Kinetics of hydrolysis of dispersions of saturated phosphatidylcholines by Crotalus atrox phospholipase A2

1978 ◽  
Vol 56 (6) ◽  
pp. 552-558 ◽  
Author(s):  
David O. Tinker ◽  
A. David Purdon ◽  
Jane Wei ◽  
Eileen Mason
Keyword(s):  
Liquid Crystalline ◽  
Phase Reaction ◽  
Reaction Products ◽  
Crotalus Atrox ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Desorption Rate Constant ◽  
Rate Of Hydrolysis ◽  
Lag Period ◽  
Lipid Surface ◽  
Hydrolysis Of

Dispersions of lamellar phase dipalmitoyl phosphatidylcholine (DDPC) and dimyristoyl phosphatidylcholine (DMPC) in 0.01 M CaCl2 were subjected to hydrolysis by phospholipase A2 (EC 3.1.1.4) from Crotalus atrox venom. The reaction was followed continuously by titrating the released fatty acids. For hydrolysis of gel phase phosphatides, the steady-state initial velocities were hyperbolic functions of bulk lipid concentrations. At the 'pre-transition' temperature (34 °C for DPPC, 15 °C for DMPC), there was a large increase in the Michaelis parameter Vmax but no change in the parameter Km. A model was devised to account for these observations, in which the enzyme desorbs from the lipid surface after hydrolysis. The desorption rate constant is postulated to increase above the pretransition temperature.For hydrolysis of liquid crystalline phosphatides, the reaction consisted of a short initial burst of hydrolysis, a long 'lag' period of very slow reaction, followed by a dramatic increase in the reaction rate. Addition of 10 mol% lysolecithin or fatty acid abolished the 'lag' period. It was postulated that the enzyme adsorbs irreversibly to the surface of the liquid crystalline phase. Reaction products are postulated to stimulate desorption of enzyme from the surface. Thus, temperature-dependent changes in the rate of hydrolysis of dispersed phosphatidylcholines are attributed to changes in the rate of desorption of the enzyme from the lipid surface.

THE EFFECT OF GLYCEROL MEDIUM ON THE ACTIVATION OF HUMAN PLASMINOGEN BY STREPTOKINASE AND ON THE ACTION OF THE ENZYME, PLASMIN

1962 ◽  
Vol 40 (1) ◽  
pp. 1203-1212 ◽  
Author(s):  
Phyllis S. Roberts
Keyword(s):  
Methyl Ester ◽  
Phosphate Buffer ◽  
Water Medium ◽  
Rate Of Hydrolysis ◽  
Human Plasminogen ◽  
Lag Period ◽  
Hydrolysis Of ◽  
Glycerol Medium

A 50% glycerol medium affected the activation of plasminogen by streptokinase (SK) and the activity of plasmin (formed by glycerol incubation) on p-toluene-sulphonyl-L-arginine methyl ester (TAMe) in the following ways:(1) TAMe interfered strongly with the activation of plasminogen by SK in a glycerol medium but not in a water medium under the same conditions.(2) SK inhibited the action of plasmin on TAMe in a glycerol medium, but in a water medium SK increased the rate of hydrolysis of TAMe, the extent of the increase depending upon pH.(3) Phosphate buffer inhibited the activation of plasminogen by SK in a glycerol medium but not in a water medium. In the glycerol medium a lag period in the formation of enzyme was found, particularly noted with a phosphate buffer, and the lag period increased with increasing concentration of the buffer.(4) Phosphate buffer inhibited the action of plasmin on TAMe in a glycerol medium but not in a water medium.Mechanisms of activation of plasminogen by SK are discussed. The data presented support the postulate that a reaction takes place between plasmin and SK to form an enzyme called "activator".

