scholarly journals Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions

1980 ◽  
Vol 185 (1) ◽  
pp. 107-114 ◽  
Author(s):  
I A Nieduszynski ◽  
J K Sheehan ◽  
C F Phelps ◽  
T E Hardingham ◽  
H Muir
Keyword(s):  
Dissociation Constant ◽  
Equilibrium Dialysis ◽  
Guanidine Hydrochloride ◽  
Dilute Solution ◽  
Binding Studies ◽  
Laryngeal Cartilage ◽  
Equilibrium Binding ◽  
Cartilage Proteoglycans ◽  
Dissociation Constant Kd

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 × 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 × 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol.wt. 670 000), an average Kd of 12 × 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

scholarly journals Binding of NAD+ by cholera toxin

1987 ◽  
Vol 244 (1) ◽  
pp. 225-230 ◽  
Author(s):  
T S Galloway ◽  
S van Heyningen
Keyword(s):  
Binding Site ◽  
Dissociation Constant ◽  
Cholera Toxin ◽  
Equilibrium Dialysis ◽  
Accurate Determination ◽  
Polyacrylamide Gel ◽  
Nad Glycohydrolase ◽  
Competitive Inhibitors ◽  
Dissociation Constant Kd

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.

scholarly journals Binding of blood coagulation factor VIII and its light chain to phosphatidylserine/phosphatidylcholine bilayers as measured by ellipsometry

1995 ◽  
Vol 310 (2) ◽  
pp. 539-545 ◽  
Author(s):  
J Spaargaren ◽  
P L A Giesen ◽  
M P Janssen ◽  
J Voorberg ◽  
G M Willems ◽  
...  
Keyword(s):  
Factor Viii ◽  
Dissociation Constant ◽  
Binding Affinity ◽  
Adsorption Kinetics ◽  
Light Chain ◽  
Maximal Surface ◽  
Binding Parameters ◽  
Binding Studies ◽  
Equilibrium Binding ◽  
Maximal Binding

Factor VIII is a plasma protein which plays an essential role in the coagulation system. When assembled with the enzyme Factor IXa on a phospholipid membrane, it functions as a cofactor in the enzyme complex that cleaves the zymogen Factor X to Factor Xa. We studied the binding of both Factor VIII and the Factor VIII light chain to planar phospholipid bilayers consisting of 25% dioleoylphosphatidylserine and 75% dioleoylphosphatidylcholine (PSPC) by ellipsometry. Equilibrium-binding studies revealed that both Factor VIII and its light chain bind with high affinity to PSPC bilayers. The binding affinity of Factor VIII, with a dissociation constant Kd of 0.24 nM, was comparable with that of the Factor VIII light chain (Kd 0.49 nM). Maximal binding was 2.3 mmol of protein per mol of PSPC for Factor VIII and 7.1 mmol of protein per mol of PSPC for the Factor VIII light chain. Adsorption kinetics of both Factor VIII and its light chain conformed to the classical Langmuir adsorption model yielding dissociation constants calculated from the rates of adsorption that were similar to those obtained by equilibrium-binding studies. In contrast, measurements of rates of desorption revealed a deviation from those expected for a single class of binding sites. The desorption rate of Factor VIII increased with increasing residence time on the lipid membrane. This indicates transition of Factor VIII to a configuration with a lower binding affinity. As this time-dependent change in affinity could affect the validity of the measurement of binding parameters, in particular equilibrium-binding determinations carried out on a long timescale, binding affinity was also estimated from adsorption kinetics at half-maximal surface coverage, a relatively rapid procedure for the determination of the affinity. A Kd of 0.087 nM was obtained under these conditions. Measurement of equilibrium binding to small PSPC vesicles, a system in which equilibrium is rapidly attained, resulted in similar binding parameters (Kd = 0.13 nM and a maximal binding of 2.8 mmol of protein per mol of PSPC). These data confirm the results of equilibrium binding to planar bilayers. Taken together, our results indicate that Factor VIII, by means of its 80 kDa light chain, binds to PSPC bilayers with a dissociation constant below the concentration of Factor VIII in plasma and therefore may readily bind to exposed phospholipid membranes under physiological conditions.

scholarly journals Self-association of proteoglycan subunits from pig laryngeal cartilage

1978 ◽  
Vol 171 (1) ◽  
pp. 109-114 ◽  
Author(s):  
J K Sheehan ◽  
I A Nieduszynski ◽  
C F Phelps
Keyword(s):  
Light Scattering ◽  
Ionic Strength ◽  
Guanidine Hydrochloride ◽  
Sedimentation Velocity ◽  
Dilute Solution ◽  
Thiol Groups ◽  
Dimer Model ◽  
Laryngeal Cartilage ◽  
Self Association ◽  
Corroborative Evidence

Proteoglycans from pig laryngeal cartilage prepared by dissociative extraction in guanidine hydrochloride were studied in dilute solution by light-scattering and ultracentrifugation. In buffered 150mM-NaCl, pH7.4, the proteoglycan particle weights were about 5×10(6) daltons, but at 100mM-, 200mM- and 300mM-NaCl particle weights of 2.5×10(6)–3.0×10(6) daltons were observed. These results, together with corroborative evidence from sedimentation-velocity experiments, were interpreted in terms of proteoglycans self-associating at physiological ionic strength. The data were examined by using a proteoglycan monomer-dimer model. Proteoglycan preparations that had thiol groups partially carboxymethylated gave particle weights of 3.2×10(6)–3.5×10(6) daltons in 150mM-NaCl, which suggested that carboxymethylation inhibited multimerization and hence that the protein core is implicated in the binding site. Further studies showed that the multimers were stable to 60 degrees C, unlike the hyaluronate-proteoglycan complex.

