scholarly journals Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers.

1988 ◽  
Vol 91 (3) ◽  
pp. 445-466 ◽  
Author(s):  
O M Sejersted ◽  
J A Wasserstrom ◽  
H A Fozzard
Keyword(s):  
Glass Electrode ◽  
Hill Coefficient ◽  
Volume Changes ◽  
Intact Cells ◽  
Intracellular Na Activity ◽  
Cardiac Purkinje Fibers ◽  
Pump Rate ◽  
Saturation Kinetics ◽  
Sequential Addition ◽  
The Hill

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.

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.

On the Measurement of Cooperativity and the Physico-Chemical Meaning of the Hill Coefficient

2019 ◽  
Vol 20 (9) ◽  
pp. 861-872 ◽  
Author(s):  
Andrea Bellelli ◽  
Emanuele Caglioti
Keyword(s):  
Ligand Binding ◽  
Hormone Receptors ◽  
Fundamental Property ◽  
Statistical Correlation ◽  
Hill Coefficient ◽  
Biological Macromolecules ◽  
Physico Chemical ◽  
Physiological Relevance ◽  
Minimum Estimate ◽  
The Hill

Cooperative ligand binding is a fundamental property of many biological macromolecules, notably transport proteins, hormone receptors, and enzymes. Positive homotropic cooperativity, the form of cooperativity that has greatest physiological relevance, causes the ligand affinity to increase as ligation proceeds, thus increasing the steepness of the ligand-binding isotherm. The measurement of the extent of cooperativity has proven difficult, and the most commonly employed marker of cooperativity, the Hill coefficient, originates from a structural hypothesis that has long been disproved. However, a wealth of relevant biochemical data has been interpreted using the Hill coefficient and is being used in studies on evolution and comparative physiology. Even a cursory analysis of the pertinent literature shows that several authors tried to derive more sound biochemical information from the Hill coefficient, often unaware of each other. As a result, a perplexing array of equations interpreting the Hill coefficient is available in the literature, each responding to specific simplifications or assumptions. In this work, we summarize and try to order these attempts, and demonstrate that the Hill coefficient (i) provides a minimum estimate of the free energy of interaction, the other parameter used to measure cooperativity, and (ii) bears a robust statistical correlation to the population of incompletely saturated ligation intermediates. Our aim is to critically evaluate the different analyses that have been advanced to provide a physical meaning to the Hill coefficient, and possibly to select the most reliable ones to be used in comparative studies that may make use of the extensive but elusive information available in the literature.

Effect of adrenomedullin on the production of endothelin-1 and on its vasoconstrictor action in resistance arteries: evidence for a receptor-specific functional interaction in patients with heart failure

2001 ◽  
Vol 101 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Chris HILLIER ◽  
Mark C. PETRIE ◽  
Michael P. LOVE ◽  
Fiona JOHNSTON ◽  
Margaret R. MACLEAN ◽  
...  
Keyword(s):  
Heart Failure ◽  
Hill Coefficient ◽  
Receptor Interaction ◽  
Resistance Arteries ◽  
Functional Interaction ◽  
High Potassium ◽  
Endothelin 1 ◽  
Patients With Heart Failure ◽  
Small Resistance ◽  
The Hill

Endothelin-1 (ET-1) and adrenomedullin (ADM) are both produced in the arterial wall, but have opposing biological actions. Evidence from experimental animals suggests a functional interaction between ET-1 and ADM. We have tested this in humans. Small resistance arteries were obtained from gluteal biopsies taken from patients with chronic heart failure (CHF) due to coronary heart disease (CHD), or with CHD and preserved ventricular function. The contractile responses to big ET-1 and to ET-1 in both sets of vessels were studied in the absence (control) and presence of ADM at 20 pmol/l (low ADM) or 200 pmol/l (high ADM), using wire myography. ADM did not affect the conversion of big ET-1 into ET-1 in vessels from patients with either CHD or CHF. Low ADM did not alter the contractile response to ET-1 in vessels from patients with CHF. Low ADM was not tested in vessels from patients with CHD, but high ADM did not affect this response in arteries from these patients. High ADM did, however, significantly reduce the vasoconstrictor effect of ET-1 in vessels from patients with CHF. The maximum response, as a percentage of the response to high potassium, was 199% (S.E.M. 25%) in the control experiments (n = 14), 205% (27%) in the low-ADM (n = 7) studies and 150% (17%) in the high-ADM (n = 6) experiments (P < 0.001). Furthermore, the Hill coefficient increased from 0.57±0.05 in the absence of ADM to 1.16±0.15 in the high-ADM experiments, indicating that ADM at 200 pmol/l specifically antagonized one receptor type in vessels from patients with CHF. We conclude that there is a one-site receptor interaction between ADM and ET-1 that is specific for vessels from patients with CHF. This functional interaction between ADM and ET-1 in resistance arteries may be of pathophysiological importance in CHF.

