scholarly journals Ca2+-dependent high-affinity complex formation between calmodulin and melittin

1983 ◽  
Vol 209 (1) ◽  
pp. 269-272 ◽  
Author(s):  
M Comte ◽  
Y Maulet ◽  
J A Cox
Keyword(s):  
Complex Formation ◽  
Gel Filtration ◽  
Peptide Hormones ◽  
Bovine Brain ◽  
Disc Electrophoresis ◽  
Hydrophobic Drugs ◽  
Basic Peptide ◽  
High Affinity ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The amphiphatic polypeptide melittin migrates as an equimolar complex with bovine brain calmodulin when monitored by gel disc electrophoresis or gel filtration in the presence of Ca2+, even in 4M-urea. The complex disassociates in the presence of EDTA and urea. The affinity is of the same order as that of calmodulin for its target enzymes, and more than 1000-fold higher than that of calmodulin for basic peptide hormones or hydrophobic drugs. The activation of brain phosphodiesterase by calmodulin is inhibited by melittin. The kinetics of inhibition suggest competition between the enzyme and melittin for calmodulin. The calmodulin-melittin interaction may constitute a model for that existing between calmodulin and its target enzymes.

scholarly journals Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain

1989 ◽  
Vol 257 (2) ◽  
pp. 541-548 ◽  
Author(s):  
P R Young ◽  
A V Briedis
Keyword(s):  
Decanoic Acid ◽  
Kinetic Mechanism ◽  
Order Rate Constant ◽  
Bovine Brain ◽  
Glutathione S Transferase ◽  
Rate Acceleration ◽  
Hydrophobic Binding ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The major glutathione S-transferase isoenzyme from bovine brain was isolated and purified approx. 500-fold. The enzyme has a pI of 7.39 +/- 0.02 and consists of two non-identical subunits having apparent Mr values of 22,000 and 24,000. The enzyme is uniformly distributed in brain, and kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate suggest a random rapid-equilibrium mechanism. The kinetics of inhibition by product, by GSH analogues and by NADH are consistent with the suggested mechanism and require inhibitor binding to several different enzyme forms. Long-chain fatty acids are excellent inhibitors of the enzyme, and values of 1nKi for hexanoic acid, octanoic acid, decanoic acid and lauric acid form a linear series when plotted as a function of alkyl chain length. A free-energy change of -1900 J/mol (-455 cal/mol) per CH2 unit is calculated for the contribution of hydrophobic binding energy to the inhibition constants. The turnover number of the purified enzyme dimer is approx. 3400/min. When compared with the second-order rate constant for the reaction between CDNB and GSH, the enzyme is providing a rate acceleration of about 1000-fold. The role of entropic contributions to this small rate acceleration is discussed.

scholarly journals Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa

1989 ◽  
Vol 262 (2) ◽  
pp. 651-658 ◽  
Author(s):  
M F Scully ◽  
V Ellis ◽  
N Shah ◽  
V Kakkar
Keyword(s):  
Rate Constant ◽  
Antithrombin Iii ◽  
Heparan Sulphate ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Order Rate Constant ◽  
Pseudo First Order Reaction ◽  
High Affinity ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.

Comparative Labelling and Biokinetic Studies of 99mTc-EDTA(Sn) and 99mTc-DTPA(Sn)

1977 ◽  
Vol 16 (01) ◽  
pp. 30-35 ◽  
Author(s):  
N. Agha ◽  
R. B. R. Persson
Keyword(s):  
Complex Formation ◽  
Significant Influence ◽  
Room Temperature ◽  
Ph Value ◽  
Maximum Activity ◽  
Reduction Step ◽  
Labelling Yield ◽  
Different Temperatures ◽  
Kinetics Of

