scholarly journals Inhibition of plasmin by fibrinogen

1990 ◽  
Vol 269 (2) ◽  
pp. 299-302 ◽  
Author(s):  
A A R Higazi ◽  
M Mayer
Keyword(s):  
Competitive Inhibition ◽  
Hill Coefficient ◽  
Coagulation System ◽  
Amidolytic Activity ◽  
Plasmin Activity ◽  
Straight Line ◽  
Non Linear ◽  
The Hill ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The kinetics of inhibition of the amidolytic activity of plasmin on D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) by fibrinogen and fibrin were determined. Reciprocal (1/v versus 1/[S]) plots of plasmin inhibition by 0.50 microM-fibrinogen showed a non-linear downward curve. The Hill coefficient (h) was 0.68, suggesting negative co-operativity. By contrast, fibrin produced a simple competitive inhibition of plasmin (Ki = 12 micrograms/ml). Addition of 0.1 mM-6-aminohexanoic acid shifted the non-linear curve obtained in the presence of fibrinogen to a straight line as for controls, indicating that 6-aminohexanoic acid abolishes the fibrinogen-induced inhibition. Transient exposure of the enzyme to pH 1.0 abrogates the ability of fibrinogen to inhibit plasmin activity. Acidification had no effect on the Vmax but increased the Km of plasmin. The present evidence for modulation of plasmin reveals a novel mechanism for control of fibrinolysis by fibrinogen, a component of the coagulation system and the precursor of the physiological substrate of plasmin.

scholarly journals Mitochondrial adenosine triphosphatase from human placenta--inhibition by free magnesium ions of ITP hydrolysis.

1994 ◽  
Vol 41 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Z Aleksandrowicz
Keyword(s):  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Negative Cooperativity ◽  
Magnesium Ions ◽  
Beef Liver ◽  
Bicarbonate Ions ◽  
The Hill ◽  
Kinetics Of ◽  
Free Magnesium

The effects of Mg2+ and bicarbonate on the kinetics of ITP hydrolysis by soluble ATPase (F1) from human placental mitochondria were studied. Increasing amounts of Mg2+ at fixed ITP concentration, caused a marked activation of F1 followed by inhibition at higher Mg2+ concentration. The appropriate substrate for the mitochondrial F1 seems to be the MgITP complex as almost no ITP was hydrolysed in the absence of magnesium. Mg2+ behaved as a competitive inhibitor towards the MgITP complex. In this respect the human placental enzyme differ from that from other sources such as yeast, beef liver or rat liver. The linearity of the plot presenting competitive inhibition by free Mg2+ of MgITP hydrolysis (in the presence of activating bicarbonate anion) suggests that both Mg2+ and MgITP bind to the same catalytic site (Km(MgITP) = 0.46 mM, Ki(Mg) = 4 mM). When bicarbonate was absent in the ITPase assay, placental F1 exhibited apparent negative cooperativity in the presence of 5 mM Mg2+, just as it did with MgATP as a substrate under similar conditions. Bicarbonate ions eliminated the negative cooperativity with respect to ITP (as the Hill coefficient of 0.46 was brought to approx. 1), and thus limited inhibition by free Mg2+. The results presented suggest that the concentration of free magnesium ions may be an important regulatory factor of the human placental F1 activity.

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 Kinetics of ADP-induced human platelet shape change: apparent positive cooperativity

1980 ◽  
Vol 58 (1) ◽  
pp. 45-52 ◽  
Author(s):  
John G. Milton ◽  
W. Yung ◽  
C. Glushak ◽  
M. M. Frojmovic
Keyword(s):  
Light Transmission ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Hill Coefficient ◽  
A Value ◽  
The Hill ◽  
Kinetics Of ◽  
Type Response ◽  
Platelet Shape

