Etude cinétique des interactions électrostatiques entre la phosphatase alcaline intestinale de veau et ses substrats

1974 ◽  
Vol 52 (17) ◽  
pp. 3087-3097 ◽  
Author(s):  
Marius Julien ◽  
Ludovic Ouellet
Keyword(s):  
Dielectric Constants ◽  
Enzyme Molecule ◽  
Ionic Charge ◽  
Substrate Molecule ◽  
Net Charge ◽  
Organic Part ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Phosphatase Alcaline ◽  
Ethanolamine Phosphate

The role played by the ionic charge of the enzyme in the kinetics of the reactions catalyzed by alkaline phosphatases has been investigated. Data obtained from the hydrolysis of p-nitrophenyl phosphate (net charge −2) indicate that the repulsion between the negatively charged enzyme molecule and the substrate molecule is sufficient to take into account, quantitatively, the variation of the Michaelis constant with the pH of the system between pH 8.5 and 10.5. However, the results obtained with ethanolamine phosphate (net charge −1) and p- or o-carboxyphenyl phosphate (net charge −3) show that either the organic part of the substrate molecule is not involved in the formation of the enzyme substrate intermediate or the charge of the enzyme does not have the role suggested from the work with p-nitrophenyl phosphate.Further investigation of this reaction in solutions of various ionic strength and dielectric constants suggests that the active center of the enzyme molecule is positively charged, but that hydrophobic effects are important.

ÉTUDE DES EFFETS ÉLECTROSTATIQUES SUR LE MÉCANISME D'ACTION CATALYTIQUE DE LA PHOSPHATASE ALCALINE INTESTINALE DE VEAU

1965 ◽  
Vol 43 (8) ◽  
pp. 2222-2235 ◽  
Author(s):  
Michel Lazdunski ◽  
Jacques Brouillard ◽  
Ludovic Ouellet
Keyword(s):  
Dielectric Constant ◽  
Ionic Strength ◽  
Maximum Activity ◽  
Enzyme Molecule ◽  
High Substrate Concentration ◽  
Nitrophenyl Phosphate ◽  
Phosphatase Alcaline ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Activated Complex

The influence of dioxane and ethanol on the rate of hydrolysis of p-nitrophenyl phosphate in the presence of an intestinal alcaline phosphatase can be interpreted as a dielectric constant effect, at high substrate concentration. The dielectric constant effect is a function of the pH of the medium and is maximum around pH 9.4 at 25 °C and pH 9.0 at 15 °C. An interpretation suggesting that the change in diameter of the enzyme molecule becoming an activated complex is minimum at a pH of maximum activity is proposed. The same model can take into account the influence of the ionic strength on the same reaction.

PHOSPHATASE ALCALINE INTESTINALE: CINÉTIQUE DE L'HYDROLYSE DU PHOSPHATE DE p-NITROPHÉNYLE

1961 ◽  
Vol 39 (6) ◽  
pp. 1298-1308 ◽  
Author(s):  
Michel Lazdunski ◽  
Ludovic Ouellet
Keyword(s):  
Active Center ◽  
Electrostatic Interactions ◽  
Enzyme Molecule ◽  
Kcal Mole ◽  
Intestinal Alkaline Phosphatase ◽  
High Substrate Concentration ◽  
System P ◽  
Nitrophenyl Phosphate ◽  
Phosphatase Alcaline ◽  
Michaelis Constants

The Michaelis constants and the rates at high substrate concentration for the system p-nitrophenyl phosphate – intestinal alkaline phosphatase have been measured at 15.3 °C and 24.4 °C from pH 7.5 to pH 10.4.The experimental data can be interpreted as indicating the presence of three acid groups in the active center of the enzyme. The observed pK of ionization of these groups are 9.02, 8.39, and 7.65 at 15.3 °C, and 9.75, 8.42, and 7.62 at 24.4 °C.These data stress the importance of electrostatic interactions between the groups in the active center and the rest of the enzyme molecule. A heat of ionization of 6 to 7 kcal/mole is attributed to each of these groups. At least one active group of the enzyme would be a thiol function and the other ones imidazole or thiol.