THE EFFECT OF GLYCEROL MEDIUM ON THE ACTIVATION OF HUMAN PLASMINOGEN BY STREPTOKINASE AND ON THE ACTION OF THE ENZYME, PLASMIN

1962 ◽  
Vol 40 (9) ◽  
pp. 1203-1212 ◽  
Author(s):  
Phyllis S. Roberts
Keyword(s):  
Methyl Ester ◽  
Phosphate Buffer ◽  
Water Medium ◽  
Rate Of Hydrolysis ◽  
Human Plasminogen ◽  
Lag Period ◽  
Hydrolysis Of ◽  
Glycerol Medium

A 50% glycerol medium affected the activation of plasminogen by streptokinase (SK) and the activity of plasmin (formed by glycerol incubation) on p-toluene-sulphonyl-L-arginine methyl ester (TAMe) in the following ways:(1) TAMe interfered strongly with the activation of plasminogen by SK in a glycerol medium but not in a water medium under the same conditions.(2) SK inhibited the action of plasmin on TAMe in a glycerol medium, but in a water medium SK increased the rate of hydrolysis of TAMe, the extent of the increase depending upon pH.(3) Phosphate buffer inhibited the activation of plasminogen by SK in a glycerol medium but not in a water medium. In the glycerol medium a lag period in the formation of enzyme was found, particularly noted with a phosphate buffer, and the lag period increased with increasing concentration of the buffer.(4) Phosphate buffer inhibited the action of plasmin on TAMe in a glycerol medium but not in a water medium.Mechanisms of activation of plasminogen by SK are discussed. The data presented support the postulate that a reaction takes place between plasmin and SK to form an enzyme called "activator".

Heterogeneous catalysis by phospholipase A2: mechanism of hydrolysis of gel phase phosphatidylcholine

1980 ◽  
Vol 58 (10) ◽  
pp. 898-912 ◽  
Author(s):  
David O. Tinker ◽  
Rosita Low ◽  
Maria Lucassen
Keyword(s):  
Equilibrium Constant ◽  
Quadratic Function ◽  
Lipid Binding ◽  
Reaction Products ◽  
Rapid Phase ◽  
Gel Phase ◽  
Desorption Equilibrium ◽  
Lipid Surface ◽  
Hydrolysis Of ◽  
Egg Pc

Hydrolysis of gel phase dipalmitoylphosphatidylcholine (DPPC) at 37 °C catalysed by Crotalus atrox phospholipase A2 (PLA) is described extremely well by the "path 1" kinetic mechanism of Tinker and Wei (1979) (Can. J. Biochem. 57, 97–106), if reversible adsorption is allowed as a side reaction. Progress curves show an initial rapid phase, the initial velocity being a Michaelis–Menten function dependent on the catalytic properties of the enzyme ([Formula: see text], [Formula: see text]), then level off to a slower rate determined by the desorption equilibrium constant [Formula: see text] and desorption rate constant [Formula: see text]. The relaxation time, τ, for the transition to the desorption-limited reaction is ~0.5 min; this large value of τ probably arises from a slow conversion of active, dimeric enzyme to an inactive protein species adsorbed to the lipid surface. At later times in the reaction there is an increase in the rate of hydrolysis, attributed to a stimulation of desorption by the products. The desorption equilibrium constant KD is a quadratic function of the surface concentration of products and increases 20- to 30-fold when all accessible substrate is hydrolysed. Both lyso-phosphatidylcholine (lyso-PC) and fatty acid were found to stimulate the desorption, but lyso-PC was also found to be a competitive inhibitor of the catalysis. Adsorption of PLA to DPPC and egg PC vesicles was directly measured using a gel partition technique. Strong binding to egg PC was observed, which was not dependent on the presence of calcium ion (essential for catalysis); PLA inhibited by acylation of up to four lysine residues per mole of monomeric enzyme with ethoxyformic anhydride was equally strongly adsorbed, indicating that lipid binding is not dependent on catalytic activity. Reaction products greatly weakened the binding of PLA to the lipid surface as expected. Cholesterol had two effects on the hydrolytic reaction: there was a striking decrease in the rate of the slower, desorption-limited phase, the rate of which decreased to almost zero at 15 mol% cholesterol, but there was also evidence for the formation of a complex with stoichiometry 1 cholesterol: 2 DPPC in which DPPC is no longer a substrate for the enzyme. Implications of the proposed mechanism for specificity and control of surface catalysis by PLA are discussed.

