scholarly journals Evidence for histidine in the triethyltin-binding site of rat haemoglobin

1969 ◽  
Vol 111 (2) ◽  
pp. 129-137 ◽  
Author(s):  
M. S. Rose
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Affinity Constant ◽  
Histidine Residues ◽  
Photo Oxidation ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

One molecule of rat haemoglobin binds two molecules of triethyltin. The binding sites are located on the globin and there is co-operativity between the sites such that the intrinsic affinity constant at pH8·0 increases from 3·5×105m−1 for the binding of the first triethyltin molecule to 5·0×105m−1 for the binding of the second. Evidence is presented, from pH studies and the kinetics of inhibition due to photo-oxidation, that each binding site contains two histidine residues.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

The uptake of atropine and related drugs by intestinal smooth muscle of the guinea-pig in relation to acetylcholine receptors

1965 ◽  
Vol 163 (990) ◽  
pp. 1-44 ◽  
Keyword(s):  
Smooth Muscle ◽  
Binding Site ◽  
Equilibrium Constant ◽  
Rate Constant ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Kinetic Properties ◽  
Kinetic Behaviour ◽  
Kinetics Of ◽  
Bound Drug

In an attempt to study the properties of acetylcholine receptors in intestinal smooth muscle, measurements have been made of the uptake of tritium-labelled atropine and methylatropinium, and of 14 C-labelled methylfurmethide by the longitudinal muscle of guinea-pig small intestine in vitro . Substantial amounts of atropine were taken up from very dilute solutions, a clearance of 160 ml. per g tissue (wet weight) being achieved at the lowest concentration tested (1.5 × 10 -10 M). Analysis of the curve relating atropine uptake at equilibrium to the bath concentration, which was explored over a concentration range 1.5 × 10 -10 M to 2.5 × 10 -3 M, enabled three components to be distinguished: (1) A binding site with a capacity of 180 pmoles/g, and equilibrium constant 1.1 × 10 -9 M. (2) A binding site of capacity about 1000 pmoles/g and equilibrium constant about 5 × 10 -7 M. (3) A compartment with a clearance of 4.7 ml./g (nonsaturable). The equilibrium constant of the first binding site agreed exactly with that measured for acetylcholine antagonism in the same tissue. Methylatropinium was taken up in rather smaller amounts than atropine, and analysis of the uptake curve showed a binding site of capacity about 90 pmoles/g with an equilibrium constant 6.5 × 10 -10 M, an ill-defined series of binding sites with much higher equilibrium constants, and a constant clearance of about 0.4 ml. /g. Analysis of this curve was much less clear cut than that of atropine. The equilibrium constant for blockade of acetylcholine receptors by methylatropinium was 4.7 × 10 -10 M. Atropine was not taken up appreciably by striated muscle, nerve or tendon of the guineapig; hydrolysed atropine was not taken up by smooth muscle (and lacks atropinic activity); cocaine and d -tubocurarine in high concentrations did not affect atropine uptake; lachesine and benzhexol blocked atropine uptake competitively at low concentrations, and with lachesine the equilibrium constant for this interaction agreed with that measured for acetylcholine antagonism (1.4 × 10 -9 M). These findings suggested that the atropine taken up could be related to receptor-bound drug. The kinetics of atropine uptake and washout were studied over the concentration range 0.5-5 × 10 -9 M. Uptake and washout took place approximately exponentially between 2½ and 50 min, and the rate constant was 4.5-5 × 10 -4 s -1 for both uptake and washout. The uptake rate constant did not increase with concentration. This contrasted with the kinetics of receptor blockade, which took place much faster, with a rate constant which increased linearly with concentration, in accordance with the theoretical kinetic behaviour of a single binding site. This finding precluded a simple identification of atropine taken up with receptor-bound drug. Studies with various metabolic inhibitors suggested that no metabolic energy was required for the accumulation of atropine, and by dialysis experiments, the atropine taken up was shown to be bound in homogenized tissue. A theoretical study, using an analogue computer, was made of the kinetic properties of three passive binding systems, in order to see whether the observed kinetic behaviour could be simulated. It was found that a system of four binding sites in series, with only one communicating directly with the surrounding medium, could show these kinetic properties, and the outermost binding site could still show the kinetic behaviour of receptors. Experimental testing of this model demands more accurate kinetic measurements than can be made by the method used in this study. The acetylcholine-like stimulant, methylfurmethide, was taken up very slowly (taking more than 24 h to reach equilibrium), reaching a clearance of about 5 ml. /g after 6 h. This uptake was unaffected by atropine in a concentration sufficient to block 80% of acetylcholine receptors, but was blocked by depolarization in high potassium solution, suggesting that it was behaving passively as a slowly permeant cation. No uptake referable to acetylcholine receptors was detected. These findings are discussed in relation to the abundance and chemical behaviour of acetylcholine receptors in smooth muscle, and in relation to current theories of drug action.

