scholarly journals Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate

1969 ◽  
Vol 115 (2) ◽  
pp. 147-162 ◽  
Author(s):  
W. N. Aldridge ◽  
Elsa Reiner
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Organophosphorus Compounds ◽  
Active Centre ◽  
Phosphorus Containing ◽  
High Concentrations ◽  
Influence Of Substrate ◽  
A Site ◽  
Kinetics Of ◽  
Related Compounds

1. The kinetics of the reaction of di-(2-chloroethyl) 3-chloro-4-methylcoumarin-7-yl phosphate (haloxon) and related compounds with acetylcholinesterase were studied and found to be unusual. 2. By a progressive reaction haloxon produces a di-(2-chloroethyl)phosphorylated enzyme. The influence of substrate on this reaction leading to a phosphorylated active centre was studied. From competition experiments between inhibitor and substrate values of Km for acetylcholine and acetylthiocholine of 0·79mm and 0·23mm respectively were derived. 3. Haloxon also combines with acetylcholinesterase by a non-progressive reaction, producing a complex that is reversible by dilution and by high concentrations of acetylcholine and acetylthiocholine. From this non-progressive reaction the competition between haloxon and substrate was studied, and it was shown that haloxon combines with a site involved in inhibition by substrate. From competition experiments the following dissociation constants were derived: for combination of haloxon and this site Ki is 4·9μm and for the combination of substrates with this site K88 values are 12mm and 3·3mm for acetylcholine and acetylthiocholine respectively. 4. The non-phosphorus-containing compound 3-chloro-7-hydroxy-4-methylcoumarin was shown to be a good reagent for the site involved in inhibition by substrate; its dissociation constant for the combination with this site is 30μm. 5. In order to interpret the experimental results, theoretical equations were derived for an enzyme with two binding sites to both of which substrate and inhibitor can combine. The equations correlate the activity of the enzyme with the concentration of substrate and inhibitor, for both progressive and non-progressive inhibition. These equations are applicable to reactions of acetylcholinesterase with organophosphorus compounds, carbamates etc. and may be applicable to other enzymes possessing two binding sites.

scholarly journals The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides

1972 ◽  
Vol 126 (4) ◽  
pp. 975-984 ◽  
Author(s):  
K. Dalziel ◽  
R. R. Egan
Keyword(s):  
Glutamate Dehydrogenase ◽  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Kinetic Studies ◽  
Binding Studies ◽  
Purine Nucleotides ◽  
Active Centre ◽  
Sodium Phosphate Buffer ◽  
Low Concentrations ◽  
High Concentrations

1. The binding of NAD+ and NADP+ to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD+ the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD+ and NADP+, in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD+ or NADP+, the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.

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.

Glucagon binding to hepatocytes isolated from two teleost fishes, the American eel and the brown bullhead

1994 ◽  
Vol 140 (2) ◽  
pp. 217-227 ◽  
Author(s):  
I Navarro ◽  
T W Moon
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Insulin Binding ◽  
American Eel ◽  
Brown Bullhead ◽  
Teleost Species ◽  
High Affinity ◽  
Glucagon Like Peptide 1 ◽  
High Concentrations

Abstract We have characterized the specific binding of glucagon in hepatocytes isolated from two teleost species, the American eel (Anguilla rostrata) and the brown bullhead (Ictalurus nebulosus). Specific glucagon binding was 9·3 and 10·7% in bullhead and eel hepatocytes respectively, after a 2-h incubation at 12 °C. Curvilinear Scatchard plots suggest the presence of two classes of binding sites with apparent dissociation constants (Kd) of 1·97 nm (high affinity) and 17·3 nm (low affinity) for bullhead and 2·68 and 22·9 nm for eel cells. The number of high-affinity binding sites per cell was significantly higher in the eel (10 413) than in the bullhead (3811). The number of high-affinity insulin-binding sites was approximately two times higher than that for glucagon in bullheads and the opposite in the eel hepatocytes. In competition experiments, insulin did not displace 125I-labelled glucagon binding in the hepatocytes of either species, while glucagon-like peptide-1(7–37) (GLP-1) displaced glucagon but only at high concentrations, suggesting separate glucagon- and GLP-1-binding sites. The rate of dissociation of hepatocyte-bound 125I-labelled glucagon was similar for both species. Preincubation of hepatocytes in 100 nm glucagon decreased the number of high-affinity glucagon-binding sites by approximately 55% in both species, while the Kd values remained unchanged. Glucagon bound to the cell surface is internalized by fish hepatocytes. These properties indicate that the glucagon binding to hepatocytes of these two teleost species is similar to that reported for mammalian hepatocytes. Journal of Endocrinology (1994) 140, 217–227

scholarly journals Kinetics of substrate hydrolysis and inhibition by mipafox of paraoxon-preinhibited hen brain esterase activity

