High-affinity substance P binding sites in rat esophagus plexus submucosus

1987 ◽  
Vol 253 (1) ◽  
pp. R167-R171 ◽  
Author(s):  
C. J. Wiedermann ◽  
K. Sertl ◽  
C. B. Pert
Keyword(s):  
Substance P ◽  
Distribution Pattern ◽  
Dissociation Constant ◽  
Binding Sites ◽  
High Affinity ◽  
Protein Dissociation ◽  
Plexus Submucosus ◽  
Dissociation Constant Kd ◽  
Substance P Receptors ◽  
Receptor Distribution

Substance P receptors were investigated in rat esophagus using 125I-Bolton-Hunter substance P as a labeling probe. Autoradiographic studies show that esophageal submucosa contains clusters of high-affinity substance P binding sites [maximum binding (Bmax) 4.2 +/- 0.28 fmol/mg protein; dissociation constant (Kd) 0.1 +/- 0.01 X 10(9) M]. The receptor distribution pattern is typical for submucous neurons. These data suggest that substance P may act as a neurotransmitter in rat esophagus.

Caffeine- and ryanodine-sensitive Ca2+ stores of canine cerebrum and cerebellum neurons

1991 ◽  
Vol 261 (6) ◽  
pp. C1048-C1054 ◽  
Author(s):  
L. G. Meszaros ◽  
P. Volpe
Keyword(s):  
Binding Sites ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Ruthenium Red ◽  
Hill Coefficient ◽  
Stopped Flow ◽  
High Affinity ◽  
Protein Dissociation ◽  
Dissociation Constant Kd ◽  
Slight Inhibition

[3H]ryanodine binding to and Ca2+ release from microsomal fractions derived from canine cerebrum (CBR) and cerebellum (CBL) were investigated. High-affinity ryanodine binding sites were detected in both cerebrum and cerebellum microsomes [CBR: maximal binding capacity (Bmax) = 446 fmol/mg protein, dissociation constant (Kd) = 9 nM, Hill coefficient (n) = 0.95; CBL: Bmax = 650, Kd = 12, n = 1.8]. Ryanodine binding in both fractions was increased by millimolar concentrations of ATP [or its nonhydrolyzable analogue beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP)] and micromolar concentrations of Ca2+ but was decreased by micromolar concentrations of ruthenium red, similar to that found in sarcoplasmic reticulum (SR) of striated muscle. The addition of caffeine or the sudden elevation of extravesicular Ca2+ induced a rapid La(3+)-sensitive Ca2+ release from both CBR and CBL microsomal fractions with rate constants of approximately 100 s-1, as determined by stopped-flow photometry of the Ca2+ indicator arsenazo III. The release of Ca2+ was activated by either millimolar ATP or AMP-PCP, blocked by micromolar concentrations of La3+, and significantly inhibited by 50 microM ryanodine. Mg2+ and ruthenium red in millimolar and micromolar concentrations, respectively, caused only a slight inhibition of Ca2+ release. These results indicate that rapid Ca2+ release occurs from caffeine-, Ca2+- and ryanodine-sensitive Ca2+ stores in both CBR and CBL microsomal fractions.

Intrinsic, apparent, and effective affinities of co- and countertransport systems

1986 ◽  
Vol 250 (4) ◽  
pp. C523-C533 ◽  
Author(s):  
W. D. Stein
Keyword(s):  
Dissociation Constant ◽  
Chemical Reaction ◽  
Binding Sites ◽  
Kinetic Schemes ◽  
High Affinity ◽  
Dependent Atpase ◽  
Conformation Changes ◽  
Rapid Equilibrium

Solutions to kinetic schemes for the simple carrier, the countertransporter (antiport, exchanger), and the rapid equilibrium cases of the cotransporter (symport) and co-chemiporter (cation-dependent ATPase) are listed. A distinction is made between the intrinsic, apparent, and effective affinities of the transporters for their substrates. Effective pumping requires that the active transporter binds the pumped substrate, at high affinity, realized at the “whence side” (from which pumping takes place) and, at low affinity, at the “whither side” (to which pumping takes place). It is demonstrated how effective pumping might be achieved by appropriate design of the transporter or chemiporter in terms of the energies of the intrinsic binding sites, the energies of the conformation changes that the pump protein undergoes, the dissociation constant of the chemical reaction that drives the co-chemiport, and the order of binding of the cosubstrates, appropriate at different prevailing levels of the driving substrate.