Hydrolysis of phosphatidylcholine liposomes by lysosomal phospholipase A is maximal at the phase transition temperature of the lipid substrate

1985 ◽  
Vol 5 (6) ◽  
pp. 477-482 ◽  
Author(s):  
Michel Vandenbranden ◽  
Georges De Gand ◽  
Robert Brasseur ◽  
Fabienne Defrise-Quertain ◽  
Jean-Marie Ruysschaert
Keyword(s):  
Phase Transition ◽  
Liquid Crystalline ◽  
Temperature Characteristic ◽  
Phosphatidylcholine Liposomes ◽  
Pancreatic Phospholipase ◽  
Pancreatic Phospholipase A2 ◽  
Rate Of Hydrolysis ◽  
Structural Analogy ◽  
Hydrolysis Of ◽  
Crystalline Phase Transition

We have measured the rate of hydrolysis of liposomes made of DL-α-dipalmitoylphosphatidylcholine (DPPC) and L-α-dimyristoylphosphatidylcholine by a soluble fraction of highly purified lysosomes isolated from rat liver. Phospholipids are hydrolyzed into lysophospho-lipids and fatty acids at a rate which is maximal near the temperature characteristic of the gel to liquid crystalline phase transition of the lipid bilayer. This strong influence of the physical properties of the substrate on the enzyme activity suggests a structural analogy between the lysosomal phospholipases of the A type (EC 3.1.1.32 and EC 3.1.1.4) and the pancreatic phospholipase A2.

The Effects of Amines on the Esterase Activities of Thrombin and Thrombokinase and on Several Clotting Tests

1974 ◽  
Vol 31 (02) ◽  
pp. 309-318
Author(s):  
Phyllis S Roberts ◽  
Raphael M Ottenbrite ◽  
Patricia B Fleming ◽  
James Wigand
Keyword(s):  
Choline Chloride ◽  
Polymerization Process ◽  
Ammonium Bromide ◽  
Bovine Thrombin ◽  
One Stage ◽  
Human Proteins ◽  
Rate Of Hydrolysis ◽  
The One ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

Summary1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis of this ester by either human or bovine thrombin. Only when 1.0 M or more choline chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly inhibited. Evidence is presented that shows that these effects are due to the quaternary amine group.2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and -n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction. In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on these reactions, however, do not follow any regular order. The tetraethyl compound is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction. The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME and the propyl compound of the thrombin-BAME reactions.3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified one-stage prothrombin test. In all cases the inhibition increases regularly as the size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025 M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage tests by the quaternary amines is not due to any effect of the com pounds on the polymerization process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary amines.

The effect of polymeric and model imidazolium halides on the rate of hydrolysis of 4-acetoxy-3-nitrobenzoic acid

1985 ◽  
Vol 50 (4) ◽  
pp. 845-853 ◽  
Author(s):  
Miloslav Šorm ◽  
Miloslav Procházka ◽  
Jaroslav Kálal
Keyword(s):  
Catalyst Concentration ◽  
Low Molecular Weight ◽  
Imidazolium Chloride ◽  
First Order ◽  
Nitrobenzoic Acid ◽  
Rate Of Hydrolysis ◽  
Acid Catalyzed ◽  
Hydrolysis Of ◽  
Ethanol In Water ◽  
Direct Attack

