Kinetics of inactivation of 4-aminobutyrate aminotransferase by 3-bromopyruvate

1992 ◽  
Vol 70 (8) ◽  
pp. 716-719 ◽  
Author(s):  
K. J. Blessinger ◽  
G. Tunnicliff
Keyword(s):  
Pseudomonas Fluorescens ◽  
Maximum Rate ◽  
Enzyme Reaction ◽  
Reversible Inhibitor ◽  
Enzyme Complex ◽  
Inhibitor Concentration ◽  
Aminobutyrate Aminotransferase ◽  
Affinity Labelling ◽  
Sulphydryl Groups ◽  
Kinetics Of

3-Bromopyruvate inhibited 4-aminobutyrate aminotransferase (EC 2.6.1.19) from Pseudomonas fluorescens, apparently irreversibly. Kinetics of this inactivation were studied by continuously monitoring the enzyme reaction at 30 °C in the presence of inhibitor. Irrespective of how high an inhibitor concentration was present, a maximum rate of inactivation was eventually achieved (5.9 × 10−3 s−1), indicating the formation of a reversible inhibitor–enzyme complex before the final inactivation step. The dissociation constant of this complex was found to be 6.5 μM. This affinity labelling by 3-bromopyruvate suggests the presence of essential sulphydryl groups on the enzyme, since this compound is known to preferentially alkylate cysteinyl residues.Key words: 4-aminobutyrate, 4-aminobutyrate aminotransferase, inactivation, 3-bromopyruvate, affinity label, Pseudomonas fluorescens.

Prothrombin Evaluation as Obtained by Kinetics Studies of Antigen-Antibody Reaction in a Laser Nephelometer

1984 ◽  
Vol 52 (01) ◽  
pp. 015-018 ◽  
Author(s):  
A Girolami ◽  
A Sticchi ◽  
R Melizzi ◽  
L Saggin ◽  
G Ruzza
Keyword(s):  
Liver Cirrhosis ◽  
Cirrhotic Patient ◽  
Maximum Rate ◽  
Serum Proteins ◽  
Clotting Factors ◽  
Kinetics Studies ◽  
Maximum Value ◽  
Time Required ◽  
Antigen Antibody ◽  
Kinetics Of

SummaryLaser nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetics of the reaction between antigen and antibody. We studied the kinetics of the reaction between prothrombin and an antiprothrombin antiserum using several prothrombins namely: Prothrombin Padua, prothrombin Molise, which are two congenital dysprothrombinemias, cirrhotic, coumarin or normal prothrombins. Different behaviors in the kinetics of the reactions were shown even when the concentration of prothrombins was about the same in all plasma tested. These differences were analyzed by means of a computer (Apple II 48 RAM) programmed to solve four unknown equations (Rodbard’s equation). From the data so obtained one can see that when voltages at the beginning and at the end of the reaction are in all cases about the same, a clear difference in the time required to reach half the maximum value of the voltage can still be demonstrated. This parameter, which is expressed in minutes, is longer in coumarin and prothrombin Molise than in controls. On the contrary it is shorter in prothrombin Padua and has about the same value of controls in the cirrhotic patient. Moreover the time at which the maximum rate is obtained is longer in coumarin and prothrombin Molise than in controls and shorter in liver cirrhosis and prothrombin Padua. In conclusion data obtained show that coumarin prothrombin behaves in a different way from cirrhotic prothrombin and also that there is a different behaviour between the two congenital dysprothrombinemias.

