Aldosterone binding in isolated tubules I. Biochemical determination in proximal and distal parts of the rabbit nephron

1982 ◽  
Vol 242 (1) ◽  
pp. F63-F68 ◽  
Author(s):  
N. Farman ◽  
A. Vandewalle ◽  
J. P. Bonvalet
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Nuclear Fraction ◽  
Lower Affinity ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Collecting Tubules ◽  
Binding Curve

Microbiochemical methods were applied to proximal tubules (PCT) and a mixture of distal and cortical collecting tubules (D + C) of rabbit kidney in order to define aldosterone binding sites. For each experiment, after incubation of kidney pyramids with [3H]aldosterone ([3H]A), either alone or in the presence of an excess unlabeled A, 100-150 mm of both categories of tubules were microdissected using collagenase. Specific binding was determined on the nuclear fraction of each sample. Aldosterone concentrations ranged from 2 X 10(-9) to 4.5 X 10(-8) M. No specific binding was detectable in PCT. Specific binding in D + C increased rapidly as a function of [3H]A concentration up to 5 X 10(-9) M and then more slowly. No plateau was reached. Both the absence of saturation of the binding curve and the curvilinear aspect of the Scatchard plot suggested the presence of two binding sites, one of high affinity, presumably a mineralocorticoid site, and the other of lower affinity, possibly a glucocorticoid site. These experiments suggest that the distal structures of the nephron, located in the cortex, are the main sites of binding of aldosterone and contain a high number of specific binding sites for this hormone.

Autoradiographic determination of dexamethasone binding sites along the rabbit nephron

1983 ◽  
Vol 244 (3) ◽  
pp. F325-F334 ◽  
Author(s):  
N. Farman ◽  
A. Vandewalle ◽  
J. P. Bonvalet
Keyword(s):  
Proximal Tubule ◽  
Binding Sites ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Nuclear Binding ◽  
Specific Binding Sites ◽  
In Vitro Incubation ◽  
Collecting Tubules

Specific binding sites of tritiated dexamethasone ([3H]dex) along the tubule of rabbit kidney were investigated using an autoradiographic method (dry film) on isolated tubular segments. After in vitro incubation of kidney pyramids with [3H]dex (0.15-53 nM) in the presence or absence of an excess (X200) of unlabeled dexamethasone, tubular segments were microdissected and processed for autoradiography. A quantitative analysis of specific labeling over cytoplasm and nuclei was performed. Specific nuclear binding was observed in all tubular segments beyond the pars recta. This binding was dose dependent and reached much higher values than those reported for aldosterone. In the proximal tubule, the specific labeling was also high but remained mostly cytoplasmic. The meaning of these drastically different intracellular localizations is still open to interpretation. Autoradiography was performed after in vivo injection of [3H]dex and [3H]aldosterone. The results were not different from those described here for dexamethasone and from those previously reported for aldosterone after in vitro incubation. We conclude that specific nuclear binding sites for dexamethasone range over the nephron except for proximal tubule, with no great difference among segments, in contrast to specific sites for aldosterone, which are restricted to distal and cortical collecting tubules. The exact significance of the proximal cytoplasmic specific binding of [3H]dex remains to be determined.

scholarly journals Binding of Calcium to Fibrinogen: Some Related Properties

1977 ◽  
Author(s):  
G. Marguerie
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Specific Binding ◽  
Equilibrium Dialysis ◽  
Structural Features ◽  
Salt Bridges ◽  
High Affinity ◽  
Direct Titration ◽  
Specific Binding Sites ◽  
Calcium Binding Sites

The calcium binding properties of bovin fibrinogen have been studied using equilibrium dialysis method. At pH 7.5 fibrinogen has 3 specific calcium binding sites of high affinity and several non specific binding sites of low affinity. Direct titration of the calcium induced proton release indicates that the binding center is a chelate. Thermal an acid denaturation is found to be markedly influenced by the presence of Ca++, suggesting that structural features are related to the binding. However the circular dichroism spectra show that no generalized conformational change is induced when Ca++ is bound to the protein.The plasminic digestion of fibrinogen is also found to be specificaly influenced by Ca++. The velocity of the initial cleavages is slightly reduced in the presence of calcium. It is therefore suggested that the C-terminal part of the Aα chain is involved in the binding.Considering the dimeric structure of the fibrinogen molecule, the presence of only 3 calcium binding sites of high affinity suggests the existence of “salt bridges” between the constitutive polypeptide chains.

