Specific binding of serotonin in rat lung

1985 ◽  
Vol 248 (1) ◽  
pp. E58-E63 ◽  
Author(s):  
D. K. Das ◽  
H. Steinberg
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Direct Action ◽  
High Capacity ◽  
Mitochondrial Fraction ◽  
Temperature Sensitive ◽  
Inner Mitochondrial Membrane ◽  
High Affinity ◽  
Single Temperature ◽  
Maximum Binding Capacity

Mammalian lungs have been shown to store and to inactivate serotonin (5-HT) by an active process involving uptake and metabolism. 5-HT has direct action on lung including constrictor effects of pulmonary vascular and tracheobronchial smooth muscle, suggesting the presence of 5-HT receptors in lung. We have identified specific 5-HT binding of high affinity to the different lung portions and have shown that there was a different capacity for this binding. Two different 5-HT-binding capacities are present in a purified mitochondrial fraction. Saturation analysis of 5-[3H]HT binding to outer mitochondrial membranes demonstrates a single, temperature-sensitive, high-affinity and high-capacity binding (Kd = 8.3 +/- 1.2 nM, maximum binding capacity = 0.819 +/- 0.046 pmol/mg protein). The dissociation constant of inner mitochondrial membrane demonstrates a low-capacity site (Kd = 25.2 +/- 2.2 nM, maximum binding capacity = 0.453 +/- 0.037 pmol/mg protein). The purified microsomal fraction of lung exhibits a high-capacity binding site for 5-[3H]HT (Kd = 14.8 +/- 1.6 nM, maximum binding capacity = 0.760 +/- 0.03 pmol/mg protein). In addition to the lung being the major site for its inactivation, the presence of several specific 5-HT receptors may be related to some of the known 5-HT actions in lung and may suggest other unknown actions of this amine.

Characterization of the binding of 3,3′-di-iodo-l-thyronine to rat liver mitochondria

1997 ◽  
Vol 154 (1) ◽  
pp. 119-124
Author(s):  
A Lombardi ◽  
M Moreno ◽  
C Horst ◽  
F Goglia ◽  
A Lanni
Keyword(s):  
Rat Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Local Environment ◽  
Liver Mitochondria ◽  
Ion Concentration ◽  
Affinity Constant ◽  
Rat Liver Mitochondria ◽  
Inner Mitochondrial Membrane ◽  
Thyroid State

Abstract The binding of labelled 3,3′-di-iodo-l-thyronine (3,3′-T2) to isolated rat liver mitochondria has been characterized. Specific binding could be detected only in the inner mitochondrial membrane, not in other mitochondrial subfractions. The composition of the incubation medium influenced the binding capacity, the best combination of high specific binding and low non-specific binding being observed in phosphate buffer, pH 6·4. The specific binding of 3,3′-T2 to mitochondria requires low ionic strength: concentrations of K+ and Na+ higher than 10 mmol/l and 0·1 mmol/l respectively resulted in a decreased binding capacity. The optimal calcium ion concentration was in the range 0·01–1·0 mmol/l. Varying magnesium ion, over the range of concentrations used (0·1–100 mmol/l), had no effect. Both ADP and ATP, at over 1 mmol/l, resulted in an inhibition of the specific binding. Incubation with protease resulted in a decrease in specific binding and an increase in non-specific binding, thus indicating the proteic nature of the binding sites. In addition to the above factors in the local environment the thyroid state of the animal might influence the 3,3′-T2-binding capacity. In fact, the thyroid state of the animal seemed not to have an influence on the affinity constant, but it did affect binding capacity. Journal of Endocrinology (1997) 154, 119–124

ATP-MgCl2 treatment after trauma-hemorrhage/resuscitation increases hepatocyte P2-purinoceptor binding capacity

1994 ◽  
Vol 266 (6) ◽  
pp. R1810-R1815
Author(s):  
M. S. Mahmoud ◽  
P. Wang ◽  
S. R. Hootman ◽  
S. S. Reich ◽  
I. H. Chaudry
Keyword(s):  
Binding Capacity ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
Beneficial Effects ◽  
High Affinity ◽  
Receptor Component ◽  
Receptor Dynamics ◽  
Maximum Binding Capacity ◽  
High Affinity Receptor ◽  
Affinity Receptor