scholarly journals Activation and Two Modes of Blockade by Strontium of Ca2+-activated K+ Channels in Goldfish Saccular Hair Cells

1998 ◽  
Vol 111 (2) ◽  
pp. 363-379 ◽  
Author(s):  
Izumi Sugihara
Keyword(s):  
Dissociation Constant ◽  
Time Constant ◽  
Hair Cells ◽  
Single Channel ◽  
Hill Coefficient ◽  
K Channels ◽  
Voltage Dependent ◽  
Time Courses ◽  
The Hill ◽  
Inside Out

Effects of internal Sr2+ on the activity of large-conductance Ca2+-activated K+ channels were studied in inside-out membrane patches from goldfish saccular hair cells. Sr2+ was approximately one-fourth as potent as Ca2+ in activating these channels. Although the Hill coefficient for Sr2+ was smaller than that for Ca2+, maximum open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca2+ and Sr2+. This suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr2+ at higher concentrations (&gt;100 μM) produced fast and slow blockade both concentration and voltage dependently. The reduction in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were 58–209 mM, 0.69–0.75, 0.45–0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr2+ lasted ∼10–200 ms and could be fitted with single-exponential curves (time constant, τl−s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time constant, τb). A significant decrease in τb and no large changes in τl−s were observed with increased Sr2+ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr2+ ions bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba2+ blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36–150 mM and 1–1.8, respectively (n = 3).

Effect of osmotic swelling and shrinkage on Na(+)-K+ pump activity in mammalian cardiac myocytes

1993 ◽  
Vol 265 (5) ◽  
pp. C1201-C1210 ◽  
Author(s):  
D. W. Whalley ◽  
L. C. Hool ◽  
R. E. Ten Eick ◽  
H. H. Rasmussen
Keyword(s):  
Ventricular Myocytes ◽  
Pump Current ◽  
Hill Equation ◽  
Cell Swelling ◽  
Intracellular Na Activity ◽  
Single Ventricular Myocytes ◽  
Hyperosmolar Solutions ◽  
Hill Coefficients ◽  
The Hill ◽  
The Relationship

The effect on the sarcolemmal Na(+)-K+ pump of exposure to anisosmolar solutions was examined using whole cell patch clamping and ion-selective microelectrodes. Na(+)-K+ pump currents were measured in single ventricular myocytes by using pipette Na+ concentrations ([Na]pip) of 0-70 mM. The relationship between [Na]pip and pump current was well described by the Hill equation. The [Na]pip for half-maximal pump current (K0.5) was 21.4 mM in isosmolar (310 mosM) solution. K0.5 was 12.8 mM during cell swelling in hyposmolar solution (240 mosM) and 39.0 mM during cell shrinkage in hyperosmolar solution (464 mosM). The maximal pump currents, derived from the best fit of the Hill equation, and the Hill coefficients were similar in isosmolar, hyposmolar, and hyperosmolar solutions. A sustained (> 20 min) decrease in the intracellular Na+ activity developed during exposure of intact papillary muscles to hyposmolar solutions, and a sustained increase developed during exposure to hyperosmolar solutions. We conclude that osmotic myocyte swelling stimulates the sarcolemmal Na(+)-K+ pump at near-physiological levels of intracellular Na+, whereas shrinkage inhibits the pump. These changes are due to increases and decreases, respectively, in the apparent affinity of the pump for Na+.

Endotoxin administration alters the force vs. pCa relationship of skeletal muscle fibers

2000 ◽  
Vol 278 (4) ◽  
pp. R891-R896 ◽  
Author(s):  
G. Supinski ◽  
D. Nethery ◽  
T. M. Nosek ◽  
L. A. Callahan ◽  
D. Stofan ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Force ◽  
Fiber Type ◽  
Contractile Proteins ◽  
Hill Coefficient ◽  
Control Group ◽  
Fiber Types ◽  
Endotoxin Administration ◽  
Generating Capacity ◽  
The Hill