A comparison of four serological tests for the detection of banana bunchy top virus in banana

1996 ◽  
Vol 47 (3) ◽  
pp. 403 ◽  
Author(s):  
ADW Geering ◽  
JE Thomas
Keyword(s):  
Alkaline Phosphatase ◽  
Completion Time ◽  
Polyclonal Antibodies ◽  
Sandwich Elisa ◽  
Nitrocellulose Membrane ◽  
Serological Tests ◽  
Banana Bunchy Top Virus ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Enzyme Amplification

Four different serological tests for detection of banana bunchy top virus (BBTV) in banana sap are compared: (i) a triple-antibody sandwich ELISA for BBTV, utilising anti-BBTV polyclonal antibodies for virus capture, and anti-BBTV monoclonal antibodies, alkaline phosphatase-labelled sheep anti-mouse antibodies, and p-nitrophenyl phosphate for detection (ELISA-NPP); (ii) an alternative enzyme-substrate system for ELISA involving an amplification step (AmpakTM enzyme amplification kit) (ELISA-A); (iii) a colorimetric dot immunobinding assay (DIBA-C), in which the enzyme-substrate system was alkaline phosphatase and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate; (iv) an enhanced chemiluminescent form (DIBA-ECL), in which the enzyme-substrate system was horseradish peroxidase and luminol. For both DIBA-C and DIBA-ECL, maximum sensitivity was obtained by pre-coating the nitrocellulose membrane with anti-BBTV polyclonal antibodies, by using 0.05 M sodium carbonate (pH 9.6) as the coating buffer, and by clarifying the sap by centrifugation and extraction with chloroform or dichloroniethane. Treatment of the sap before centrifugation by snap-freezing at -70�C, or heating at either 30 or 50�C for 10 min, had no effect on sensitivity; heating at 70�C for 10 min eliminated antigenicity. ELISA-NPP, ELISA-A, and DIBA-ECL had equivalent sensitivity, but DIBA-C was up to 8-fold less sensitive than the former 3 assays. ELISA-NPP was adjudged to be the best compromise between sensitivity, cost and completion time.

Influence of Decreasing Solvent Polarity (Dioxane-Water Mixtures) on the Stability of Metal Ion Complexes Formed with Phosphate Monoesters

1989 ◽  
Vol 44 (5) ◽  
pp. 538-542 ◽  
Author(s):  
Guogang Liang ◽  
Nicolas A. Corfù ◽  
Helmut Sigel
Keyword(s):  
Metal Ion ◽  
Equilibrium Constants ◽  
Solvent Polarity ◽  
Bulk Water ◽  
Dielectric Constants ◽  
Metal Ion Binding ◽  
Nitrophenyl Phosphate ◽  
Phosphate Monoesters ◽  
The Stability ◽  
Straight Lines

The acidity constants of H(R-MP)-, where R-MP2- = 4-nitrophenyl phosphate (NPhP2-), phenyl phosphate (PhP2-) and D-ribose 5′-monophosphate (RibMP2-), and the stability constants of the binary Cu(R-MP) complexes were determined by potentiometric pH titrations in aqueous solution and in 20, 30, 40 and 50% (v/v) dioxane-water mixtures. The solvent influence on the corresponding equilibrium constants is compared with the same influence on previously studied systems containing uridine 5′-triphosphate, formate or acetate. The influence of the solvent composition on the various ligand (L) systems was evaluated by constructing log KM(L) versus pKH(L)H plots; in all cases straight lines are obtained with slopes close to 1. This indicates that in all these systems, despite the different negative charges of the involved ligands, the solvent effect on proton binding and on metal ion binding is approximately of the same size: A decreasing solvent polarity resulting from the addition of increasing amounts of organic solvent to the aqueous solutions favors the affinity of the negatively charged ligands for protons and metal ions as well. Information of this type is considered important because the ‘effective’ or ‘equivalent solution’ dielectric constants in active-site cavities of enzymes are reduced compared with the dielectric constant of bulk water; i. e., in protein cavities also a decreased ‘solvent polarity’ is occurring and this is expected to affect the stability of metal ion-ligand bonds.

scholarly journals Kinetic characterization of enzyme forms involved in metal ion activation and inhibition of myo-inositol monophosphatase

1995 ◽  
Vol 307 (2) ◽  
pp. 585-593 ◽  
Author(s):  
F Strasser ◽  
P D Pelton ◽  
A J Ganzhorn
Keyword(s):  
Metal Ions ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Inositol Monophosphatase ◽  
Inositol 1 ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Preferential Binding ◽  
Sugar Phosphates ◽  
High Concentrations