Vulcanization of Rubber in the Presence of N,N-Diethyl-2-Benzothiazolylsulfenamide as Accelerator

1960 ◽  
Vol 33 (2) ◽  
pp. 361-372 ◽  
Author(s):  
B. A. Dogadkin ◽  
O. N. Beliatskaya ◽  
A. B. Dobromyslova ◽  
M. S. Feldshtein
Keyword(s):  
Induction Period ◽  
Maximum Rate ◽  
Initial Period ◽  
Pure Oxygen ◽  
Butadiene Rubber ◽  
Sulfur Vulcanization ◽  
Analogous Manner ◽  
Chemical Crosslinks ◽  
Main Period ◽  
Kinetics Of

Abstract 1. The vulcanization of rubber in the presence of N,N-diethyl-2-benzothiazolylsulfenamide is characterized by an S-shaped curve for the addition of sulfur with an initial induction period in the reaction. The modulus and number of crosslinks are changed in an analogous manner to the structure of the vulcanizate. 2. The energy of activation of the addition of sulfur in the initial period is equal to 30 kcal per mole as against 14 kcal per mole in the main period. 3. The induction period is increased if the sodium-butadiene rubber is purified from alkali. 4. Molecular oxygen present in the compound being vulcanized decreases the induction period and increases the rate of the addition of the sulfur in the main period. An induction period is not observed when vulcanization is carried out in an atmosphere of pure oxygen. 5. The interaction of N,N-diethyl-2-benzothiazolylsulfenamide with rubber (in the absence of sulfur) at vulcanization temperatures is accompanied by the formation of MBT, diethylamine, and the addition of the elements of the accelerator to the rubber. The kinetics of this process were studied. 6. The interaction of N,N-diethyl-2-benzothiazolyl sulfenamide with rubber leads to the formation of chemical crosslinks between the molecules of rubber (the effect of vulcanization). 7. The change of N,N-diethyl-2-benzothiazolyl sulfenamide under the conditions of normal sulfur vulcanization has the same character as in the interaction of it with rubber. The kinetics of the formation of MBT have a maximum which coincides with the maximum rate of the addition of sulfur to the rubber. 8. A mechanism is presented for the vulcanization and acceleration actions of N,N-diethyl-2-benzothiazolyl sulfenamide which provides for the extraction of hydrogen by the accelerator radicals from the molecular chains of the rubber with the formation of MBT, diethylamine and polymer radicals which are able to interact with the sulfur.

scholarly journals Inhibition of staphylococcal α-toxin. A kinetic evaluation of aromatic polysulphonic acids as inhibitors of haemolysis

1968 ◽  
Vol 108 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Margaret Robson Wright ◽  
J. P. Arbuthnott ◽  
I. R. W. Lominski
Keyword(s):  
Inhibitory Activity ◽  
Maximum Rate ◽  
Lag Time ◽  
Test Compound ◽  
Rabbit Erythrocyte ◽  
Compound I ◽  
Kinetic Evaluation ◽  
Ph Values ◽  
Inhibitory Compounds ◽  
Kinetics Of

1. The effect of a number of aromatic polysulphonic acids on the kinetics of haemolysis of rabbit erythrocyte suspensions by crude staphylococcal α-toxin was studied at pH8·6 and 6·8. 2. All of the inhibitory compounds caused an increase in the prelytic lag time (τ) of the sigmoid haemolysis curves, an increase in the time to reach 50% haemolysis (t½) and a decrease in the maximum rate of haemolysis (Rmax.). The most inhibitory compounds caused a 50% decrease in Rmax. at concentrations between 0·1 and 0·2mm. 3. The effect of pH varied considerably: compounds (I) and (II) were almost equally inhibitory at both pH values, compounds (IV) and (IX) were more inhibitory at pH6·8 than at pH8·6, and compounds (VII), (VIII), (X), (XI) and (XII) were more inhibitory at pH8·6. 4. Increased time of premixing α-toxin with compound (I) caused increased inhibition. 5. An attempt was made, where possible, to relate the inhibitory activity to the structure of the test compound.