Recent work indicates that endotoxemia elicits severe reductions in skeletal muscle force-generating capacity. The subcellular alterations responsible for these decrements have not, however, been fully characterized. One possibility is that the contractile proteins per se are altered in endotoxemia and another is that the mechanism by which these proteins are activated is affected. The purpose of the present study was to assess the effects of endotoxin administration on the contractile proteins by examining the maximum calcium-activated force (Fmax) and calcium sensitivity of single Triton-skinned fibers of diaphragm, soleus, and extensor digitorum longus (EDL) muscles taken from control and endotoxin-treated (8 mg/kg) rats. Fibers were mounted on a force transducer and sequentially activated by serial immersion in solutions of increasing Ca2+ concentration (i.e., pCa 6.0 to pCa 5.0); force vs. pCa data were fit to the Hill equation. All fibers were typed at the conclusion of studies using gel electrophoresis. Fmax, the calcium concentration required for half-maximal activation (Ca50), and the Hill coefficient were compared as a function of muscle and fiber type for the control and endotoxin-treated animals. Control group Fmax was similar for diaphragm, soleus, and EDL fibers, i.e., 112.34 ± 2.64, 111.55 ± 3.66, and 104.05 ± 4.33 kPa, respectively. Endotoxin administration reduced the average Fmax for fibers from all three muscles to 80.25 ± 2.30, 72.47 ± 2.97, and 78.32 ± 2.43 kPa, respectively ( P < 0.001 for comparison of each to control). All fiber types in diaphragm, soleus, and EDL muscles manifested similar endotoxin-related reductions in Fmax. The Ca50 and the Hill coefficient for all fiber types and all muscles were unaffected by endotoxin administration. We speculate that these alterations in the intrinsic properties of the contractile proteins represent a major mechanism by which endotoxemia reduces muscle force-generating capacity.

SERCA1a can functionally substitute for SERCA2a in the heart

1999 ◽  
Vol 276 (1) ◽  
pp. H89-H97 ◽  
Author(s):  
Yong Ji ◽  
Evgeny Loukianov ◽  
Tanya Loukianova ◽  
Larry R. Jones ◽  
Muthu Periasamy
Keyword(s):  
Turnover Rate ◽  
Ph Dependence ◽  
Fold Increase ◽  
Enzymatic Properties ◽  
Hill Coefficient ◽  
Maximal Velocity ◽  
Apparent Affinity ◽  
Serca Pump ◽  
Fast Twitch ◽  
The Hill

We recently generated a transgenic (TG) mouse model in which the fast-twitch skeletal muscle sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) is overexpressed in the heart. Ectopic overexpression of SERCA1a results in remodeling of the cardiac SR containing 80% SERCA1a and 20% endogenous SERCA2a with an ∼2.5-fold increase in the total amount of SERCA protein (E. Loukianov et al. Circ. Res. 83: 889–897, 1998). We have analyzed the Ca2+ transport properties of membranes from SERCA1a TG hearts in comparison to control hearts. Our data show that the maximal velocity of SR Ca2+ transport was significantly increased (∼1.9-fold) in TG hearts, whereas the apparent affinity of the SERCA pump for Ca2+ was not changed. Addition of phospholamban antibody in the Ca2+ uptake assays increased the apparent affinity for Ca2+ to the same extent in TG and non-TG (NTG) hearts, suggesting that phospholamban regulates the SERCA1a pump in TG hearts. Analysis of SERCA enzymatic properties in TG hearts revealed that the SERCA pump affinity for ATP, the Hill coefficient, the pH dependence of Ca2+ uptake, and the effect of acidic pH on Ca2+ transport were similar to those of NTG hearts. Interestingly, the rate constant of phosphoenzyme decay (turnover rate of SERCA enzyme) was also very similar between TG and NTG hearts. Together these findings suggest that 1) the SERCA1a pump can functionally substitute for SERCA2a and is regulated by endogenous phospholamban in the heart and 2) SERCA1a exhibits several enzymatic properties similar to those of SERCA2a when expressed in a cardiac setting.

scholarly journals Kinetic studies on the regulation of rabbit liver pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 287-301 ◽  
Author(s):  
M. G. Irving ◽  
J. F. Williams
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Rabbit Liver ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Low Concentrations ◽  
Ammonium Sulphate Fractionation ◽  
The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

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 SENSIBILIDAD DE LA GARRAPATA COMÚN DEL BOVINO Rhipicephalus (Boophilus) microplus A IVERMECTINA ESTIMADA CON EL MODELO SIGMOIDEO DE RESPUESTA MÁXIMA O DE HILL

2021 ◽  
Vol 19 (suplemento) ◽  
Author(s):  
J Torrents
Keyword(s):  
Statistical Analysis ◽  
Boophilus Microplus ◽  
Susceptible Strain ◽  
Maximum Response ◽  
Hill Coefficient ◽  
Concentration Effect ◽  
Hill Model ◽  
Low Concentration ◽  
Immersion Technique ◽  
The Hill

The aim of this study was to obtain pharmacodynamics parameters to detect resistance or susceptibility of R. microplus strains to ivermectin (IVM). Two larvae samples; a susceptible strain (S) and field isolation (T) were treated with increasing concentrations of IVM using the larvae immersion technique the efficacy values measured at 24 hours were analysed with the sigmoidal maximum response so called Hill model as statistical analysis. The results obtained showed that the IVM have an all or nothing response represented by the Hill coefficient value >1 in both samples. Additionally, a low concentration effect was observed as E0 de 12.83% (S) and 9.91% (T). The field isolation larvae were susceptible to IVM in comparison with the susceptible strain by the resistance ratio (RR) which in one case was not significantly greater that one (RR50= 0.756 and RR90=1.009).

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