scholarly journals Kinetic Characterisation of Phosphofructokinase Purified from Setaria cervi: A Bovine Filarial Parasite

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Bechan Sharma
Keyword(s):  
Adult Female ◽  
Binding Sites ◽  
Inhibitory Concentration ◽  
Product Inhibition ◽  
Hill Coefficient ◽  
Enzyme Protein ◽  
Setaria Cervi ◽  
The Hill ◽  
Inhibition Studies ◽  
Cooperative Kinetics

Phosphofructokinase (PFK), a regulatory enzyme in glycolytic pathway, has been purified to electrophoretic homogeneity from adult female Setaria cervi and partially characterized. For this enzyme, the Lineweaver-Burk's double reciprocal plots of initial rates and D-fructose-6-phosphate (F-6-P) or Mg-ATP concentrations for varying values of cosubstrate concentration gave intersecting lines indicating that Km values for F-6-P (1.05 mM) and ATP (3 μM) were independent of each other. S. cervi PFK, when assayed at inhibitory concentration of ATP (>0.1 mM), exhibited sigmoidal behavior towards binding with F-6-P with a Hill coefficient (n) value equal to 1.8 and 1.7 at 1.0 and 0.33 mM ATP, respectively. D-fructose-1,6-diphosphate (FDP) competitively inhibited the filarial enzyme: Ki and Hill coefficient values being 0.18 μM and 2.0, respectively. Phosphoenolpyruvate (PEP) also inhibited the enzyme competitively with the Ki value equal to 0.8 mM. The Hill coefficient values (>1.5) for F-6-P (at inhibitory concentration of ATP) and FDP suggested its positive cooperative kinetics towards F-6-P and FDP, showing presence of more than one binding sites for these molecules in enzyme protein and allosteric nature of the filarial enzyme. The product inhibition studies gave us the only compatible mechanism of random addition process with a probable orientation of substrates and products on the enzyme surface.

Cooperative Binding of the Organophosphate Paraoxon to the (Na+ + K+)-ATPase

1995 ◽  
Vol 50 (9-10) ◽  
pp. 660-663 ◽  
Author(s):  
Janusz Blasiak
Keyword(s):  
Active Sites ◽  
Microsomal Fraction ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Cooperative Binding ◽  
Enzyme Protein ◽  
The Hill ◽  
The Relationship ◽  
Dose Dependent ◽  
Substrate Kinetics

Abstract Paraoxon. the main active metabolite of the organophosphorus insecticide parathion. exerted a dose-dependent inhibitory effect on the activity of pig kidney (Na+ + K+)-ATPase contained in microsomal fraction and purified from it. Substrate kinetics studies revealed the existence of two active sites with high and low affinity to ATP. The Dixon analysis of the mode of the inhibition indicated its noncompetitive character. The purified enzyme was more affected than enzyme contained in the microsomal fraction. The inhibition constant K, ranged from 73 to 246 μm depending on the type of preparation. The Hill coefficient (n) fulfilled the relationship 1<n<3. These properties of the interaction suggest the cooperative binding of paraoxon to the enzyme. An indirect mechanism of the interaction was proposed: paraoxon could inhibit the activity of the (N a+ + K+)-ATPase by excluding the enzyme protein from its normal lipid milieu

scholarly journals Allosteric effects of Mg2+ on the gating of Ca2+-activated K+ channels from mammalian skeletal muscle

1986 ◽  
Vol 124 (1) ◽  
pp. 5-13
Author(s):  
J. Golowasch ◽  
A. Kirkwood ◽  
C. Miller
Keyword(s):  
Binding Sites ◽  
Hill Coefficient ◽  
K Channel ◽  
Activation Process ◽  
Mammalian Skeletal Muscle ◽  
Activation Curve ◽  
K Channels ◽  
The Hill ◽  
Cytoplasmic Side ◽  
High Conductance

Ca2+-activated K+ channels from rat muscle transverse tubule membranes were inserted into planar phospholipid bilayers, and the activation of these channels by Ca2+ was studied. On the cytoplasmic side of the channel, calcium ions (in the range 10–100 mumol l-1) increase the opening probability of the channel in a graded way. This ‘activation curve’ is sigmoid, with an average Hill coefficient of about 2. Magnesium ions, in the range 1–10 mmol l-1, increase the apparent affinity of the channel for Ca2+ and greatly enhance the sigmoidicity of the Ca2+ activation curve. In the presence of 10 mmol l-1 Mg2+, the Hill coefficient for Ca2+ activation is about 4.5. This effect depends upon Mg2+ concentration but not upon applied voltage. Mg2+ is effective only when added to the cytoplasmic side of the channel. The results argue that this high-conductance, Ca2+-activated K+ channel contains at least six Ca2+-binding sites involved in the activation process.