Although our studies indicate that P2-purinoceptor binding capacity decreases after hemorrhage and resuscitation, it is not known whether ATP-MgCl2 administration after hemorrhage has any beneficial effects on the receptor dynamics. To study this, we performed laparotomy (i.e., trauma induced) on rats and bled them to and maintained them at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). The animals were then resuscitated with 3 times the volume of maximum bleedout with RL over 45 min followed by 2 times RL along with ATP-MgCl2 (50 mumol/kg body wt) over 95 min. Hepatocytes were isolated at 4, 17, and 27 h after resuscitation. P2-purinoceptor binding characteristics were determined by using [alpha-35S]ATP. Scatchard analysis revealed high-affinity and low-affinity receptor components in the hepatocytes isolated from sham-operated or hemorrhaged animals with or without ATP-MgCl2 infusion. ATP-MgCl2 ameliorated and subsequently restored the decreased maximum binding capacity (Bmax) of the high-affinity receptor component and significantly improved Bmax of the low-affinity receptor component. ATP-MgCl2 administration also produced a progressive enhancement in the affinity of the low-affinity receptor component. Thus the beneficial effects of ATP-MgCl2 observed after trauma-hemorrhage and resuscitation may be, in part, due to the restoration of P2-purinoceptor binding capacity and the enhancement of the receptor affinity.

Characterization of galanin receptor in canine small intestinal circular muscle synaptosomes

1994 ◽  
Vol 266 (1) ◽  
pp. G106-G112 ◽  
Author(s):  
C. K. Chen ◽  
T. J. McDonald ◽  
E. E. Daniel
Keyword(s):  
Small Intestine ◽  
Binding Site ◽  
G Protein ◽  
Binding Capacity ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Circular Muscle ◽  
Galanin Receptor ◽  
High Affinity

We used 125I-galanin (porcine) as ligand to study the galanin receptors in circular muscle and deep muscular plexus from canine small intestine. Specific binding sites were found in both nerve and muscle membranes. On synaptosomal membranes, the equilibrium binding study showed a high-affinity (dissociation constant, Kd = 1.1 +/- 0.13 nM; maximum binding capacity, Bmax = 244 +/- 2.1 fmol/mg) binding site. The specific binding of 125I-galanin to nerve membrane was inhibited by galanin or NH2-terminal galanin fragments but not by the COOH-terminal fragment. Computer analysis suggested a two-site model (inhibitor constants, Ki1 = 0.02 +/- 0.005 nM and Ki2 = 1.05 +/- 0.3 nM) for competition by galanin-(1-29). Kinetic and competition studies using guanosine 5'-O-(3-thiotriphosphate) or pertussis toxin (PTX) suggested that the high-affinity binding site involved a PTX-sensitive G protein which acted to slow dissociation of bound galanin from the receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the galanin receptor complex revealed a radioactive band at 50 kDa. We conclude that, in canine small intestine, galanin may act as an inhibitory neuromodulator by a PTX-sensitive G protein-coupled interaction of galanin and its specific receptor on enteric nerve synaptosomes

Uptake of 3,5,3′-l-tri-iodothyronine in human erythrocytes

1989 ◽  
Vol 121 (3) ◽  
pp. 585-591 ◽  
Author(s):  
K. Yamauchi ◽  
R. Horiuchi ◽  
H. Takikawa
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Human Erythrocytes ◽  
The Other ◽  
Transport Pathway ◽  
High Affinity ◽  
High Affinity Site ◽  
Maximum Binding Capacity ◽  
Energy Dependent ◽  
Dependent Pathway

ABSTRACT The mechanisms of 3,5,3′-l-tri-iodothyronine (T3) uptake into human erythrocytes were examined. Purified membranes of human erythrocytes were shown to have two classes of T3-binding sites with one being a high-affinity site (dissociation constant, 59·2±17·8 nmol/l; maximum binding capacity, 344·3 ± 95·5 fmol/μg protein). Furthermore, it was shown that there were two pathways for T3 uptake in human erythrocytes; one was saturable, stereospecific (T3»thyroxine > 3,5,3′-d-tri-iodothyronine), energydependent and dominant at 15 °C; the other was not displaced by unlabelled T3 and was energyindependent but did not occur by passive diffusion. The former pathway which, it is suggested, is a receptor-mediated transport pathway, was inhibited by monodansylcadaverine, phloretin or oligomycin at 15 or 37 °C, but the latter pathway was not inhibited by these inhibitors. Our results strongly suggest that uptake of T3 by the energy-independent pathway became predominant over the energy-dependent pathway at 37 °C and accounted for 83% of total T3 uptake of human erythrocytes. Journal of Endocrinology (1989) 121, 585–591

Effect of prolonged anaerobiosis on 125I-insulin binding to rat soleus muscle: permissive effect of ATP.