Kinetics of reversible DIDS inhibition of chloride self exchange in human erythrocytes

1989 ◽  
Vol 257 (4) ◽  
pp. C601-C606 ◽  
Author(s):  
T. Janas ◽  
P. J. Bjerrum ◽  
J. Brahm ◽  
J. O. Wieth
Keyword(s):  
Transport System ◽  
Time Course ◽  
Order Rate Constant ◽  
Anion Transport ◽  
Disulfonic Acid ◽  
Reversible Inhibitor ◽  
Apparent Dissociation Constant ◽  
Anion Transport System ◽  
Relative Change ◽  
Kinetics Of

The capnophorin (band 3)-mediated chloride self exchange flux in intact erythrocytes and in resealed erythrocyte ghosts was determined at pH 7.3 by measuring the unidirectional efflux of 36Cl-. The time-dependent irreversible inactivation of the anion transport system by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was measured as the relative change of the unidirectional 36Cl efflux rate. The rate of covalent DIDS binding under conditions of excess DIDS in solution that ensure a complete irreversible inhibition followed an exponential time course with a rate coefficient Kcov (min-1). The Arrhenius activation enthalpy of Kcov was constant, 114 kJ/mol, at 0-38 degrees C. At 38 and 0 degrees C, Kcov was 0.5 min-1 [half time (T1/2) = ln2/Kcov = 1.3 min] and 0.004 min-1 (T1/2 = 178 min), respectively. The slow irreversible DIDS binding to the anion transport system at 0 degrees C allows a determination of the kinetics of the reversible DIDS reaction. The pseudo first-order rate constant for binding, kon, was 3.5 X 10(5) (M.s)-1. The apparent dissociation constant, KD, determined from the steady-state binding to the erythrocyte membrane was 3.1 X 10(-8) M at an equal internal and external Cl- concentration of 165 mM (0 degrees C). The value of KD shows that DIDS is the most efficient reversible inhibitor among the stilbene derivatives so far studied. Maximum reversible inhibition by DIDS was obtained by binding of a minimum of approximately 10(6) molecules/cell membrane. The number is similar to that obtained from studies of irreversible DIDS binding.

The Frank-Starling mechanism is not mediated by changes in rate of cross-bridge detachment

1997 ◽  
Vol 273 (5) ◽  
pp. H2428-H2435 ◽  
Author(s):  
Thomas Wannenburg ◽  
Paul M. L. Janssen ◽  
Dongsheng Fan ◽  
Pieter P. De Tombe
Keyword(s):  
Cardiac Muscle ◽  
Maximum Rate ◽  
Sarcomere Length ◽  
Myosin Head ◽  
Isometric Force ◽  
Force Development ◽  
Cross Bridge ◽  
Average Slope ◽  
The Cross ◽  
Kinetics Of

We tested the hypothesis that the Frank-Starling relationship is mediated by changes in the rate of cross-bridge detachment in cardiac muscle. We simultaneously measured isometric force development and the rate of ATP consumption at various levels of Ca2+ activation in skinned rat cardiac trabecular muscles at three sarcomere lengths (2.0, 2.1, and 2.2 μm). The maximum rate of ATP consumption was 1.5 nmol ⋅ s−1 ⋅ μl fiber vol−1, which represents an estimated adenosinetriphosphatase (ATPase) rate of ∼10 s−1 per myosin head at 24°C. The rate of ATP consumption was tightly and linearly coupled to the level of isometric force development, and changes in sarcomere length had no effect on the slope of the force-ATPase relationships. The average slope of the force-ATPase relationships was 15.5 pmol ⋅ mN−1 ⋅ mm−1. These results suggest that the mechanisms that underlie the Frank-Starling relationship in cardiac muscle do not involve changes in the kinetics of the apparent detachment step in the cross-bridge cycle.