SummaryGelchromatography column scanning has been used to study the fractions of 99mTc-pertechnetate, 99mTcchelate and reduced hydrolyzed 99mTc in preparations of 99mTc-EDTA(Sn) and 99mTc-DTPA(Sn). The labelling yield of 99mTc-EDTA(Sn) chelate was as high as 90—95% when 100 μmol EDTA · H4 and 0.5 (Amol SnCl2 was incubated with 10 ml 99mTceluate for 30—60 min at room temperature. The study of the influence of the pH-value on the fraction of 99mTc-EDTA shows that pH 2.8—2.9 gave the best labelling yield. In a comparative study of the labelling kinetics of 99mTc-EDTA(Sn) and 99mTc- DTPA(Sn) at different temperatures (7, 22 and 37°C), no significant influence on the reduction step was found. The rate constant for complex formation, however, increased more rapidly with increased temperature for 99mTc-DTPA(Sn). At room temperature only a few minutes was required to achieve a high labelling yield with 99mTc-DTPA(Sn) whereas about 60 min was required for 99mTc-EDTA(Sn). Comparative biokinetic studies in rabbits showed that the maximum activity in kidneys is achieved after 12 min with 99mTc-EDTA(Sn) but already after 6 min with 99mTc-DTPA(Sn). The long-term disappearance of 99mTc-DTPA(Sn) from the kidneys is about five times faster than that for 99mTc-EDTA(Sn).

Anticoagulants Produced by Thrombin from Fibrin, the Effect on Blood Coagulation, Some Physical Characteristics

1971 ◽  
Vol 26 (02) ◽  
pp. 211-223 ◽  
Author(s):  
Ch R. Muirhead ◽  
D. C Triantaphyllopoulos
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fibrin Clot ◽  
Disc Electrophoresis ◽  
Physical Characteristics ◽  
Advanced Stage ◽  
Kallikrein Inhibitor ◽  
Shed Blood ◽  
Complete Lysis

SummaryChromatographed thrombin in the presence of both 50 Kallikrein inhibitor units of Trasylol per ml and 0.1 M E-ACA solubilized fibrin and the products of lysis possessed anticoagulant properties. The peak of the antithrombic activity coincided with the time of complete lysis of the fibrin clot, plasmin lysed fibrin exhibited the peak of its antithrombic activity much earlier. The effect of thrombin lysed fibrin on the prothrombin consumption of shed blood was found to be inhibitory.The products of the digestion of fibrin by thrombin and by plasmin, isolated at an advanced stage of proteolysis were compared by gel filtration, disc electrophoresis and DEAE cellulose chromatography. Differences in physical characteristics of these fibrin breakdown products offer evidence that they were produced by two different enzymes.

The Kinetics of Inhibition of the Action of Thrombin by the Fibrinopeptides Formed during the Clotting of Fibrinogen

1966 ◽  
Vol 16 (01/02) ◽  
pp. 277-295 ◽  
Author(s):  
A Silver ◽  
M Murray
Keyword(s):  
Amino Acids ◽  
Competitive Inhibition ◽  
Amorphous Material ◽  
Peptide Bond ◽  
Initial Reaction ◽  
Reaction Products ◽  
Coagulation Mechanism ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Burk Plot

SummaryVarious investigators have separated the coagulation products formed when fibrinogen is clotted with thrombin and identified fibrinopeptides A and B. Two other peaks are observed in the chromatogram of the products of coagulation, but these have mostly been dismissed by other workers. They have been identified by us as amino acids, smaller peptides and amorphous material (37). We have re-chromatographed these peaks and identified several amino acids. In a closed system of fibrinogen and thrombin, the only reaction products should be fibrin and peptide A and peptide B. This reasoning has come about because thrombin has been reported to be specific for the glycyl-arginyl peptide bond. It is suggested that thrombin also breaks other peptide linkages and the Peptide A and Peptide B are attacked by thrombin to yield proteolytic products. Thrombin is therefore probably not specific for the glycyl-arginyl bond but will react on other linkages as well.If the aforementioned is correct then the fibrinopeptides A and B would cause an inhibition with the coagulation mechanism itself. We have shown that an inhibition does occur. We suggest that there is an autoinhibition to the clotting mechanism that might be a control mechanism in the human body.The experiment was designed for coagulation to occur under controlled conditions of temperature and time. Purified reactants were used. We assembled an apparatus to record visually the speed of the initial reaction, the rate of the reaction, and the density of the final clot formed after a specific time.The figures we derived made available to us data whereby we could calculate and plot the information to show the mechanism and suggest that such an inhibition does exist and also further suggest that it might be competitive.In order to prove true competitive inhibition it is necessary to fulfill the criteria of the Lineweaver-Burk plot. This has been done. We have also satisfied other criteria of Dixon (29) and Bergman (31) that suggest true competitive inhibition.