1986 ◽  
Vol 236 (2) ◽  
pp. 503-507 ◽  
Author(s):  
C D Carrington ◽  
M B Abou-Donia
Keyword(s):  
Kinetic Parameters ◽  
Organophosphorus Compounds ◽  
Model Fit ◽  
Component Model ◽  
Low Concentrations ◽  
Two Component ◽  
High Concentrations ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Organophosphorus Inhibitor

For the purpose of assessing the neurotoxic potential of organophosphorus compounds, it has been determined that paraoxon-preinhibited hen brain has both neurotoxicant (mipafox)-sensitive (neurotoxic esterase; NTE) and -insensitive esterase components. Several experiments designed to investigate the kinetic parameters governing the reaction of these esterases with two substrates and one organophosphorus inhibitor are presented. First, kinetic parameters for the hydrolysis of phenyl valerate and phenyl phenylacetate were measured. At 37 degrees C, the Km values of NTE for phenyl valerate and phenyl phenylacetate were found to be about 1.4 × 10(-3) and 1.6 × 10(-4) M respectively. At 25 degrees C, the Km of NTE for phenyl valerate was determined to be about 2.4 × 10(-3) M. Secondly, the kinetic constants of NTE for mipafox were measured at both 25 degrees C and 37 degrees C. With either phenyl valerate or phenyl phenylacetate as substrate, the Km at 37 degrees C was determined to be about 1.8 × 10(-4) M, and the phosphorylation constant (k2) was about 1.1 min-1. For phenyl valerate only, the Km at 25 degrees C was found to be about 6 × 10(-4) M, and the k2 was about 0.7 min-1. The data obtained at 25 degrees C were analysed by using a two-component model without formation of Michaelis complex, a two-component model with formation of Michaelis complex on the second component (NTE), or a three-component model without formation of Michaelis complex. The fact that the Michaelis model fit the data significantly better than either of the other two models indicates that the higher apparent Ki values that occur with low concentrations of mipafox are due to formation of Michaelis complex at high concentrations, rather than because of the presence of two NTE isoenzymes, as has been suggested by other investigators.

INTERACTIONS OF HUMAN PLATELETS WITH COAGULATION FACTORS IX AND IXa

1987 ◽  
Author(s):  
S S Ahmad ◽  
R Rawala ◽  
P N Walsh
Keyword(s):  
Binding Sites ◽  
Molecular Mechanisms ◽  
Dissociation Constants ◽  
Coagulation Factors ◽  
Human Platelets ◽  
Factor Ix ◽  
Binding Studies ◽  
Activated Platelets ◽  
Ca Ions ◽  
A Site

To elucidate the molecular mechanisms underlying the platelet contribution to the activation of factor IX (FIX) and FX, we have previously reported the presence of specific, high-affinity binding sites on activated platelets for FIX and FIXa (Blood 66:300a, 1985; Circulation, 74:11-238, 1986). Since both FIX and FIXa bind to a common site on thrombin-activated platelets in the presence of Ca++ ions, it is important to determine whether saturation of platelet binding sites with FIX would prevent F-IXa binding. We have therefore made direct comparisons of F-IX and F-IXa binding and examined the functional consequences of F-IXa binding in F-X activation. Gel-filtered platelets (GFP) were incubated with 125I-FIX or 125I-FlXa and centrifuged through silicone barriers to separate GFP from unbound proteins. Optimal binding of both FIX and FIXa occurred in the presence of CaCl2 (5 mM) and thrombin (0.1 U/ml), with maximal binding in 10-15 min at 37°C. Binding of both proteins was specific since a 130-fold excess of FIX or FIXa inhibited FIX binding >85% and excess FIXa inhibited FIXa binding >85%, whereas excess FIX inhibited FIXa binding to a lesser extent, and excess high Mr kininogen, FXI, FX, prothrombin, and prekallikrein inhibited binding <30%. Binding of both FIX and FIXa was rapidly reversible and saturable. The number of FIXa binding sites (551 + 48 per platelet) was significantly (p < 0.001) higher than for FIX (306 + 57). Dissociation constants (Kd) were 2.57 +0.14 nM for FIXa and 2.68 + 0.25 nM for FIX. However, the concentration of FIXa required for half-maximal rates of FX activation in the presence of FVIIIa and thrombin-activated GFP was approximately 0.1 nM. This suggests that the FIXa receptors functionally active in FX activation may bind FIXa >20-fold more tightly than indicated by equilibrium binding studies, and that FIXa binding studies should be carried out in the presence of large molar excesses of FIX. We conclude that FIX and FIXa bind to a coupon site on thrombin-activated platelets in the presence of Ca ions, and that FIXa also binds to a site distinct from that for FIX. FIXa binding to platelets appears to contribute to F-X activation.