Kinetics of hepatic alanine uptake and urea synthesis in pigs

1988 ◽  
Vol 254 (4) ◽  
pp. G602-G609
Author(s):  
H. Vilstrup ◽  
L. T. Skovgaard
Keyword(s):  
Maximum Rate ◽  
Maximum Likelihood Method ◽  
Blood Flow Rate ◽  
Likelihood Method ◽  
Synthesis Rate ◽  
Urea Synthesis ◽  
Half Maximum ◽  
Concentration Gradients ◽  
Saturation Kinetics ◽  
Kinetics Of

The kinetics of hepatic alanine uptake and urea synthesis in relation to sinusoid alanine concentration was investigated in seven anesthetized pigs weighing 63 kg, using liver vein catheterizations. Each experiment consists of four steady-state periods of 40 min with alanine concentrations in the range of 0.4-27 mmol/l. The process rates were measured as the products of transhepatic concentration gradients and hepatic blood flow rate, determined by indocyanine green. The data suggest that both processes follow saturation kinetics, that there exists a sinusoidal concentration of alanine below which net removal is limited, and that urea synthesis consists of two components: one alanine independent and one depending on alanine concentration according to Michaelis-Menten kinetics. The kinetic parameters were estimated iteratively by the maximum likelihood method. The maximum rate of alanine uptake was 1.13 +/- 0.74 mmol.min-1.kg liver wt-1 (mean +/- SD), the alanine concentration resulting in half-maximum alanine uptake rate was 1.69 +/- 0.99 mmol/l, and the removal-limiting alanine concentration was 0.27 +/- 0.09 mmol/l. The maximum rate of urea-N synthesis was 1.49 +/- 0.87 mmol.min-1.kg liver wt-1, the alanine concentration resulting in half-maximum urea-N synthesis rate was 2.32 +/- 1.11 mmol/l, and the alanine concentration-independent urea-N synthesis rate was 0.13 +/- 0.10 mmol.min-1.kg liver wt-1.

scholarly journals A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase

1976 ◽  
Vol 159 (3) ◽  
pp. 563-570 ◽  
Author(s):  
W A Laing ◽  
J T Christeller
Keyword(s):  
Initial Rate ◽  
Equilibrium Constants ◽  
Co2 Concentration ◽  
Time Dependent ◽  
Enzyme Complex ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Bisphosphate Carboxylase ◽  
Rate Of Reaction ◽  
Kinetics Of

Further evidence for time-dependent interconversions between active and inactive states of ribulose 1,5-bisphosphate carboxylase is presented. It was found that ribulose bisphosphate oxygenase and ribulose bisphosphate carboxylase could be totally inactivated by excluding CO2 and Mg2+ during dialysis of the enzyme at 4 degrees C. When initially inactive enzyme was assayed, the rate of reaction continually increased with time, and the rate was inversely related to the ribulose bisphosphare concentration. The initial rate of fully activated enzyme showed normal Michaelis-Menten kinetics with respect to ribulose bisphosphate (Km = 10muM). Activation was shown to depend on both CO2 and Mg2+ concentrations, with equilibrium constants for activation of about 100muM and 1 mM respectively. In contrast with activation, catalysis appeared to be independent of Mg2+ concentration, but dependent on CO2 concentration, with a Km(CO2) of about 10muM. By studying activation and de-activation of ribulose bisphosphate carboxylase as a function of CO2 and Mg2+ concentrations, the values of the kinetic constants for these actions have been determined. We propose a model for activation and catalysis of ribulose bisphosphate carboxylase: (see book) where E represents free inactive enzyme; complex in parentheses, activated enzyme; R, ribulose bisphosphate; M, Mg2+; C, CO2; P, the product. We propose that ribulose bisphosphate can bind to both the active and inactive forms of the enzyme, and slow inter-conversion between the two states occurs.

SPECIFIC DEGRADATION OF CRYPTOCOCCUS NEOFORMANS 3723 CAPSULAR POLYSACCHARIDE BY A MICROBIAL ENZYME: I. ISOLATION, PARTIAL PURIFICATION, AND PROPERTIES OF THE ENZYME

1961 ◽  
Vol 7 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Hans H. Gadebusch ◽  
John D. Johnson
Keyword(s):  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Enzyme Reaction ◽  
Partial Purification ◽  
Intracellular Enzyme ◽  
Purification And Properties ◽  
Microbial Enzyme ◽  
Specific Degradation ◽  
Kinetics Of ◽  
Enzyme I