The kinetics of ADP-induced human platelet shape change have been examined. Initial velocities of platelet shape change were estimated by two methods: (1) the slope of the initial decrease in light transmission through stirred, citrated platelet-rich plasma, and (2) direct examination of platelet morphologies by phase-contrast microscopy. In both cases, a value of the Hill coefficient, NH, significantly greater than 1 is obtained (2.0 ± 0.2 and 1.8 ± 0.2, respectively). The observed elevated value of NH is not due to a substantial fraction of the ADP being platelet bound, the presence of factors in the plasma, platelet heterogeneity, or the influence of the rate of platelet shape change reversion. Our observations suggest that ADP-induced platelet shape change may be a positively cooperative or "threshold" type response.

scholarly journals Effects of penicillins and 6-aminohexanoic acid on the kinetics of human plasmin

1989 ◽  
Vol 260 (2) ◽  
pp. 609-612 ◽  
Author(s):  
A A A Higazi ◽  
M Mayer
Keyword(s):  
Active Site ◽  
Dependent Manner ◽  
Chromogenic Substrate ◽  
Regulatory Site ◽  
Amidolytic Activity ◽  
Plasmin Activity ◽  
Maximal Inhibition ◽  
Sigmoidal Curve ◽  
Kinetics Of ◽  
Dose Dependent

Human plasmin activity is inhibited by various penicillins in a dose-dependent manner. Ampicillin and cloxacillin produce a 50% inhibition of the globinolytic activity of plasmin at 4.5 and 5.3 mM respectively. A lower inhibitory capacity is displayed by carbenicillin. Assay of plasmin by its amidolytic activity on D-valyl-L-leucyl-L-lysine p-nitroanilide dihydrochloride showed that ampicillin at a concentration producing half-maximal inhibition converted the hyperbolic activity-substrate concentration curve into a sigmoidal curve. A similar conversion occurred in the presence of ampicillin when plasmin was assayed with an alternative chromogenic substrate, L-pyroglutamyl-glycyl-L-arginine p-nitroanilide hydrochloride 6-Aminohexanoic acid at 7.5 microM abolished the inhibition of plasmin induced by ampicillin. The present observations suggest that ampicillin interacts with plasmin at a regulatory site different from the active site of the enzyme. The effect of 6-aminohexanoic acid indicates that the lysine-binding site may be part of a regulatory site. It is possible that modulation of plasmin activity by ligands plays a role in the control of fibrinolysis.

The anthranilate aggregate of Escherichia coli: kinetics of inhibition by tryptophan of phosphoribosyltransferase

1986 ◽  
Vol 64 (7) ◽  
pp. 681-691 ◽  
Author(s):  
Jose E. Gonzalez ◽  
Ronald L. Somerville
Keyword(s):  
Feedback Inhibition ◽  
Kinetic Mechanism ◽  
Ternary Complexes ◽  
Hill Coefficient ◽  
Anthranilate Synthase ◽  
Active Centers ◽  
Catalytic Sites ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Tryptophan Molecule

The kinetic mechanism of the phosphoribosyltransferase reaction is shown to be rapid equilibrium random bi bi with an enzyme–anthranilate–pyrophosphate abortive complex. We present a rate equation that not only predicts the observed kinetic patterns but also accomodates the fact that feedback inhibition is partial, even though tryptophan (Ki = 0.5 μM) and phosphoribosylpyrophosphate (Km = 50 μM) are competitive. Neither ligand completely abolishes the effect of the other. Instead, the binding of one ligand leads to a mutual elevation in the dissociation constant of the opposing ligand by a factor of two to three. Tryptophan inhibition is noncompetitive with respect to anthranilate (Km = 0.58 μM) and does not diminish the rate of interconversion of ternary complexes. Tryptophan cooperativity, with respect to the inhibition of phosphoribosyltransferase, conforms to the concerted Monod–Wyman–Changeux formulation (kinetic Hill coefficient = 2), whereas tryptophan as an inhibitor of anthranilate synthase more closely conforms to a Koshland model of sequential cooperativity with a kinetic Hill coefficient of 1.4. The aggregrate contains only one class of tryptophan sites. Thus the first tryptophan molecule bound to the aggregate maximally inhibits both phosphoribosyltransferase active centers and one of the two anthranilate synthase catalytic sites. The remaining anthranilate synthase subunit thereupon is converted into a form with less (but not zero) affinity for chorismate and a greater affinity for a second molecule of tryptophan.