EFFECT OF Ca2+ and Mg2+ ON PLATELET ACTIVATING FACTOR (PAF) INDUCED AGGREGATION AND SPECIFIC BINDING TO HUMAN PLATELETS

1987 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Calcium Channel ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Human Platelets ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Significant Difference ◽  
Induced Aggregation

Gel filtered human platelets (GFP) collected in Tyrode's buffer containing 0.5 mM Ca+2, ImM Mg+2, and 0.35% albumin exhibit high affinity binding of 3H-PAF with a Kd of 0.109 α 0.029 nM (mean α SD; n=13) and 267 α 70 sites per platelet. When fibrinogen (1.67 mg/ml final concentration) is added to these GFP preparations biphasic aggregation is observed with PAF (4 nM). Normal aggregation is also observed with other platelet agonists including ADP, epinephrine, collagen, arachidonic acid, A23187 and thrombin. If GFP is prepared without added Ca+2 or Mg+2 in the presence of 3mM EDTA, platelets do not aggregate in response to PAF. However the number of specific binding sites remains unchanged (387 per platelet) with some decrease in affinity of binding (Kd = 0.2l4nM). In the presence of ImM Mg+2 there is no significant difference in binding kinetics over a range of Ca+2 concentrations (0-2mM). On the other hand the calcium channel blocker verapamil (5-10uM) exhibits competitive inhibition of 3H-PAF as analyzed by Lineweaver-Burk plots. Specific binding of 3H-PAF to GFP in the presence of ImM Mg+2 and ImM EGTA shows Kd of 0.l66nM but with increase in specific binding sites to 665. Despite increase in number of sites and no change in binding affinity, GFP under these conditions does not exhibit platelet aggregation with PAF in doses up to 80 nM.From these data it appears that external Ca+2 is not necessary for specific binding of 3H-PAF to its high affinity receptor. However, calcium does appear to be necessary for second wave aggregation with PAF. While Mg+2 appears to enhance 3H-PAF binding to platelets Mg+2 cannot substitute for Ca+2 in PAF induced platelet aggregation. Although verapamil appears to competitively inhibit binding of PAF to GFP it is not clear whether the inhibition is due to competition at or near the actual PAF receptor or at a site involving the calcium channel.

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.

Evidence for Presynaptic High Affinity Imidazoline-Specific Binding Sites in the Bovine Brainstem

1999 ◽  
Vol 881 (1 IMIDAZOLINE R) ◽  
pp. 185-188 ◽  
Author(s):  
F. M. J. HEEMSKERK ◽  
M. DONTENWILL ◽  
H. GRENEY ◽  
C. VONTHRON ◽  
P. BOUSQUET
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
High Affinity ◽  
Specific Binding Sites

Specific binding sites in the rabbit kidney for prostaglandin a

1973 ◽  
Vol 4 (5) ◽  
pp. 703-709 ◽  
Author(s):  
Ahmad A. Attallah ◽  
James B. Lee
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Specific Binding Sites

scholarly journals The Ins(1,4,5)P3 binding site of bovine adrenocortical microsomes: function and regulation

1989 ◽  
Vol 260 (2) ◽  
pp. 593-596 ◽  
Author(s):  
S Palmer ◽  
M J O Wakelam
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Specific Binding ◽  
Cell Signalling ◽  
Single Population ◽  
Lower Affinity ◽  
Specific Binding Sites ◽  
Kinase C ◽  
And Function ◽  
Gamma S

Adrenocortical microsomes possess a single population of Ins(1,4,5)P3-specific binding sites [IC50 5.9 +/- 0.9 nM; Palmer, Hughes, Lee & Wakelam (1988) Cell. Signalling 1, 147-156]. Competition studies showed that Ins(1:2-cyclic,4,5)P3 exhibits a 21-fold lower affinity for the site than Ins(1,4,5)P3 (IC50 124 +/- 16 nM). The affinity of the binding sites for Ins(1,4,5)P3 was not influenced by the non-hydrolysable GTP analogues GTP gamma S and Gpp[NH]p or by preincubation of the binding protein with a preparation of partially purified protein kinase C in the presence of ATP and TPA (12-O-tetradecanoylphorbol 13-acetate). These observations are discussed with reference to the identify and function of the Ins(1,4,5)P3 binding site.