A partially purified intracellular enzyme from a species of Alcaligenes is described which specifically initiates the degradation of the he heteropolysaccharide of Cryptococcus neoformans, isolate 3723, The enzyme is active in the presence of serum and can be inactivated by heating at 45 °C for 10 minutes, The kinetics of the enzyme reaction are similar to those of other enzymes. Recovery and identification of the four known monosaccharides from enzymatic hydrolyzates suggest the presence of a number of other enzymes in these preparations.

scholarly journals The kinetics of the lysis of Bacterium coli by glycine

1951 ◽  
Vol 49 (2-3) ◽  
pp. 169-174 ◽  
Author(s):  
J. Gordon ◽  
R. A. Hall ◽  
L. H. Stickland
Keyword(s):  
High Temperature ◽  
Latent Period ◽  
Temperature Coefficient ◽  
Maximum Rate ◽  
Bacterial Suspension ◽  
Glycine Concentration ◽  
Wide Range ◽  
Kinetics Of ◽  
Very High

The lysis of Bacterium coli suspensions brought about by glycine shows the following characteristics:(1) There is a latent period of 2 hr., followed by a rapid lysis reaching a maximum in about 8 hr.(2) The extent of the lysis is independent of the dilution of the bacterial suspension over a wide range.(3) The extent of the lysis increases with the glycine concentration up to 10M, but is approaching a limit at this concentration.(4) The lysis is negligible below pH 5 and above pH 10, and shows a maximum rate in the region of pH 6–5–8–5.(5) The rate of lysis has a very high temperature coefficient (Q10 of the order of 5).

Inhibition of the chlorinating activity of myeloperoxidase by tempol: revisiting the kinetics and mechanisms

2011 ◽  
Vol 439 (3) ◽  
pp. 423-434 ◽  
Author(s):  
Raphael F. Queiroz ◽  
Sandra M. Vaz ◽  
Ohara Augusto
Keyword(s):  
Hypochlorous Acid ◽  
Tissue Injury ◽  
Kinetic Modelling ◽  
Oxidation Kinetic ◽  
Reversible Inhibitor ◽  
Binding Interaction ◽  
Oxidative Potential ◽  
Oxidant Production ◽  
A Minor ◽  
Kinetics Of

The nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of inflammation by mechanisms that are not completely understood. MPO (myeloperoxidase), which plays a fundamental role in oxidant production by neutrophils, is an important target for anti-inflammatory action. By amplifying the oxidative potential of H2O2, MPO produces hypochlorous acid and radicals through the oxidizing intermediates MPO-I [MPO-porphyrin•+-Fe(IV)=O] and MPO-II [MPO-porphyrin-Fe(IV)=O]. Previously, we reported that tempol reacts with MPO-I and MPO-II with second-order rate constants similar to those of tyrosine. However, we noticed that tempol inhibits the chlorinating activity of MPO, in contrast with tyrosine. Thus we studied the inhibition of MPO-mediated taurine chlorination by tempol at pH 7.4 and re-determined the kinetic constants of the reactions of tempol with MPO-I (k=3.5×105 M−1·s−1) and MPO-II, the kinetics of which indicated a binding interaction (K=2.0×10−5 M; k=3.6×10−2 s−1). Also, we showed that tempol reacts extremely slowly with hypochlorous acid (k=0.29 and 0.054 M−1·s−1 at pH 5.4 and 7.4 respectively). The results demonstrated that tempol acts mostly as a reversible inhibitor of MPO by trapping it as MPO-II and the MPO-II–tempol complex, which are not within the chlorinating cycle. After turnover, a minor fraction of MPO is irreversibly inactivated, probably due to its reaction with the oxammonium cation resulting from tempol oxidation. Kinetic modelling indicated that taurine reacts with enzyme-bound hypochlorous acid. Our investigation complements a comprehensive study reported while the present study was underway [Rees, Bottle, Fairfull-Smith, Malle, Whitelock and Davies (2009) Biochem. J. 421, 79–86].

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