormones may exert direct effects on skeletal growth at the epiphysial growth plate, the specific binding of hormones to the epiphysial cartilage of growing dogs and rabbits was studied. Membrane fractions obtained by centrifugation of homogenates prepared from dog and rabbit growth plate cartilage at 600, 15 000 and 105 000 g showed significant specific binding of serum insulin-like activity and insulin. Binding of growth hormone and prolactin by the three membrane fractions was negligible. Saturable binding sites for triiodothyronine could be demonstrated in nuclei from the dog growth plate. Nuclear binding showed an apparent Kd of 11 ±3·6 nmol/l and a maximum binding capacity of 4·1 ± 1·6 pmol/mg DNA, a level comparable to dog liver. Using a viable chondrocyte suspension prepared from dog epiphysial cartilage, specific steroid binding in the cells could be demonstrated for [3H]dexamethasone but not 17α-methyltrienolone, oestradiol-17β or 1α,25-di-hydroxycholecalciferol. Scatchard analysis of dexamethasone binding showed high affinity binding sites having a Kd of 1·2 ± 0·35 nmol/l and a capacity of 1700 sites/cell, and a low affinity binding with a Kd of 109 ± 57 nmol/l and a capacity of 24 000 sites/cell. Steroid competition for the specific binding showed the following sequence of affinity: dexamethasone > corticosterone > 11-deoxycortisol > testosterone > oestradiol-17β. Although all of the hormones examined except prolactin have well-established physiological effects on skeletal growth, our present results suggest that some of the hormonal effects observed in intact animals are secondary and do not involve receptor–hormone interaction in cartilage as such. J. Endocr. (1984) 103, 125–131

Direct effect of endothelin-1 on the granulosa cells of the porcine ovary

1992 ◽  
Vol 134 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S. Kamada ◽  
T. Kubota ◽  
Y. Hirata ◽  
M. Taguchi ◽  
S. Eguchi ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Binding Sites ◽  
Inositol Phosphate ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
Endothelin 1 ◽  
Porcine Granulosa Cells

ABSTRACT Specific binding sites for endothelin-1 (ET-1), a novel potent vasoconstrictor peptide, as well as the effects of ET-1 on cytosolic free Ca2+ concentration ([Ca2+]i), intracellular total inositol phosphate (IP) generation and steroidogenesis were studied in cultured porcine granulosa cells. Scatchard analysis of a binding study using 125I-labelled ET-1 indicated the presence of a single class of high-affinity binding sites with almost equal affinity for ET-1 and ET-3: the apparent dissociation constant was 0·59 nmol/l and the maximal binding capacity was 1·84 pmol/mg protein. Affinitylabelling of 125I-labelled ET-1 to the membranes using disuccinimidyl tartarate as a cross-linker revealed one major and one minor band with the apparent molecular weights of 32 kDa and 49 kDa respectively. ET-1 dose-dependently (1−100 nmol/l) induced rapid and transient increases in [Ca2+]i in fura-2-labelled cells. ET-1 also dose-dependently stimulated total IPs in cells prelabelled with myo-[3H]inositol. ET-1 had a slight stimulatory effect on the secretion of progesterone but not of oestradiol from porcine granulosa cells. The present data clearly demonstrate the presence of a non-selective ET receptor (ETB) in porcine granulosa cells coupled with phosphoinositide hydrolysis and [Ca2+]i mobilization, and suggest that ET-1 may play some role in the production of progesterone by porcine granulosa cells. Journal of Endocrinology (1992) 134, 59–66

Fasting alters somatostatin binding to liver membranes of rainbow trout (Oncorhynchus mykiss)

1996 ◽  
Vol 150 (2) ◽  
pp. 179-186 ◽  
Author(s):  
M J Pesek ◽  
M A Sheridan
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
High Affinity ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
C Terminus ◽  
Liver Membranes

Abstract Somatostatins are a diverse family of peptides that influence various aspects of animal growth, development, and metabolism. Recent work in our laboratory has shown that somatostatins stimulate hepatic lipolysis in rainbow trout. In this study we characterized somatostatin-binding sites in trout hepatic membrane preparations. We also examined changes in binding characteristics brought about by food deprivation. Binding of [Tyr11]-somatostatin-14 (SS-14) was saturable, reversible, and time- and temperature-dependent. Under optimal conditions, [Tyr11]-SS-14 specific binding averaged 5·7 ± 0·3%. While SS-14 and SS-28 (an N-terminally extended form of SS-14 and derived from the same gene as SS-14) displaced [Tyr11]-SS-14 specific binding (ED50 values of approximately 50 nm and 100 nm respectively), salmon SS-25 (containing [Tyr7,Gly10]-SS-14 at its C terminus and presumably derived from a gene different from that giving rise to SS-14/SS-28), except at pharmacological concentrations, did not. Significant specific binding was also detected in brain, esophagus, stomach, upper and lower intestine, pancreas, and adipose tissue. Scatchard analysis suggested the existence of two classes of hepatic somatostatin-binding sites: a high-affinity site with a Kd of 23 nm and Bmax of 1·4 pmol/mg protein and a low-affinity site with a Kd of 379 nm and Bmax of 4·9 pmol/mg protein. Fasting resulted in reduced growth and elevated plasma levels of SS-14 compared with fed animals. SS-14 binding capacity of the high-affinity class in liver membranes isolated from fasted fish increased by 120% over that from fed counter-parts. No difference in Kd for the high-affinity binding class or in either Kd or Bmax of the low-affinity class was noted between fasted and fed animals. These data support the role of the liver as a target of somatostatin and suggest that fasting enhances hepatic sensitivity to SS-14 binding. Journal of Endocrinology (1996) 150, 179–186

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