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 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.

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 Pharmacodynamics of TRPV1 Agonists in a Bioassay Using Human PC-3 Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Daniel Alvarez-Berdugo ◽  
Marcel Jiménez ◽  
Pere Clavé ◽  
Laia Rofes
Keyword(s):  
Sensory Neurons ◽  
Fluorescence Emission ◽  
Hill Equation ◽  
Hill Coefficient ◽  
Response Curves ◽  
Trpv1 Receptor ◽  
Specific Antagonist ◽  
Emission Changes ◽  
The Hill

Purpose. TRPV1 is a multimodal channel mainly expressed in sensory neurons. We aimed to explore the pharmacodynamics of the TRPV1 agonists, capsaicin, natural capsaicinoids, and piperine in anin vitrobioassay using human PC-3 cells and to examine desensitization and the effect of the specific antagonist SB366791.Methods. PC-3 cells expressing TRPV1 were incubated with Fluo-4. Fluorescence emission changes following exposition to agonists with and without preincubation with antagonists were assessed and referred to maximal fluorescence following the addition of ionomycin. Concentration-response curves were fitted to the Hill equation.Results. Capsaicin and piperine had similar pharmacodynamics (Emax204.8 ± 184.3% piperine versus 176.6 ± 35.83% capsaicin,P=0.8814, Hill coefficient 0.70 ± 0.50 piperine versus 1.59 ± 0.86 capsaicin,P=0.3752). In contrast, capsaicinoids had lowerEmax(40.99 ± 6.14% capsaicinoids versus 176.6 ± 35.83% capsaicin,P<0.001). All the TRPV1 agonists showed significant desensitization after the second exposition and their effects were strongly inhibited by SB366791.Conclusion. TRPV1 receptor is successfully stimulated by capsaicin, piperine, and natural capsaicinoids. These agonists present desensitization and their effect is significantly reduced by a TRPV1-specific antagonist. In addition, PC-3 cell bioassays proved useful in the study of TRPV1 pharmacodynamics.

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 MODELING OF THE SPONTANEOUS POLYPROPYLENE SORBENTS IMBIBITION WITH EMULSIONS

2014 ◽  
Vol 23 (2) ◽  
pp. 83-93 ◽  
Author(s):  
Jerzy SĘK ◽  
Olga S. SHTYKA ◽  
Kamila SZYMCZAK
Keyword(s):  
Porous Material ◽  
Aqueous Media ◽  
Hill Equation ◽  
Negative Consequences ◽  
Physical Processes ◽  
Precise Description ◽  
Oil In Water ◽  
Oil Penetration ◽  
The Hill ◽  
Kinetics Of

Oil penetration into aqueous media results in various chemical and physical processes including formation of “oil-in-water” emulsions, that have devastating effects on the environment. This research was conducted to evaluate the kinetics of permeation of “oil-in-water” emulsions into synthetic porous material. It gives the ability to develop more effective sorbents, decreasing risks of negative consequences and environmental contamination. The following publication focuses on assessment of the possibility to describe and predict a process of porous medium imbibition with emulsions by means of the Hill equation. Its adequacy is compared with the classical Lucas–Washburn equation and its modified version. The advanced approach is to substitute the commonly used parameter of the height of an imbibed emulsion (h(t)) by the parameter mh(t), which is the mass of an imbibed emulsion in the case of the classical and modified Washburn equations. According to the obtained results, the Hill equation provides the most appropriate and precise description of the porous material imbibition with “oil-in-water” emulsions and oil in comparison with others, and shows the highest correlation values (Rav = 0.995±0.001) and the lowest normalized root mean square error (NRMSEav 1.95±0.138).

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.

Modulation of rod, but not cone, cGMP-gated photoreceptor channels by calcium-calmodulin

1997 ◽  
Vol 14 (2) ◽  
pp. 233-239 ◽  
Author(s):  
Lawrence W. Haynes ◽  
Stephanie C. Stotz
Keyword(s):  
Hill Equation ◽  
Hill Coefficient ◽  
Β Subunit ◽  
Open Probability ◽  
Apparent Affinity ◽  
Maximum Current ◽  
Voltage Dependent ◽  
The Hill ◽  
Inside Out ◽  
Rod And Cone Photoreceptors