Decreased Reactivity of Plasmin-Streptokinase Towards α-2-Antiplasmin

1979 ◽  
Author(s):  
S Cederholm-Williams ◽  
H Lijnen ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Dissociation Constant ◽  
Active Center ◽  
Rate Constant ◽  
Binding Sites ◽  
Reaction Rate ◽  
Kd Values ◽  
Dissociation Constant Kd

Plasmin (P) is very rapidly (rate constant 2 x 107M-1s-1) inactivated by α-2-antiplasmin (A), and blocking of the lysine binding sites (LSB) of plasmin reduses this rate 50 fold (Eur.J.Biochem.84, 573-578, 1978). Using purified reactants and the plasmin substrate D-Val-Leu-Lys-pNA (S 2251) the influence of streptokinase (SK) on the reaction between P and A was examined. Addition of SK to P reduces the reaction rate with A in a sigmoidal fashion. Assuming the formation of a reversible bimolecular P:SK complex which is unreactive towards A then the dissociation constant Kd of this complex is less than 10-9M. The LSB of P does not play a role in this interaction since similar Kd values are found with modified P, lacking LSB and in the presence of 0.3 mM 6-aminohexanoic acid which blocks the LSB. The P:SK complex is only very slowly inhibited by A with a rate constant < 5 x 102M-1s-1.It is concluded that the α-2-antiplasmin is less reactive towards the plasmin streptokinase complex due to a modification of the active center of plasmin rather than an effect upon the binding sites.

[35S]ATP gamma S binding sites in the purified heart sarcolemma membrane

1990 ◽  
Vol 258 (1) ◽  
pp. C185-C188 ◽  
Author(s):  
D. Y. Zhao ◽  
N. S. Dhalla
Keyword(s):  
Binding Sites ◽  
Heart Cell ◽  
High Affinity ◽  
Heart Sarcolemma ◽  
Kd Values ◽  
Specific Manner ◽  
High Affinity Binding Site ◽  
Heart Cell Membrane ◽  
Gamma S ◽  
Dissociation Constant Kd

Purified heart sarcolemma membranes were found to bind a slowly hydrolyzable analogue of ATP [35S-labeled adenosine 5'-(gamma-thio)triphosphate [( 35S]ATP gamma S)] in a specific manner and exhibited two apparent affinity sites. The high-affinity site had a dissociation constant (KD) of 4.7-8.3 nM [maximum binding (Bmax) = 9.5-18.4 pmol/mg protein], whereas the low-affinity site had a KD of 655-1,257 nM (Bmax = 812-2,955 pmol/mg protein). Like ATP, other nucleotides such as GTP, UTP, ITP, and CTP were effective in displacing [35S]ATP gamma S binding. Although crude membrane preparations from different tissues also exhibited both high- and low-affinity sites for [35S]ATP gamma S, KD values for the high affinity sites were severalfold higher than that for the purified heart membranes. It is proposed that the high-affinity binding site for nucleotides may represent the ATP receptor in the heart cell membrane.

Platelet 3H Ketanserin Binding in Migraine

Cephalalgia ◽  
2001 ◽  
Vol 21 (5) ◽  
pp. 567-572 ◽  
Author(s):  
R Shukla ◽  
VK Khanna ◽  
S Pradeep ◽  
M Husain ◽  
R Tandon ◽  
...  
Keyword(s):  
Dissociation Constant ◽  
Clinical Features ◽  
Binding Sites ◽  
Scatchard Analysis ◽  
Healthy Controls ◽  
Equilibrium Dissociation Constant ◽  
Binding Characteristics ◽  
Number Of Binding Sites ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd

Platelet 3H ketanserin binding was studied in 33 patients of migraine and 30 healthy controls. The binding characteristics: equilibrium dissociation constant (Kd) and maximal number of binding sites (Bmax) determined by Scatchard analysis revealed a significant decrease in Kd and no change in Bmax in migraine cases. No correlation was observed between the Kd and Bmax with the clinical features of migraine. The findings of the present study show that there is a decreased affinity of platelet 5-HT2 receptors in migraine.