scholarly journals Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols

1977 ◽  
Vol 167 (3) ◽  
pp. 799-810 ◽  
Author(s):  
M Shipton ◽  
K Brocklehurst
Keyword(s):  
High Electron ◽  
Thiol Groups ◽  
Active Centre ◽  
Enzyme Substrate ◽  
Mechanistic Basis ◽  
A Site ◽  
Kinetics Of ◽  
State Concentration ◽  
Imidazolium Ion ◽  
Active Centres

1. The characteristics of benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) that relate to its application as a reactivity probe for the study of environments of thiol groups are discussed. 2. To establish a kinetic and mechanistic basis for its use as a probe, a kinetic study of its reaction with 2-mercaptoethanol was carried out. 3. This reaction appears to proceed by a rate-determining attack of the thiolate ion on one of the electrophilic centres of benzofuroxan (possibly C-6) to provide a low steady-state concentration of an intermediate adduct; rapid reaction of this adduct with a second molecule of thiol gives the disulphide and o-benzoquinone dioxime. 4. The effects of the different types of environment that proteins can provide on the kinetic characteristics of reactions of thiol groups with benzofuroxan are delineated. 5. Benzofuroxan was used as a thiolspecific reactivity probe to investigate the active centres of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). The results support the concept that the active centres of all three enzymes either contain a nucleophilic thiolate ion whose formation is characterized by a pKa of 3-4 and whose reaction with an electrophile can be assisted by interaction of a site of high electron density in the electrophile with active-centre imidazolium ion of pKa 8-9, or can provide such ions by protonic redistribution in enzyme-reagent or enzyme-substrate complexes.

Protamine Neutralization of the Release of Tissue Factor Pathway Inhibitor Activity by Heparins

1993 ◽  
Vol 70 (06) ◽  
pp. 0942-0945 ◽  
Author(s):  
Job Harenberg ◽  
Marietta Siegele ◽  
Carl-Erik Dempfle ◽  
Gerd Stehle ◽  
Dieter L Heene
Keyword(s):  
Tissue Factor ◽  
Binding Sites ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Protamine Sulfate ◽  
Low Molecular Weight ◽  
Factor X ◽  
Rebound Phenomenon ◽  
Tissue Factor Pathway ◽  
Kinetics Of

SummaryThe present study was designed to investigate the action of protamine on the release of tissue factor pathway inhibitor (TFPI) activity by unfractionated (UF) and low molecular weight (LMW) heparin in healthy individuals. 5000 IU UF-heparin or 5000 IU LMW-heparin were given intravenously followed by saline, 5000 U protamine chloride or 5000 U protamine sulfate intravenously after the 10 min blood sample. Then serial blood samples for the measurement of TFPI activity and anti-factor Xa- activity were taken, in order to detect a possible relation between the remaining anti-factor X a activity after neutralization of LMW-heparin with protamine and TFPI activity and to establish whether or not a rebound phenomenon of plasmatic TFPI occurs.There was no difference in the release and in the kinetics of TFPI by UF- and LMW-heparin with subsequent administration of saline. After administration of protamine TFPI activity decreased immediately and irreversibly to pretreatment values. There were no differences between protamine chloride and protamine sulfate on the effect of TFPI induced by UF- or LMW-heparin. No rebound phenomenon of TFPI activity occurred. In contrast anti-factor Xa- activity, as measured by the chromogenic S2222-assay, issued the known differences between UF- and LMW-heparin. The half-life of the aXa-effect of LMW-heparin was twice as long as of UF-heparin. Protamine antagonized UF-heparin completely and about 60% of the anti-factor Xa activity of LMW-heparin, using chromogenic S2222-method. No differences could be detected for protamine chloride and sulfate form of protamineIt is assumed that protamine displaces heparins from the binding sites of TFPI. There were no differences between UF- and LMW-heparin. The data indicate that the sustained antifactor Xa activity after antagonization of LMW-heparins as well as heparin rebound phenomena are not mediated by TFPI activity.