1978 ◽  
Vol 235 (6) ◽  
pp. E606
Author(s):  
K T Yu ◽  
M K Gould
Keyword(s):  
Membrane Protein ◽  
Soleus Muscle ◽  
Specific Binding ◽  
High Capacity ◽  
Insulin Binding ◽  
High Affinity ◽  
Dependent Process ◽  
Xylose Uptake ◽  
High Affinity Site ◽  
Rat Soleus Muscle

The specific binding of 125I-insulin by rat soleus muscle was depressed when muscle ATP was depleted, either by prolonged anoxia or more rapidly with 2,4-dinitrophenol. Insulin binding was not eliminated in ATP-depleted muscle, but was reduced by 70--80%. Insulin binding by aerobic muscle could be resolved into two components; a high-affinity, low-capacity site (KD = 7.8 nM) and a low-affinity, high-capacity site (KD = 390 nM). The stimulatory effect of insulin on xylose uptake could be correlated with binding to the high-affinity site. These results indicate that there is some ATP-dependent process involved in the regulation of insulin binding by soleus muscle. It is suggested that this could be a phosphorylation-dephosphorylation system, acting either on the receptor itself or on some closely related membrane protein.

PLASMA CORTICOSTEROIDS AND THE EFFECT OF ADRENOCORTICOTROPHIN IN A MACROPODID MARSUPIAL (SETONIX BRACHYURUS, QUOY & GAIMARD)

1977 ◽  
Vol 75 (3) ◽  
pp. 409-418 ◽  
Author(s):  
I. R. McDONALD ◽  
S. D. BRADSHAW
Keyword(s):  
Plasma Proteins ◽  
Binding Capacity ◽  
Control Value ◽  
High Affinity ◽  
Peripheral Plasma ◽  
Constant Rate ◽  
Moderate Disturbance ◽  
Maximum Binding Capacity ◽  
Repeated Sampling ◽  
The Mean

The total corticosteroid concentrations in the peripheral plasma of unanaesthetized, undisturbed quokkas were 0·75 ± 0·10 (s.e.m.) and 0·93 ± 0·14 μg/100 ml in male and female quokkas respectively. Repeated sampling for periods of 36–49 h disclosed irregular fluctuations over the range 0·4–5·0 μg/100 ml, but no evidence for a regular periodicity. The major corticosteroid was usually cortisol but corticosterone contributed 25–50% of the total unstimulated corticosteroid concentration. Relatively minor concentrations of 11-deoxycortisol were detected. Constant-rate i.v. infusion of ACTH caused a significant increase in the concentration of total corticosteroids in the plasma; this increase was detectable at a dose of 0·05 i.u. ACTH/kg/h, and rose to approximately 15 times the control value at a dose of 2·0 i.u./kg/h. This increase was due mainly to a change in the concentration of cortisol. Synthetic (β1–24) and porcine ACTH were equipotent. The sensitivity of the quokka to ACTH was approximately one-tenth that of another marsupial (Trichosurus vulpecula) or 1/160 that of man. Moderate disturbance increased the concentration of corticosteroids in the plasma to the same level as that caused by infusion of 0·1 i.u. ACTH/kg/h, the increase being mainly in the cortisol fraction. High-affinity binding of cortisol and corticosterone by plasma proteins was demonstrated. The maximum binding capacities for cortisol were 3·89 ± 0·5 and 3·02 ± 0·6 μg/100 ml in female and male quokkas respectively. The mean association constant was 3·2 × 1081/mol at 4 °C and 5·5 × 1071/mol at 36 °C. The maximum binding capacity for corticosterone was approximately one-third that of cortisol.

scholarly journals A subclass of glutathione S-transferases as intracellular high-capacity and high-affinity steroid-binding proteins

1986 ◽  
Vol 235 (3) ◽  
pp. 763-768 ◽  
Author(s):  
H Homma ◽  
H Maruyama ◽  
Y Niitsu ◽  
I Listowsky
Keyword(s):  
Binding Capacity ◽  
High Capacity ◽  
Gel Permeation Chromatography ◽  
Hormone Metabolism ◽  
High Affinity ◽  
Glutathione S Transferases ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
Binding Component