The course of hydrolysis of an ester, 4-acetoxy-3-nitrobenzoic acid catalyzed with poly(1-methyl-3-allylimidazolium bromide) (IIa), poly[l-methyl-3-(2-propinyl)imidazolium chloride] (IIb) and poly[l-methyl-3-(2-methacryloyloxyethyl)imidazolium bromide] (IIc) in a 28.5% aqueous ethanol was investigated as a function of pH and compared with low-molecular weight models, viz., l-methyl-3-alkylimidazolium bromides (the alkyl group being methyl, propyl, and hexyl, resp). Polymers IIb, IIc possessed a higher activity at pH above 9, while the models were more active at a lower pH with a maximum at pH 7.67. The catalytic activity at the higher pH is attributed to an attack by the OH- group, while at the lower pH it is assigned to a direct attack of water on the substrate. The rate of hydrolysis of 4-acetoxy-3-nitrobenzoic acid is proportional to the catalyst concentration [IIc] and proceeds as a first-order reaction. The hydrolysis depends on the composition of the solvent and was highest at 28.5% (vol.) of ethanol in water. The hydrolysis of a neutral ester, 4-nitrophenyl acetate, was not accelerated by IIc.

Kinetics of hydrolysis of peroxodisulphate ions in acidic medium to peroxomonosulphuric acid

1981 ◽  
Vol 46 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
Jan Balej ◽  
Milada Thumová
Keyword(s):  
Ionic Strength ◽  
Rate Constants ◽  
Acidic Medium ◽  
Measured Data ◽  
Molar Ratios ◽  
Starting Solution ◽  
Kinetics Of Hydrolysis ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

The rate of hydrolysis of S2O82- ions in acidic medium to peroxomonosulphuric acid was measured at 20 and 30 °C. The composition of the starting solution corresponded to the anolyte flowing out from an electrolyser for production of this acid or its ammonium salt at various degrees of conversion and starting molar ratios of sulphuric acid to ammonium sulphate. The measured data served to calculate the rate constants at both temperatures on the basis of the earlier proposed mechanism of the hydrolysis, and their dependence on the ionic strength was studied.

Polarographic study of hydrolysis of [8-lysine]vasopressin and its derivatives with blood serum of pregnant women

1980 ◽  
Vol 45 (4) ◽  
pp. 1099-1108 ◽  
Author(s):  
Mikuláš Chavko ◽  
Michal Bartík ◽  
Evžen Kasafírek
Keyword(s):  
Blood Serum ◽  
Pregnant Women ◽  
Degree Of Hydrolysis ◽  
Polarographic Study ◽  
Ph Optimum ◽  
Lysine Vasopressin ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Vasopressin Analogues

A polarographic study of the hydrolysis of [8-lysine]vasopressin and some hormonogens of the vasopressin series with the blood serum of women in the last week of pregnancy was studied. The dependence of hydrolysis on pH (pH optimum: 7.4-7.50, substrate concentration (Km 1.2 . 10-5M), pH stability and thermal stability were determined. The rate of hydrolysis of individual vasopressin analogues decreases in the order: [8-lysine]vasopressin > Nα-glycyl-prolyl[8-lysine]-vasopressin > Nα-leucyl-[8-lysine]vasopressin > Nα-alanyl-[8-lysine]vasopressin > Nα-phenyl alanyl-[8-lysine]vasopressin > Nα-diglycyl-[8-lysine]vasopressin > Nα-prolyl-[8-lysine]vasopressin > Nα-triglycyl-[8-lysine]vasopressin > Nα-sarcosyl-glycyl-[8-lysine]vasopressin. The degree of hydrolysis gradually increases to a multiple with the length of the pregnancy in consequence of the presence of oxytocine. However, vasopressin is also hydrolysed to a small extent with the enzymes from the blood sera of non-pregnant women. Under similar analytical conditions oxytocin was not hydrolysed with the sera of non-pregnant women and therefore oxytocin is a more suitable substrate than vasopressin for polarographic determination of serum oxytocinase.

Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

1985 ◽  
Vol 108 (4) ◽  
pp. 511-517 ◽  
Author(s):  
Nandalal Bagchi ◽  
Birdie Shivers ◽  
Thomas R. Brown
Keyword(s):  
Time Course ◽  
Period 2 ◽  
Iodotyrosine Deiodinase ◽  
Rate Of Hydrolysis ◽  
Underlying Mechanisms ◽  
Excess Iodide ◽  
Sites Of Action ◽  
Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

Sign in / Sign up

Export Citation Format

Share Document