Activation and inhibition of recombinant bovine myo-inositol monophosphatase by metal ions was studied with two substrates, D,L-inositol 1-phosphate and 4-nitrophenyl phosphate. Mg2+ and Co2+ are essential activators of both reactions. At high concentrations, they inhibit hydrolysis of inositol 1-phosphate, but not 4-nitrophenyl phosphate. Mg2+ is highly selective for inositol 1-phosphate (kcat. = 26 s-1) compared with the aromatic substrate (kcat. = 1 s-1), and follows sigmoid activation kinetics in both cases. Co2+ catalyses the two reactions at similar rates (kcat. = 4 s-1), but shows sigmoid activation only with the natural substrate. Li+ and Ca2+ are uncompetitive inhibitors with respect to inositol 1-phosphate, but non-competitive with respect to 4-nitrophenyl phosphate. Both metal ions are competitive inhibitors with respect to Mg2+ with 4-nitrophenyl phosphate as the substrate. With inositol 1-phosphate, Ca2+ is competitive and Li+ non-competitive with respect to Mg2+. Multiple inhibition studies indicate that Li+ and Pi can bind simultaneously, whereas no such complex was detected with Ca2+ and Pi. Several sugar phosphates were also characterized as substrates of myo-inositol monophosphatase. D-Ribose 5-phosphate is slowly hydrolysed (kcat. = 3 s-1), but inhibition by Li+ is very efficient (Ki = 0.15 mM), non-competitive with respect to the substrate and competitive with respect to Mg2+. Depending on the nature of the substrate, Li+ inhibits by preferential binding to free enzyme (E), the enzyme-substrate (E.S) or the enzyme-phosphate (E.Pi) complex. Ca2+, on the other hand, inhibits by binding to E and E.S, in competition with Mg2+. The results are discussed in terms of a catalytic mechanism involving two metal ions.

The Kinetics of Reactions Catalyzed by Alkaline Phosphatase: The Effects of Added Nucleophiles

1972 ◽  
Vol 50 (12) ◽  
pp. 1360-1368 ◽  
Author(s):  
Irwin Hinberg ◽  
Keith J. Laidler
Keyword(s):  
Alkaline Phosphatase ◽  
Preceding Paper ◽  
The Other ◽  
Intestinal Alkaline Phosphatase ◽  
Michaelis Constant ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Kinetics Of Formation ◽  
Kinetics Of

An experimental study was made of the hydrolyses of phenyl phosphate and p-nitrophenyl phosphate catalyzed by chicken intestinal alkaline phosphatase. The work was done at pH 8.0 and 10.0, 25.0 °C, and an ionic strength of 0.1 M, and particular attention was paid to the kinetics of formation of the products in the presence of Tris and ethanolamine. It was found that the rates of formation of phenol or p-nitrophenol (P1) and of the phosphorylated nucleophile (P3) were dependent on the concentration of added nucleophile; on the other hand the rate of formation of phosphate (P2) and the Michaelis constant were independent of nucleophile concentration. This result cannot be reconciled with any of the mechanisms discussed in the preceding paper with the exception of mechanism VI, which is an elaboration of one proposed by Trentham and Gutfreund; mechanism VI is[Formula: see text]where W is water and N the alternative nucleophile. ES and E*S are two conformers of the enzyme–substrate complex, and E*S′ and ES′ two forms of the phosphorylated enzyme; only the latter can react with water and only the former with nucleophile.

scholarly journals Resistance to inactivation by EGTA of the enzyme-substrate and enzyme-phosphate complexes of alkaline phosphatase

1987 ◽  
Vol 244 (3) ◽  
pp. 781-785 ◽  
Author(s):  
S J Pike ◽  
R G Duggleby
Keyword(s):  
Alkaline Phosphatase ◽  
Steady State ◽  
Chelating Agent ◽  
Enzyme Substrate ◽  
Steady State Kinetic ◽  
Nitrophenyl Phosphate ◽  
Comparison Of The Results ◽  
State Kinetic ◽  
Substrate Concentrations ◽  
Stability Measurements

Bovine intestinal mucosal alkaline phosphatase is inactivated by the chelating agent EGTA. Several concentrations of the enzyme were incubated with EGTA and a range of concentrations of the substrate p-nitrophenyl phosphate to determine the substrate concentration as a function of time. As predicted by a recently developed theory [Duggleby (1986) J. Theor. Biol. 123, 67-80], catalysis ceases before all substrate is exhausted. An analysis of these final substrate concentrations according to the theory revealed that, whereas the free enzyme is unstable, the effect of EGTA is counteracted when either the substrate or product (phosphate) is bound. Comparison of the results with those obtained by direct stability measurements and steady-state kinetic experiments gave a qualitatively and quantitatively consistent body of evidence in support of this interpretation.