Specific somatostatin-binding to cytosol of bovine gallbladder mucosa

1986 ◽  
Vol 6 (3) ◽  
pp. 283-291 ◽  
Author(s):  
E. Arilla ◽  
B. Colás ◽  
J. C. Prieto
Keyword(s):  
Substance P ◽  
Vasoactive Intestinal Peptide ◽  
Binding Sites ◽  
High Capacity ◽  
Nerve Endings ◽  
Cytosolic Fraction ◽  
Binding Reaction ◽  
Gallbladder Mucosa ◽  
High Affinity

The binding of somatostatin was studied in the cytosolic fraction of bovine gallbladder mucosa. The binding reaction depended on time, temperature and pH, and was reversible, saturable and specific. Stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (Kd=23.6 n M) and low capacity (3.7 pmol somatostatin/mg protein) and a class with low affinity (Kd=284.6 n M) and high capacity (85.0 pmol somatostatin/mg protein) at 37°C and pH 7.4. The binding sites were highly specific for somatostatin since peptides such as [Leu]enkephalin, neurotensin, vasoactive intestinal peptide and substance P showed practically no effect upon somatostatin binding. The presence of somatostatin-binding sites in the cytosolic fraction of gallbladder mucosa, together with the known occurrence of somatostatin nerve endings in the gallbladder strongly suggests that this peptide may be involved in the physiology and physiopathology of gallbladder mucosa.

scholarly journals Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 999-1006 ◽  
Author(s):  
SS Husain ◽  
AA Hasan ◽  
AZ Budzynski
Keyword(s):  
Dissociation Constant ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Aminocaproic Acid ◽  
Factor Xiiia ◽  
Single Type ◽  
Affinity Binding ◽  
High Affinity ◽  
Tissue Plasminogen ◽  
Fibrin Clots

Abstract Interaction of tissue plasminogen activator (t-PA) with fibrin plays a key role in regulation of plasminogen activation and clot dissolution. Previous investigations of t-PA-fibrin interaction, using incorporation of t-PA into polymerizing fibrin clots, have suggested that no significant differences exist in the binding of one-chain or two-chain t-PA to non-cross-linked or cross-linked fibrin. In the present study, binding of 125I-labeled and affinity-purified one-chain and two-chain forms of t-PA to preformed non-cross-linked or cross-linked, sonicated suspension of fibrin was investigated. Interaction of one-chain t-PA with cross-linked fibrin involved a single type of binding site with dissociation constant (kd) of 0.58 mumol/L and a stoichiometry (n) of 1.5. Interaction of one-chain t-PA with non-cross-linked fibrin, however, involved two classes of binding sites with dissociation constants of 0.32 and 1.5 mumol/L and corresponding number of binding sites equal to 0.57 and 2.0, respectively. In contrast to the binding of one-chain t-PA to cross-linked fibrin by a limited number of sites, two-chain t-PA appeared to involve a considerably greater number of sites (minimum six) whose dissociation constant was 3.2 mumol/L. Interaction of two-chain t-PA with non-cross-linked fibrin also showed the presence of many binding sites (minimum seven) with approximate dissociation constant of 6.4 mumol/L, as well as a few (n = 0.012) high- affinity sites with a kd of 0.011 mumol/L epsilon-Aminocaproic acid did not completely reverse the binding of either one-chain t-PA or two- chain t-PA to fibrin. The present findings suggest that the fibrin- binding properties of t-PA undergo considerable changes on proteolytic conversion from one-chain to two-chain t-PA, catalyzed under physiologic conditions by plasmin. The cleavage of one-chain t-PA to two-chain t-PA allows to bind to a large number of low-affinity binding sites on fibrin. Cross-linking of fibrin by factor XIIIa results in masking of high-affinity binding sites that are present in non-cross- linked fibrin. We propose that both plasmin and factor XIIIa play an important regulatory role in dissolution of blood clots by modulating t- PA-fibrin interaction.