The High Affinity Calcium Ion Bending Sites of Fibrinogen

1981 ◽  
Author(s):  
Joan Ross ◽  
Graham D Kemp
Keyword(s):  
Calcium Ions ◽  
Binding Sites ◽  
Calcium Ion ◽  
Specific Binding ◽  
Ion Release ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Scatchard Plots ◽  
Systems Studies ◽  
Flow Dialysis

There is considerable evidence that fibrinogen contains a number of strongly bound calcium ions and these appear to have a significant role in the structure and properties of the molecule. Most of the evidence suggests that there are three such strongly bound calcium ions in fibrinogen and each of the two fragments D contains one of these. It has been suggested that the section of the (A) α chain which is the region of the molecule first attacked by plasnin is involved in binding calcium ions. Should this constitute the third site it follows that this calcium ion must link the two (A) α chains and the site may well be destroyed by minimal plasnin attack. The figure of three calcium ions bound, however, must be open to sane doubt due to the difficulty in evaluating data from Scatchard plots prepared from. a system, such as fibrinogen, which contains a number of identical ligands with more than one binding affinity. Accordingly we have developed methods to prepare fibrinogen in as intact a form as possible, and used such fibrinogen in flow dialysis systems. Studies of calcium ion release during proteolytic degradation of fibrinogen lead us to conclude that there are probably only two high affinity, calcium ion specific binding sites in fibrinogen.

Somatostatin binding sites in cytosolic fractions of parietal and non-parietal cells from rabbit fundic mucosa

1985 ◽  
Vol 5 (4) ◽  
pp. 321-328 ◽  
Author(s):  
Eduardo Arilla ◽  
M. Pilar Löpez-Ruiz ◽  
Luis Gonzalez-Guijarro ◽  
Juan C. Prieto
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Parietal Cells ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Fundic Mucosa ◽  
High Affinity ◽  
Specific Binding Sites

Specific binding sites for somatostatin have been found in the cytosolic fractions of both parietal and non-parietal cells from rabbit gastric fundic mucosa. The stoichiometric data suggested the presence of two classes of binding sites in both types of ceils. The number of low-affinity binding sites was significantly higher in parietal cells than in non-parietal cells. The reverse was true for the high-affinity binding sites. However, the affinity of each class of binding sites was similar in the cytosolic fractions of both parietal and non-parietal ceils. It thus appears that low-affinity somatostatin binding sites are mainly located in the parietal ceils whereas the high-affinity sites occur principally in the non-parietal cells.

HIGH-AFFINITY BINDING OF REVERSE T3 AND TETRAC IN NUCLEI OF HUMAN LYMPHOCYTES

1978 ◽  
Vol 87 (3) ◽  
pp. 516-524 ◽  
Author(s):  
T. Lemarchand-Béraud ◽  
A.-C. Holm ◽  
G. Bornand ◽  
A. Burger
Keyword(s):  
Human Lymphocyte ◽  
Binding Sites ◽  
Specific Binding ◽  
Human Lymphocytes ◽  
Affinity Constant ◽  
Reverse T3 ◽  
Intact Cells ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Direct Measurements

ABSTRACT In a previous study, human lymphocyte nuclei were found to possess high affinity, low capacity binding sites for triiodothyronine (T3) and thyroxine (T4). The number of receptors per cell was similar for T3 and T4 (115±20), but the equilibrium affinity constant (Ka) for T3 (2.20±0.23 1010m−1) was twice that for T4 (1.05 ± 0.25 1010m−1). The present study shows that human lymphocyte nuclei also bind highly purified [125I]tetrac and [125I]rT3. The number of specific binding sites was 60 for tetrac and 40 for rT3. The Ka for tetrac (2.12 ± 0.29 1010m−1) was similar to that of T3, whereas that of rT3 (1.31 ± 0.2110m−1) was similar to that of T4. The Ka was the same when measured in intact cells and in nuclei isolated after incubation. Despite the similar Ka for tetrac, rT3 and T3, as obtained by direct measurements, tetrac had only 2 % and rT3 0.1 % of the T3 potency in T3 displacement studies. [125I] tetrac was displaced 50% by 20 fmol of T3 and [125I]rT3 by 8 fmol. These results show that tetrac and rT3 do bind as strongly to nuclear receptors as T3 and T4, but that when competing with T3 the apparent affinities decrease considerably for tetrac and rT3. Thus, the nuclear binding of these two analogues probably has no significance under physiological conditions, but may play some role under pathological conditions when the formation of T3 is decreased and that of rT3 and tetrac is increased. This may represent an adaptive mechanism in T4 inactivation.

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