AbstractInside-out patches containing cGMP-gated channels were excised from catfish rod or cone outer segments and held under voltage clamp. The net cGMP-dependent currents elicited by saturating and subsaturating concentrations of cGMP at ±30 mV were measured and the dependence of current upon cGMP concentration was determined. The apparent affinity of the channel for its ligand was estimated by fitting these data with the Hill equation. The concentration of cGMP required to give half the maximum current (K1/2) in rod and cone channels at +30 mV was ~28 μM and ~37 μM, respectively, and was weakly voltage dependent. Thus, cone channels have an intrinsically higher K1/2 than rod channels. For both types of channel, the Hill coefficient was ~2.3. In the presence of calcium-calmodulin, the apparent affinity of the rod channel for cGMP decreased by about twofold, but the apparent affinity of the cone channels was unaffected. These results indicate that the open probability of the cone channel for its ligand cannot be modulated by calmodulin. This represents the first significant departure between rod and cone photoreceptors in mechanisms used by phototransduction and suggests that the β subunit of the cone channel must be different from that of the rod channel.

scholarly journals Kinetic analysis of cAMP-activated Na+ current in the molluscan neuron. A diffusion-reaction model.

1991 ◽  
Vol 98 (4) ◽  
pp. 835-848 ◽  
Author(s):  
R C Huang ◽  
R Gillette
Keyword(s):  
Rate Constant ◽  
Response Amplitude ◽  
Diffusion Reaction ◽  
Hill Coefficient ◽  
Hydrolysis Rate ◽  
Equation Modeling ◽  
Na Current ◽  
Phosphodiesterase Inhibition ◽  
The Hill ◽  
Kinetics Of

cAMP-activated Na+ current (INa,cAMP) was studied in voltage-clamped neurons of the seaslug Pleurobranchaea californica. The current response to injected cAMP varied in both time course and amplitude as the tip of an intracellular injection electrode was moved from the periphery to the center of the neuron soma. The latency from injection to peak response was dependent on the amount of cAMP injected unless the electrode was centered within the cell. Decay of the INa,cAMP response was slowed by phosphodiesterase inhibition. These observations suggest that the kinetics of the INa,cAMP response are governed by cAMP diffusion and degradation. Phosphodiesterase inhibition induced a persistent inward current. At lower concentrations of inhibitor, INa,cAMP response amplitude increased as expected for decreased hydrolysis rate of injected cAMP. Higher inhibitor concentrations decreased INa,cAMP response amplitude, suggesting that inhibitor-induced increase in native cAMP increased basal INa,cAMP and thus caused partial saturation of the current. The Hill coefficient estimated from the plot of injected cAMP to INa,cAMP response amplitude was close to 1.0. An equation modeling INa,cAMP incorporated terms for diffusion and degradation. In it, the first-order rate constant of phosphodiesterase activity was taken as the rate constant of the exponential decay of the INa,cAMP response. The stoichiometry of INa,cAMP activation was inferred from the Hill coefficient as 1 cAMP/channel. The equation closely fitted the INa,cAMP response and simulated changes in the waveform of the response induced by phosphodiesterase inhibition. With modifications to accommodate asymmetric INa,cAMP activation, the equation also simulated effects of eccentric electrode position. The simple reaction-diffusion model of the kinetics of INa,cAMP may provide a useful conceptual framework within which to investigate the modulation of INa,cAMP by neuromodulators, intracellular regulatory factors, and pharmacological agents.

scholarly journals Kinetics of hexokinase D (‘glucokinase’) with inosine triphosphate as phosphate donor. Loss of kinetic co-operativity with respect to glucose

1987 ◽  
Vol 245 (3) ◽  
pp. 625-629 ◽  
Author(s):  
D Pollard-Knight ◽  
A Cornish-Bowden
Keyword(s):  
High Glucose ◽  
Kinetic Models ◽  
Binding Sites ◽  
Ternary Complex ◽  
Hill Coefficient ◽  
Phosphate Donor ◽  
Km Value ◽  
Number Of Binding Sites ◽  
The Hill ◽  
Kinetics Of

When ATP, the normal phosphate donor for hexokinase D (‘glucokinase’), is replaced by ITP, the positive co-operativity with respect to glucose disappears. This may be rationalized in relation to kinetic models for hexokinase D co-operativity, which assume that with the normal substrates the chemical reaction and subsequent release of products occur so rapidly that binding of substrates cannot approach equilibrium and is therefore not constrained by the thermodynamic requirement that the Hill coefficient for substrate binding cannot exceed the number of binding sites. ITP is a much poorer substrate than ATP, however: its Km value at high glucose concentrations is 24 times the value for ATP, whereas the value of the limiting rate V is decreased about 8-fold. Consequently it is no longer possible for the ternary complex to be converted into products rapidly enough to generate kinetic co-operativity. The negative co-operativity with respect to glucose observed in 2H2O with ATP as phosphate donor also disappears when ITP is used instead of ATP.

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