Equilibrium and kinetic studies of the interaction of site-specific ligands with acetylcholinesterase from Electrophorus electricus

1978 ◽  
Vol 56 (12) ◽  
pp. 1133-1140 ◽  
Author(s):  
George Tomlinson ◽  
Bulent Mutus ◽  
W. John Rutherford
Keyword(s):  
Steady State ◽  
Dissociation Constant ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Competitive Inhibitor ◽  
Binding Studies ◽  
Electrophorus Electricus ◽  
Equilibrium Binding ◽  
Low Ionic Strength ◽  
Hydrolysis Of

The interactions of edrophonium chloride, gallamine triiodide, and propidium diiodide with affinity-purified acetylcholinesterase from Electrophorus electricus have been examined under conditions of low ionic strength (0.001 M Tris, pH 8.0) using kinetic and fluorescence titration techniques. Edrophonium is a competitive inhibitor of the steady-state hydrolysis of acetylthiocholine, with an inhibition constant, Kcomp, of 1.2 × 10−8 M. Double reciprocal plots in the presence of either gallamine or propidium are nonlinear. Similarly, the pre-steady-state carbamoylation of the enzyme by 7-(dimethylcarbamoyloxy)-N-methyl quinolinium iodide is competitively inhibited by edrophonium, whereas the intercepts of the double reciprocal plots of pseudo-first-order rate constant of carbamoylation versus substrate concentration are displaced downwards in the presence of gallamine or propidium. These results, and those of equilibrium binding studies utilizing the fluorescence properties of bound propidium, suggest that gallamine and propidium compete for a peripheral class of anionic sites on the enzyme, whereas edrophonium binds to the anionic subsite of the catalytic site. The characteristics of propidium binding to the eel enzyme differ from those previously observed with enzyme isolated from Torpedo californica. Whereas the tetrameric Torpedo enzyme possesses four binding sites of equal affinity for propidium, the eel enzyme appears to have two classes of propidium binding site. One set of approximately two sites per tetramer is characterized by a dissociation constant of approximately 2–5 × 10−8 M; a second set of two sites bind propidium with a dissociation constant of 4 × 10−6 M. Possible reasons for these differences are discussed.

Plasminogen-Plasmin System IX. Specific Binding of Tranexamic Acid to Plasmin

1975 ◽  
Vol 33 (03) ◽  
pp. 573-585 ◽  
Author(s):  
Masahiro Iwamoto
Keyword(s):  
Tranexamic Acid ◽  
Affinity Chromatography ◽  
Dissociation Constant ◽  
Factor Xiii ◽  
Specific Binding ◽  
The Other ◽  
Inhibitory Mechanism ◽  
Binding Studies ◽  
Similar Affinity ◽  
Fibrin Structure

SummaryInteractions between tranexamic acid and protein were studied in respect of the antifibrinolytic actions of tranexamic acid. Tranexamic acid did neither show any interaction with fibrinogen or fibrin, nor was incorporated into cross-linked fibrin structure by the action of factor XIII. On the other hand, tranexamic acid bound to human plasmin with a dissociation constant of 3.5 × 10−5 M, which was very close to the inhibition constant (3.6 × 10−5 M) for this compound in inhibiting plasmin-induced fibrinolysis. The binding site of tranexamic acid on plasmin was not the catalytic site of plasmin, because TLCK-blocked plasmin also showed a similar affinity to tranexamic acid (the dissociation constant, 2.9–4.8 × 10−5 M).In the binding studies with the highly purified plasminogen and TLCK-plasmin preparations which were obtained by affinity chromatography on lysine-substituted Sepharose, the molar binding ratio was shown to be 1.5–1.6 moles tranexamic acid per one mole protein.On the basis of these and other findings, a model for the inhibitory mechanism of tranexamic acid is presented.

scholarly journals Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Gianluca Trinco ◽  
Valentina Arkhipova ◽  
Alisa A. Garaeva ◽  
Cedric A. J. Hutter ◽  
Markus A. Seeger ◽  
...  
Keyword(s):  
Kinetic Mechanism ◽  
Protein Quantification ◽  
Detergent Solution ◽  
Sodium Ions ◽  
Binding Studies ◽  
Equilibrium Binding ◽  
Uptake Rates ◽  
Bona Fide ◽  
Inside Out

AbstractIt is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.

scholarly journals Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

1974 ◽  
Vol 141 (2) ◽  
pp. 365-381 ◽  
Author(s):  
Christopher W. Wharton ◽  
Athel Cornish-Bowden ◽  
Keith Brocklehurst ◽  
Eric M. Crook
Keyword(s):  
Methyl Ester ◽  
Dissociation Constant ◽  
Computational Methods ◽  
Rate Constant ◽  
Guanidine Hydrochloride ◽  
Ester Hydrolysis ◽  
Binary Complex ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Ester Substrate

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S0]/vi (initial substrate concn./initial velocity) versus [S0] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S0]/vi versus [S0] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S0]/vi versus [S0] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25°C is characterized by Km1 (the dissociation constant of ES)=1.22±0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57×10-2±0.32×10-2s-1, Ka2 (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38±0.06m, and k′ (the rate constant for the breakdown of SES to E+P+S)=0.45±0.04s-1. 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of α-N-benzoyl-l-arginine ethyl ester and of α-N-benzoyl-l-arginine amide; Km1 and k for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine amide hydrolysis and Kas and k′ for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of kcat. and Km for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (Km=52±4mm, kcat.=2.80±0.1s-1 at pH7.0, I=0.1, 25.0°C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.

Sign in / Sign up

Export Citation Format

Share Document