The Kinetics of Activated Thrombin-Activatable Fibrinolysis Inhibitor with Respect to Plasminogen Binding Site Removal From Fibrin Degradation Products.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3185-3185
Author(s):  
Jonathan H. Foley ◽  
Michael E. Nesheim
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Degradation Products ◽  
Catalytic Efficiency ◽  
Plasminogen Activation ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Fibrin Degradation Products ◽  
Fibrinolysis Inhibitor ◽  
Arginine Residues ◽  
Kinetics Of

Abstract Abstract 3185 Poster Board III-122 TAFI (thrombin activatable fibrinolysis inhibitor, or carboxypeptidase U) is a plasma zymogen that can be activated by thrombin, thrombin-thrombomodulin or plasmin. When activated, TAFIa cleaves C-terminal lysine and arginine residues from plasmin modified fibrin (Fn'). Fn' as a cofactor increases the rate of plasminogen activation by 3-fold over intact fibrin and 3000-fold compared to in the absence of fibrin. Upon extensive treatment with TAFIa, the cofactor activity of TAFIa modified fibrin decreases by approximately 97%. Determining the kinetics of TAFIa will give insight into how much TAFIa is required to efficiently inhibit plasminogen activation and fibrinolysis. The kinetics of TAFIa on its primary physiological substrate were measured by exploiting the binding of plasminogen to fibrin degradation products (FDPs). Fluorescently labeled plasminogen (5IAF-Pg) was equilibrated with FDPs labeled with a quencher, QSY C5-maleimide (QSY-FDP). When 5IAF-Pg is bound to QSY-FDP a baseline fluorescence reading is obtained. When treated with TAFIa, plasminogen binding sites are removed from the QSY-FDP and the fluorescence increases. A model was used to convert the rate of fluorescence increase into the rate of Plasminogen binding site removal. The model includes two distinct binding sites on QSY-FDPs (C-terminal and internal lysines), only one of which is susceptible to removal by TAFIa (C-terminal lysine). 5IAF-Glu-Pg (fluorescent native plasminogen) binds to QSY-FDP with a Kd of 176nM and when QSY-FDP are treated with TAFIa the Kd increases to 1.06μM. It appears that 5IAF-Glu-Pg has the ability to weakly bind TAFIa-treated QSY-FDP, however, the capacity is greatly reduced. Similar binding constants were obtained for 5IAF-Lys-Pg (fluorescent plasmin-cleaved plasminogen) (Kd=92nM; Kd (+TAFIa)=1.55μM). The increase in Kd upon treatment of the QSY-FDP with TAFIa is similar to that observed with 5IAF-Glu-Pg, however, the capacity of the FDPs to bind 5IAF-Lys-Pg is relatively unchanged. The calculated rate of 5IAF-Glu-Pg binding site removal by TAFIa was determined at various QSY-FDP concentrations (0-2 μM). The data are hyperbolic in nature and when fit using the Michaelis-Menten model the kcat and Km of plasminogen binding site removal were 2.34 s-1 and 142.6nM, respectively, implying a catalytic efficiency of 16.41 μM-1s-1. The rate is sensitive to the TAFIa concentration with all TAFIa concentrations (50, 75 and 100pM) yielding similar kinetic parameters. The data described here suggest that TAFIa is very efficient in removing plasminogen binding sites. The catalytic efficiency of TAFIa toward QSY-FDP is 60-fold higher than reported for bradykinin, which was previously the best known substrate of TAFIa. This increased catalytic efficiency is due to a much lower Km (0.146 μM compared to 70.6 μM). These data are reflective of plasminogen site removal and not every C-terminal lysine or arginine cleaved by TAFIa is expected to be involved in plasminogen binding. Therefore, the catalytic efficiency of TAFIa reported here (16.41 μM-1s-1) is likely a lower limit for the true value. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization of the Binding of Adenosine Diphosphate to Human Platelet Membranes

1977 ◽  
Author(s):  
C. Legrand ◽  
B. Bauvois ◽  
J. P. Caen
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Adenosine Diphosphate ◽  
Affinity Constant ◽  
Membrane Bound ◽  
High Concentrations ◽  
Kinetics Of