Abnormal Platelet Serotonin Uptake and Binding Sites in Myeloproliferative Disorders

1984 ◽  
Vol 51 (03) ◽  
pp. 349-353 ◽  
Author(s):  
C Caranobe ◽  
P Sié ◽  
F Fernandez ◽  
J Pris ◽  
S Moatti ◽  
...  
Keyword(s):  
Binding Sites ◽  
High Capacity ◽  
Specific Inhibitor ◽  
Myeloproliferative Disorders ◽  
Normal Subjects ◽  
Binding Data ◽  
Full Explanation ◽  
Number Of Binding Sites ◽  
5 Ht Uptake ◽  
Kinetics Of

SummaryA simultaneous investigation of the kinetics of serotonin (5 HT) uptake and of binding sites was carried out in the platelets of normal subjects and of 10 patients affected with various types of myeloproliferative disorders (MD). The 5 HT uptake was analysed according to the Lineweaver-Burk and the Eadie-Hofstee methods. With the two methods, the patient’s platelets exhibited a dramatic reduction of the Vi max and of the Km; in some patients the Eadie-Hofstee analysis revealed that a passive diffusion phenomenon is superimposed on the active 5 HT uptake at least for the higher concentration used. The binding data were analysed with the Scatchard method. Two classes of binding sites (high affinity - low capacity, low affinity - high capacity) were found in normal subjects and patients. Pharmacological studies with imipramine, a specific inhibitor of 5 HT uptake, suggested that both the sites are involved in 5 HT uptake. The number of both binding sites was significantly decreased in patient’s platelets while the affinity constants of these binding sites were not significantly reduced in comparison with those of the control subjects. No correlations were found between Vi max, Km and the number of binding sites. These results suggest that a reduction in the number of platelet membrane acceptors for 5 HT commonly occurs in myeloproliferative disorders but does not provide a full explanation of the uptake defect.

m-[o-(2-Chloro-5-Fluorosulfonylphenylureido)Phenoxybutoxy]Benzamidine: Affinity Labeling Reagent Which Can Identify Secondary Binding Sites of Coagulation Complement and Fibrinolytic Serine Proteases

1979 ◽  
Author(s):  
D Bing ◽  
D Robison ◽  
J Andrews ◽  
R Laura
Keyword(s):  
Binding Sites ◽  
Serine Proteases ◽  
Enzyme Inhibitor ◽  
Molecular Model ◽  
Factor Xa ◽  
Affinity Labeling ◽  
Catalytic Center ◽  
Inhibitor Complex ◽  
Polypeptide Chains ◽  
Kinetics Of

We have determined that m-[o-(2-chloro-5-fluorosulfonylphenylureido)phenoxybutoxy]benza-midine [mCP(PBA)-F] is an affinity labeling reagent which labels both polypeptide chains of thrombin, factor Xa, complement component CIS and plasmin. As this means it is reacting outside of the catalytic center, we have called this reagent an exo-site affinity labeling reagent. Progressive irreversible inhibition of these enzymes by this reagent is rapid (k1st 2.5-4.6 x 10-3sec-1), the kinetics of inactivation are consistent with inhibition proceding via formation of a specific enzyme-inhibitor complex analogous to a Michaelis-Menton complex (KL - 115-26 μM), and diisopropylfluorophosphate or p-amidino-phenylmethanesulfonyfluoride Prevent labeling by [3H]mCP(PBA)-F. A molecular model of mCP(PBA)-F shows that the reactive SO2F group can be 17 A from the cationic amidine. The data are consistent with the hypothesis that both peptide chains are required for the specific proteolytic activity exhibited by these proteases and that the peptide chain which does not contain the active site serine is close to the catalytic center. (Supported by NIH and AHA grants

Functional Involvement of Thrombospondin in Platelet Aggregation Induced by Low Versus High Concentrations of Thrombin

1986 ◽  
Vol 55 (01) ◽  
pp. 136-142 ◽  
Author(s):  
K J Kao ◽  
David M Shaut ◽  
Paul A Klein
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Inhibitory Effect ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Platelet Binding ◽  
Functional Involvement ◽  
Thrombin Activation ◽  
Platelet Aggregates ◽  
High Concentrations

SummaryThrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (≤0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (≥60 μg/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (≥60 μg/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin. In conclusion, our findings demonstrate that TSP is functionally important for platelet aggregation induced by low (≤0.05 U/ml) but not high (≥0.1 U/ml) concentrations of thrombin. High concentrations of exogenous TSP may univalently saturate all its platelet binding sites consequently interfering with TSP-crosslinking of thrombin-activated platelets.

Sign in / Sign up

Export Citation Format

Share Document