scholarly journals The binding of human and bovine thrombin to human platelets

Blood ◽  
1976 ◽  
Vol 47 (1) ◽  
pp. 43-54 ◽  
Author(s):  
MA Shuman ◽  
DM Tollefsen ◽  
PW Majerus
Keyword(s):  
Dissociation Constant ◽  
Platelet Function ◽  
Primary Structure ◽  
Binding Sites ◽  
Human Platelets ◽  
Serotonin Release ◽  
Bovine Thrombin ◽  
High Affinity ◽  
Human Thrombin ◽  
Specific Receptors

Abstract Human thrombin binds to specific receptors on the surface of human platelets in a manner analogous to bovine thrombin. Thus, two classes of binding are observed--high affinity with a dissociation constant (Kdiss) of 0.02 U/ml and low affinity with a Kdiss of 5 U/ml. Bovine and human thrombin bind to the same platelet receptors, although bovine thrombin binds with slightly greater affinity. When the amount of thrombin bound to platelets is related to the extent of 14C-serotonin release, bovine and human thrombin are equally effective. Antibodies to human and bovine thrombin were found to differ markedly in their ability to precipitate thrombin of the two species. Thus, antibovine thrombin precipitated eightfold more bovine thrombin than human thrombin, while antihuman thrombin precipitated tenfold more human thrombin than bovine thrombin. Similar differences were found in the ability of Fab fragments of these antibodies to block the interaction of thrombin of each species with human platelets. The finding that both species of thrombin, despite significant evolutionary differences in primary structure, retain essentially identical binding sites to platelets suggests that this part of the thrombin molecule is physiologically important and supports our hypothesis of a role for thrombin binding to platelets in platelet function and hemostasis.

Presence of NK1 receptors on a mucosal-like mast cell line, RBL-2H3 cells

1998 ◽  
Vol 76 (2) ◽  
pp. 188-193 ◽  
Author(s):  
Helen J Cooke ◽  
Paula Fox ◽  
Lisa Alferes ◽  
Charity C Fox ◽  
Seth A Wolfe, Jr.
Keyword(s):  
Mast Cell ◽  
Substance P ◽  
Binding Sites ◽  
Neurokinin A ◽  
Competition Binding ◽  
Polymerase Chain ◽  
Mast Cell Line ◽  
Neurokinin 1 ◽  
Receptor Agonists And Antagonists ◽  
Substance P Receptors

Reverse transcription - polymerase chain reaction of mRNA from rat RBL-2H3 cells yielded a 316 base pair band consistent with that predicted for the neurokinin-1 (NK1) receptor. Saturation and competition binding with 125I-labeled Bolton-Hunter substance P, substance P fragments, and a series of selective tachykinin receptor agonists and antagonists demonstrated that RBL-2H3 cells express high affinity binding sites for substance P on their surfaces with the kinetic and pharmacological properties of NK1 receptors. The pharmacology of these 125I-labeled substance P binding sites was (from most to least potent) [Sar9,Met(O2)11]substance P > substance P 4-11 >> GR82334 >> MEN 10,376. However, substance P 1-4, substance P 8-11, substance P 9-11, and [Trp7, beta -Ala8]neurokinin A 4-10 failed to compete for binding. The metabolically stable NK1 receptor agonist, [Sar9,Met(O2)11] substance P, caused a 49% increase in 5-hydroxytryptamine release above basal levels. The results demonstrate the presence of functional NK1 receptors on RBL-2H3 cells, a mucosal-like mast cell line.Key words: substance P, receptors, mast cells, 5-hydroxytryptamine, tachykinins.

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