scholarly journals Thermodynamic and detailed kinetic study of the formation of orthophthalaldehyde-agmatine complex by fluorescence intensities

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Khémesse Kital ◽  
Moumouny Traoré ◽  
Diégane Sarr ◽  
Moussa Mbaye ◽  
Mame Diabou Gaye Seye ◽  
...  
Keyword(s):  
Complex Formation ◽  
Thermodynamic Parameters ◽  
Initial Rate ◽  
Reaction Medium ◽  
Standard Entropy ◽  
Step Size ◽  
Formation Reaction ◽  
Different Temperatures ◽  
Kinetics Of ◽  
Complex Formation Process

Abstract The aim of this work is to determine the thermodynamic parameters and the kinetics of complex formation between orthophthalaldehyde (OPA) and agmatine (AGM) in an alkaline medium (pH 13). Firstly, the association constant (Ka) between orthophthalaldehyde and agmatine was determined at different temperatures (between 298 K and 338 K) with a step size of 10 K. Secondly, the thermodynamic parameters such as standard enthalpy (ΔH°), standard entropy (ΔS°),and Gibbs energy (∆G) were calculated, where a positive value of ΔH° (+45.50 kJ/mol) was found, which shows that the reaction is endothermic. In addition, the low value of ΔS°(+0.24 kJ/mol) indicates a slight increase in the disorder in the reaction medium. Furthermore, the negative values of ΔG between −35.62 kJ/mol and −26.02 kJ/mol show that the complex formation process is spontaneous. Finally, the parameters of the kinetics of the reaction between OPA and AGM were determined as follows: when the initial concentration of AGM (5 × 10−6 M) is equal to that of the OPA, the results show that the reaction follows an overall 1.5 order kinetics with an initial rate of 5.1 × 10−7Mmin−1 and a half-life of 8.12 min. The partial order found in relation to the AGM is 0.8. This work shows that the excess of OPA accelerates the formation reaction of the complex.

INHIBITION BY HUMAN SERUM OF THE ADIPOKINETIC EFFECT OF A HUMAN PITUITARY LIPID-MOBILIZING FACTOR

1972 ◽  
Vol 71 (3) ◽  
pp. 443-453 ◽  
Author(s):  
Olav Trygstad ◽  
Irene Foss
Keyword(s):  
Human Serum ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Disc Electrophoresis ◽  
Inhibitory Protein ◽  
Human Pituitary ◽  
Albumin Fraction ◽  
Pituitary Glands ◽  
Normal Human

ABSTRACT A lipid-mobilizing factor (LMF) with an adipotrophic effect in human and animal fat tissue has been prepared from human pituitary glands. The addition of normal human serum to LMF reduced its lipolytic effect, and it was completely abolished by serum from a group of obese patients, whereas the lipolysis was not influenced by serum from patients with generalized lipodystrophy. By DEAE-cellulose chromatography of human serum the inhibitory effect on LMF was found to be present in a protein fraction less acidic than the main serum albumin fraction. The inhibitory fraction was deprived of some contaminants by Sephadex gel filtration. Disc electrophoresis demonstrated the presence of three components in the inhibitory protein (IP), and they were identified as albumin, transferin, and haemopexin by immuno-electrophoresis. Precipitation of these proteins by their rabbit antisera demonstrated that the inhibitory effect was present in the albumin fraction. Insulin like activity was not observed in IP. A protein binding of LMF by IP could not be demonstrated. Incubation at 37°C for one hour of a mixture of LMF and IP eliminated the electrophoretic picture of LMF. It is concluded that the inhibitory effect of human serum may be due to proteolysis of LMF.

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