ADP-mediated platelet aggregation is a routinely employed test but its mechanism is poorly understood. The aim of this study was to compare the binding of ADP to plasma membranes isolated from normal platelets and thrombasthenic platelets (which do not aggregate with ADP). Binding of ADP to isolated membranes was assayed by incubation with 14C-ADP followed by Mill i pore filtration. In standard conditions, 14C-ADP was not transformed and non specific binding represented lessthan 3 % of the total binding. Using 1 μM 14C-ADP, the binding has been shown to be a rapid (t 1/2 = 2 mn 30 sec), saturable and reversible phenomenon at 37° C. The existence of a major population of binding sites, with an affinity constant Ka = 0.43 (+ 0.1) χ 106M-1, has been demonstrated. The kinetics of the binding was normal with membranes Tsolated from the platelets of 4 thrombasthenic patients and the affinity constant, when determined, was in the normal range. Dissociation of the membrane-bound 14C-ADP occurred rapidly at 37° C (t l/2c≃3mn) when samples were diluted enough (dilution 1 : 100 was currently employed) to avoid rebinding of the radioligand. Accelerated dissociation (t 1/2 ≃ 1 mn) was observed when the dilution was performed in the presence of an excess of unlabeled ADP, suggesting the existence of negatively cooperative site-site interactions among the ADP binding sites. This effect was only observed at high concentrations of ADP (> 10–5M) and its eventual role in vivo remains to be established. Two thrombasthenic membrane preparations studied in the same way dissociated as did the control membranes.

scholarly journals Mechanisms of Barbiturate Inhibition of Acetylcholine Receptor Channels

1997 ◽  
Vol 109 (3) ◽  
pp. 401-414 ◽  
Author(s):  
James P. Dilger ◽  
Rebecca Boguslavsky ◽  
Martin Barann ◽  
Tamir Katz ◽  
Ana Maria Vidal
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Single Channel ◽  
Inhibitory Effects ◽  
Channel Current ◽  
State Dependent ◽  
Rapid Perfusion ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Acetylcholine Receptor Channel

We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts is 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 μM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 μM, n = 1.09; for barbital, Ki = 1900 μM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. <0.1 ms at the Ki). Pentobarbital binds ≥10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at >100 μM pentobarbital.

scholarly journals Histidine residues of zinc ligands in β-lactamase II

1978 ◽  
Vol 175 (2) ◽  
pp. 441-447 ◽  
Author(s):  
G S Baldwin ◽  
A Galdes ◽  
H A O Hill ◽  
B E Smith ◽  
S G Waley ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Binding Site ◽  
Binding Sites ◽  
Zinc Binding ◽  
Histidine Residue ◽  
Beta Lactamase ◽  
Histidine Residues ◽  
Zinc Enzyme ◽  
Zinc Binding Site ◽  
Ph Range

1. The Zn(II)-requiring beta-lactamase from Bacillus cereus 569/H/9, which has two zinc-binding sites, was examined by 270 MHz 1H n.m.r. spectroscopy. Resonances were assigned to five histidine residues. 2. Resonances attributed to three of the histidine residues in the apoenzyme shift on the addition of one equivalent of Zn(II). 3. Although these three histidine residues are free to titrate in the apoenzyme, none of them titrates over the pH range 6.0–9.0 in the mono-zinc enzyme. 4. The ability of the C-2 protons of these three histidine residues to exchange with solvent (2H2O) is markedly decreased on Zn(II) binding. 5. It is proposed that these three histidine residues act as zinc ligands at the tighter zinc-binding site. 6. Resonances attributed to a fourth histidine residue shift on addition of further zinc to the mono-zinc enzyme. It is proposed that this histidine residue acts as a Zn(II) ligand at the second zinc-binding site.

Evidence for histidine residues in the immunoglobulin-binding site of human C1q

1983 ◽  
Vol 3 (2) ◽  
pp. 135-140 ◽  
Author(s):  
S. B. Easterbrook-Smith
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Ionic Interactions ◽  
Histidine Residues ◽  
First Order ◽  
First Order Kinetics ◽  
Inactivation Process ◽  
Immunoglobulin Binding ◽  
Modification Treatment

The immunoglobulin-binding activity of subcomponent Clq of human complement is lost following treatment with diethylpyrocarbonate; the inactivation showed first-order kinetics with respect to time and modifier concentration. Soluble IgG oligomers protected Clq against diethylpyrocarbonate modification. Treatment of modified Clq with hydroxylamine resulted in an 85% recovery of its ability to bind to aggregated immuno-globulin. The inactivation process was associated with modification of 12.1±0.7 histidine residues per Clq molecule. These data are consistent with the presence of histidine residues in the immunoglobulin-binding sites of Clq; these residues may participate in ionic interactions with the carboxyl groups known to be in the Clq binding site of IgG.