scholarly journals Adenine nucleotides and magnesium ions in relation to control of mammalian cerebral-cortex hexokinase

1969 ◽  
Vol 112 (5) ◽  
pp. 579-586 ◽  
Author(s):  
H S Bachelard ◽  
P. S. G. Goldfarb
Keyword(s):  
Mixed Type ◽  
Competitive Inhibition ◽  
Adenine Nucleotides ◽  
Effective Control ◽  
Magnesium Ions ◽  
Soluble Enzyme ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

1. The kinetics of inhibition of brain soluble cytoplasmic hexokinase by ADP were examined in relation to variations in the concentrations of Mg2+ and ATP. The type of inhibition observed was dependent on the Mg2+/ATP ratio. 2. ADP at Mg2+/ATP ratios 2:1 exhibited inhibition of the ‘mixed’ type; at Mg2+/ATP ratios 1:1 the inhibition appeared to be competitive with regard to ATP. 3. Inhibition by free ATP was observed when the Mg2+/ATP ratio was less than 1:1. The inhibition was also of the ‘mixed’ type with respect to MgATP2−. 4. The inhibitions due to ADP and to free ATP were not additive. The results suggested that there may be up to four sites in the soluble enzyme: for glucose, glucose 6-phosphate, ADP and MgATP2−. 5. The ‘free’ non-particulate intracellular Mg2+ concentration was measured and concluded to be about 1·5mm. 6. The concentrations in vivo of Mg2+ and ATP likely to be accessible to a cytoplasmic enzyme are suggested to be below those that yield maximum hexokinase rates in vitro. The enzymic rates were measured at relevant suboptimum concentrations of Mg2+ and ATP in the presence of ADP. Calculations that included non-competitive inhibition due to glucose 6-phosphate (56–65% at 0·25mm) resulted in net rates very similar to the measured rates for overall glycolysis. This system may therefore provide a basis for effective control of cerebral hexokinase.

Kinetics of Inhibition of ADP-Induced Platelet Aggregation by Dihomo-β-Linolenic Acid (DHLA).

1979 ◽  
Author(s):  
M. Zuzel ◽  
A. Spencer
Keyword(s):  
Fatty Acids ◽  
Platelet Aggregation ◽  
Competitive Inhibition ◽  
Human Platelets ◽  
Inhibition Of Platelet Aggregation ◽  
Kinetics Of ◽  
Dose Dependent ◽  
Kinetics Of Inhibition ◽  
Primary Platelet

In the study of the effects on platelets of thromboxanes and prostaglandins (PG) large amounts of precursor fatty acids have frequently been added to platelet suspensions. In the case of DHLA, this results in a general inhibition of platelet reactions. We have employed kinetic analysis of the inhibition of ADP-induced primary platelet aggregation to estimate the potency, specificity and mode of action of DHLA on human platelets in citrated PRP. Platelet PG production was estimated from formation of malondialdehyde (MDA) and aspirin (ASA) was used to inhibit production-Inhibition of aggregation and MEA formation were dose-dependent and both were observed at DHLA concentrations of 0.1 mM and above. Inhibition of aggregation was of mixed type, consisting of an ASA-sensitive competitive component (K1 ≈ 0.2mM) and an ASA-insensitve component which was non-competitive and dominated inhibition at higher concentrations of DHLA. The KI (DHLA) of the non-competitive component varied from 0.4 to 1.5 mM with different batches of PRP. Other polyunsaturated fatty acids which are not PG precursors, did not cause competitive inhibition but were as potent as DHLA in the non-competitive inhibition of aggregation. The results show that a large part of the inhibition of platelet aggregation by DHLA in vitro is not due to its transformation into an inhibitor of platelet function in the PG production pathway.

scholarly journals The Hill analysis and co-ion–driven transporter kinetics

2015 ◽  
Vol 145 (6) ◽  
pp. 565-574 ◽  
Author(s):  
Juke S. Lolkema ◽  
Dirk-Jan Slotboom
Keyword(s):  
Protein Complex ◽  
Kinetic Data ◽  
Hill Equation ◽  
Hill Coefficient ◽  
Ion Binding ◽  
Exact Number ◽  
Widespread Phenomenon ◽  
Multiple Ligands ◽  
The Hill ◽  
Kinetics Of