The distribution of glucocorticoids incubated with rat liver cytosol preparations or administered in vivo to adrenalectomized rats was analysed by chromatographic procedures. Corticosterone or dexamethasone was co-eluted with Yb-type GSH S-transferases in anion-exchange and gel-permeation chromatography systems, and these glucocorticoids also were bound to Yb forms in analyses by immunoadsorbent and lysyl-GSH affinity matrices. Pretreatment of cytosol with lysyl-GSH to extract GSH S-transferases or incubation with excess bilirubin, which is expected to compete with steroids for binding to the protein, yielded preparations that were devoid of this major steroid-binding component. In mixtures of the multiple rat GSH S-transferases, corticosterone preferentially interacted with Yb forms rather than Ya and Yc subgroups. All of the multiple Yb forms resolved by chromatofocusing procedures retained the steroid-binding capacity. It is suggested that these abundant proteins can account for a considerable share of intracellular glucocorticoid binding and represent a high-affinity non-saturable binding component with potential to function in steroid-hormone metabolism and action.

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)

Binding of bumetanide to microsomes from optic ganglia of the squid, Loligo pealei

1990 ◽  
Vol 258 (5) ◽  
pp. C933-C943 ◽  
Author(s):  
A. A. Altamirano ◽  
B. A. Watts ◽  
J. M. Russell
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Affinity Constant ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Relative Affinity ◽  
Microsomal Membranes ◽  
Loligo Pealei ◽  
Dissociation Constant Kd

Saturable high-affinity binding of [3H] bumetanide [dissociation constant (KD) = 80 nM] was measured in microsomal membranes prepared from squid optic ganglia. Under control conditions, the maximal specific binding of labeled bumetanide (Bmax) was approximately 6-7 pmol/mg protein. Binding had a higher relative affinity for bumetanide than for furosemide and depended on the presence of Cl- and K+, but not Na+, in the incubation media. In the case of K+, [3H]bumetanide binding was half-saturated at [K+] = 100 mM. The Cl- effect was biphasic. At [Cl-] between 0 and 150 mM, [3H]bumetanide binding increased with increasing [Cl-]. However, when [Cl-] was increased above 150 mM, [3H]bumetanide binding was progressively reduced. ATP acted as a nonessential activator [mean affinity constant (K0.5) approximately 1 microM] of the ion-dependent [3H]bumetanide binding by increasing the apparent binding capacity. The activation by ATP did not require Mg2+. Other adenosine analogues also stimulated the binding of bumetanide.

scholarly journals Identification of Novel High Molecular Weight Insulin-Like Growth Factor-Binding Protein-3 Association Proteins in Human Serum1

1998 ◽  
Vol 83 (8) ◽  
pp. 2843-2848
Author(s):  
Paulo F. Collett-Solberg ◽  
Steven E. Nunn ◽  
Tara Beers Gibson ◽  
Pinchas Cohen
Keyword(s):  
Growth Factor ◽  
Human Serum ◽  
Serum Proteins ◽  
Binding Capacity ◽  
Specific Binding ◽  
Insulin Like Growth Factor ◽  
Igf Binding Proteins ◽  
High Affinity ◽  
Acid Labile ◽  
Igfbp 3

abstract The insulin-like growth factor (IGF)-binding proteins (IGFBPs) carry IGFs in serum and regulate their activity and bioavailability. The main IGFBP in serum, IGFBP-3, is known to form a 150-kDa complex with IGFs and the acid-labile subunit (ALS). We investigated the binding of IGFBP-3 to additional association proteins in human serum (IGFBP-3 APs). Ligand blots, column chromatography, and affinity cross-linking experiments revealed the specific binding of IGFBP-3 to at least three novel serum proteins. These techniques demonstrated the presence of proteins with molecular masses of 70, 100, and 150 kDa that bind IGFBP-3 with high affinity. Serum ALS migrated separately (at 88 kDa) from the novel IGFBP-3 APs (as evident by Western immunoblot), and bound IGFBP-3 weakly (by reverse ligand blots). We also demonstrated that large amounts of one of the IGFBP-3 APs and small amounts of ALS were coimmunoprecipitated with IGFBP-3 from human serum. Similar to ALS, these IGFBP-3 APs are acid labile and lose their IGFBP-3 binding capacity after exposure to low pH. We conclude that there are several serum proteins in addition to ALS and IGFs that bind IGFBP-3 with high affinity. These IGFBP-3 APs may serve as an additional reservoir of IGFBP-3 or modulate its functions.

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