scholarly journals Binding of triethyltin to cat haemoglobin and modification of the binding sites by diethyl pyrocarbonate

1977 ◽  
Vol 163 (3) ◽  
pp. 583-589 ◽  
Author(s):  
B M Elliott ◽  
W N Aldridge
Keyword(s):  
Methylene Blue ◽  
Binding Site ◽  
Binding Sites ◽  
Chemical Nature ◽  
Photo Oxidation ◽  
Diethyl Pyrocarbonate

Cat haemoglobin binds 2 mol of triethyltin/mol of haemoglobin. Pretreatment of the haemoglobin with diethyl pyrocarbonate at pH6.0 prevents binding to one site only, whereas photo-oxidation with Methylene Blue removes both sites. Pretreatment of rat haemoglobin with diethyl pyrocarbonate also leads to the loss of one binding site. The possibility is discussed that the two binding sites for triethyltin on both cat and rat haemoglobin have a different chemical nature.

scholarly journals Kinetics of Activated Thrombin-activatable Fibrinolysis Inhibitor (TAFIa)-catalyzed Cleavage of C-terminal Lysine Residues of Fibrin Degradation Products and Removal of Plasminogen-binding Sites

2011 ◽  
Vol 286 (22) ◽  
pp. 19280-19286 ◽  
Author(s):  
Jonathan H. Foley ◽  
Paul F. Cook ◽  
Michael E. Nesheim
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Degradation Products ◽  
Catalytic Efficiency ◽  
Tissue Type ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Fibrin Degradation Products ◽  
Lysine Residues ◽  
Fibrinolysis Inhibitor ◽  
Kinetics Of

Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the kcat and Km of Glu1-plasminogen (Glu-Pg)-binding site removal are 2.34 s−1 and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 μm−1 s−1. The corresponding values of Lys77/Lys78-plasminogen (Lys-Pg)-binding site removal are 0.89 s−1 and 96 nm implying a catalytic efficiency of 9.23 μm−1 s−1. These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 μm−1 s−1. Interestingly, this value increases to 3.85 μm−1 s−1 in the presence of Glu-Pg. These changes are due to a decrease in Km. This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg.

scholarly journals Investigation of the mechanism by which glucose analogues cause translocation of glucokinase in hepatocytes: evidence for two glucose binding sites

2000 ◽  
Vol 346 (2) ◽  
pp. 413-421 ◽  
Author(s):  
Loranne AGIUS ◽  
Mark STUBBS
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
The Other ◽  
Hill Coefficient ◽  
Site Model ◽  
Glucose Binding ◽  
Glucose Analogues ◽  
Hill Coefficients ◽  
Effect Of Glucose ◽  
Kinetics Of

Glucokinase translocates between the cytoplasm and nucleus of hepatocytes where it is bound to a 68 kDa protein. The mechanism by which glucose induces translocation of glucokinase from the nucleus was investigated using glucose analogues that are not phosphorylated by glucokinase. There was strong synergism on glucokinase translocation between effects of glucose analogues (glucosamine, 5-thioglucose, mannoheptulose) and sorbitol, a precursor of fructose 1-phosphate. In the absence of glucose or glucose analogues, sorbitol had a smaller effect than glucose on translocation. However, sorbitol potentiated the effects of glucose analogues. In the absence of sorbitol the effect of glucose on glucokinase translocation is sigmoidal with a Hill coefficient of 1.9 suggesting involvement of two glucose-binding sites. The effects of glucosamine and 5-thioglucose were also sigmoidal but with lower Hill Coefficients. In the presence of sorbitol, the effects of glucose, glucosamine and 5-thioglucose were hyperbolic. Mannoheptulose, unlike the other glucose analogues, had a hyperbolic effect on glucokinase translocation in the absence of sorbitol suggesting interaction with one site and was synergistic rather than competitive with glucose. The results favour a two-site model for glucokinase translocation involving either two glucose-binding sites or one binding-site for glucose and one for fructose 1-phosphate. The glucose analogues differed in their effects on the kinetics of purified glucokinase. Mannoheptulose caused the greatest decrease in co-operativity of glucokinase for glucose whereas N-acetylglucosamine had the smallest effect. The anomalous effects of mannoheptulose on glucokinase translocation and on the kinetics of purified glucokinase could be explained by a second glucose-binding site on glucokinase.

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