Interaction of multiple ligands with a protein or protein complex is a widespread phenomenon that allows for cooperativity. Here, we review the use of the Hill equation, which is commonly used to analyze binding or kinetic data, to analyze the kinetics of ion-coupled transporters and show how the mechanism of transport affects the Hill coefficient. Importantly, the Hill analysis of ion-coupled transporters can provide the exact number of transported co-ions, regardless of the extent of the cooperativity in ion binding.

scholarly journals Regulation of phosphoenolpyruvate carboxylase of Zea mays by metabolites

1973 ◽  
Vol 131 (3) ◽  
pp. 451-458 ◽  
Author(s):  
K. F. Wong ◽  
D. D. Davies
Keyword(s):  
Zea Mays ◽  
Phosphoenolpyruvate Carboxylase ◽  
Competitive Inhibition ◽  
Energy Charge ◽  
Dicarboxylic Acids ◽  
Ph Optimum ◽  
Km Value ◽  
Sugar Phosphates ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

Crude preparations of phosphoenolpyruvate carboxylase obtained from aetiolated seedlings of Zea mays are unstable but can be stabilized with glycerol. At the pH optimum of 8.3, the Km value for phosphoenolpyruvate is 80μm. When assayed at 30°C, the enzyme shows normal Michaelis–Menten kinetics, but when assayed at 45°C sigmoid kinetics are exhibited. At pH7.0 the enzyme is inhibited by a number of dicarboxylic acids and by glutamate and aspartate. d and l forms of the hydroxy acids and amino acids are inhibitory and the kinetics approximate to simple non-competitive inhibition. The same compounds produce less inhibition at pH7.6 than at pH7.0 and the kinetics of inhibition are more complex. The enzyme is activated by Pi, by SO42- and by a number of sugar phosphates. Maximum activation occurs at acid pH values, where enzyme activity is lowest. The enzyme is activated by AMP and inhibited by ADP and ATP so that the response to energy charge is of the R type and is thus at variance with Atkinson's (1968) concept of energy charge. The physiological significance of the response to metabolites is discussed.

scholarly journals UMP kinase from Streptococcus pneumoniae: evidence for co-operative ATP binding and allosteric regulation

2004 ◽  
Vol 384 (3) ◽  
pp. 619-627 ◽  
Author(s):  
Florence FASSY ◽  
Odile KREBS ◽  
Maryse LOWINSKI ◽  
Paul FERRARI ◽  
Jacques WINTER ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Active Site ◽  
Hydrodynamic Radius ◽  
Ph Dependence ◽  
Hill Coefficient ◽  
High Affinity ◽  
Saturation Kinetics ◽  
Ump Kinase ◽  
The Hill ◽  
Kinetics Of

UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP and ADP. In prokaryotes, the reaction is carried out by a hexameric enzyme, activated by GTP and inhibited by UTP. In the present study, Streptococcus pneumoniae UMP kinase was studied as a target for antibacterial research and its interest was confirmed by the demonstration of the essentiality of the gene for cell growth. In the presence of MnCl2 or MgCl2, the saturation kinetics of recombinant purified UMP kinase was hyperbolic for UMP (Km=0.1 mM) and sigmoidal for ATP (the substrate concentration at half-saturation S0.5=9.4±0.7 mM and n=1.9±0.1 in the presence of MgCl2). GTP increased the affinity for ATP and decreased the Hill coefficient (n). UTP decreased the affinity for ATP and only slightly increased the Hill coefficient. The kcat (175±13 s−1 in the presence of MgCl2) was not affected by the addition of GTP or UTP, whose binding site was shown to be different from the active site. The hydrodynamic radius of the protein similarly decreased in the presence of ATP or GTP. There was a shift in the pH dependence of the activity when the ATP concentration was switched from low to high. These results support the hypothesis of an allosteric transition from a conformation with low affinity for ATP to a form with high affinity, which would be induced by the presence of ATP or GTP.

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