THE INFLUENCE OF pH ON THE HYDROLYSIS OF BENZOYLCHOLINE BY PSEUDOCHOLINESTERASE OF HUMAN PLASMA

1964 ◽  
Vol 42 (2) ◽  
pp. 161-168 ◽  
Author(s):  
W. Kalow
Keyword(s):  
Human Plasma ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Enzyme Molecule ◽  
Acid Base ◽  
Enzyme Substrate ◽  
Michaelis Constants ◽  
Automatic Titrator ◽  
Influence Of Ph ◽  
Hydrolysis Of

The study was performed with an automatic titrator and purified human pseudocholinesterase prepared from pooled plasma. The data obtained are compatible with the assumption that each enzyme molecule contains two binding sites for benzoylcholine which are unlike in their dependence on pH. Michaelis constants and maximum hydrolysis velocities were derived for each of the two binding sites, and acid–base dissociation constants of the enzyme substrate complexes were estimated.

scholarly journals The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli

1968 ◽  
Vol 106 (2) ◽  
pp. 455-460 ◽  
Author(s):  
D. R. Trentham ◽  
H. Gutfreund
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Ph Dependence ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Steady State Rate ◽  
State Rate ◽  
Rate Of Hydrolysis ◽  
Ph Range

1. The steady-state rate of hydrolysis of 2,4-dinitrophenyl phosphate catalysed by Escherichia coli phosphatase is identical with that of 4-nitrophenyl phosphate over the pH range 5·5–8·5. 2. The increase in the rate of the enzyme-catalysed decomposition of nitrophenyl phosphates in the presence of tris at pH8·1 and 5·9 is consistent with the hypothesis that tris increases the rate of decomposition of a phosphoryl-enzyme intermediate. At pH8·1 the rate of decomposition of the phosphoryl-enzyme is approximately twice as fast as the rate of its formation, whereas at pH5·9 the rate of formation of the phosphoryl-enzyme is considerably faster than its decomposition. 3. Pre-steady-state measurements of the initial transient of the liberation of 2,4-dinitrophenol during the reaction of the enzyme with 2,4-dinitrophenyl phosphate confirmed the above pH-dependence of the ratio of the rates of phosphorylation and dephosphorylation of the enzyme. At optimum pH (above pH8), when the phosphorylation of the enzyme by the substrate is rate-determining, this step must be controlled by a rearrangement of the enzyme or enzyme–substrate complex.

Characteristics of an Electrostatic Phase Plate

1972 ◽  
Vol 30 ◽  
pp. 618-619
Author(s):  
William Krakow ◽  
Benjamin Siegel
Keyword(s):  
Phase Contrast ◽  
Objective Lens ◽  
Phase Plate ◽  
Net Charge ◽  
Phase Plates ◽  
Beam Currents ◽  
Contrast Image ◽  
Bright Phase ◽  
Additional Phase Shift ◽  
Additional Phase

Unwin has used a metallized non-conducting thread in the back focal plane of the objective lens that stops out a portion of the unscattered beam, takes on a localized positive charge and thus produces an additional phase shift to give a different transfer function of the lens. Under the particular conditions Unwin used, the phase contrast image was shifted to bright phase contrast for optimum focus.We have investigated the characteristics of this type of electrostatic phase plate, both analytically and experimentally, as functions of the magnitude of charge and defocus. Phase plates have been constructed by using Wollaston wire to mount 0.25μ diameter platinum wires across apertures ranging from 50 to 200μ diameter and vapor depositing SiO and gold on the mounted wires to give them the desired charging characteristics. The net charge was varied by adjusting only the bias on the Wehnelt shield of the gun, and hence the beam currents and effective size of the source.

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