A comparative study of the activation of the cholinergic receptor by various agonists

1983 ◽  
Vol 218 (1211) ◽  
pp. 241-251 ◽  
Keyword(s):  
Dose Response ◽  
Binding Sites ◽  
Nicotinic Receptor ◽  
Response Curve ◽  
Cholinergic Receptor ◽  
Hill Coefficient ◽  
Frog Neuromuscular Junction ◽  
Cholinergic Agonists ◽  
The Mean ◽  
The Hill

At the frog neuromuscular junction, a dose-response curve for the activation of the nicotinic receptor by carbachol has been determined under conditions where desensitization could be estimated and corrected for. The value of the Hill coefficient was 2 and that of the dissociation constant for carbachol was 400 μm, the two binding sites of the receptor being assumed identical. The properties of six cholinergic agonists were then compared. The potencies and mean open times of these agonists are ranked in the same order, but the range of the potencies is much larger (1–200) than that of the mean open times (1-4). The differences in the properties of the different agonists could simply be due to differences in the rate of dissociation of the agonist, if it is assumed that one apparent opening of the channel is in fact a burst of several oscillations between the open and closed conformations, such that a burst is interrupted by the dissociation of one agonist molecule.

scholarly journals Functional Characterization of a Novel ArgA from Mycobacterium tuberculosis

2005 ◽  
Vol 187 (9) ◽  
pp. 3039-3044 ◽  
Author(s):  
James C. Errey ◽  
John S. Blanchard
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Initial Velocity ◽  
Inhibitory Concentration ◽  
Functional Characterization ◽  
Initial Step ◽  
Kinetic Mechanism ◽  
Hill Coefficient ◽  
Arginine Biosynthesis ◽  
Inhibition Studies

ABSTRACT The Mycobacterium tuberculosis gene Rv2747 encodes a novel 19-kDa ArgA that catalyzes the initial step in l-arginine biosynthesis, namely the conversion of l-glutamate to α-N-acetyl-l-glutamate. Initial velocity studies reveal that Rv2747 proceeds through a sequential kinetic mechanism, with Km values of 280 mM for l-glutamine and 150 μM for acetyl-coenzyme A and with a k cat value of 200 min−1. Initial velocity studies with l-glutamate showed that even at concentrations of 600 mM, saturation was not observed. Therefore, only a k cat/Km value of 125 M−1 min−1 can be calculated. Inhibition studies reveal that the enzyme is strongly regulated by l-arginine, the end product of the pathway (50% inhibitory concentration, 26 μM). The enzyme was completely inhibited by 500 μM arginine, with a Hill coefficient of 0.60, indicating negatively cooperative binding of l-arginine.

scholarly journals The oxygen-linked zinc-binding site of human haemoglobin

1978 ◽  
Vol 169 (3) ◽  
pp. 625-632 ◽  
Author(s):  
J G Gilman ◽  
G J Brewer
Keyword(s):  
Binding Sites ◽  
Zinc Ion ◽  
Bohr Effect ◽  
Zinc Binding ◽  
Hill Coefficient ◽  
Human Haemoglobin ◽  
Equilibrium Binding ◽  
Zinc Binding Site ◽  
Alkaline Bohr Effect ◽  
The Hill

Zn2+ is known to increase the 02 affinity of human haemoglobin. Previous data suggested that Zn2+ exerts its effect by directly binding to haemoglobin, rather than by competing with or binding to 2,3-bisphosphoglycerate. It was also shown that there are two 02-linked zinc-binding sites in haemoglobin, and that Zn2+ does not significantly alter haemoglobin co-operativity or the alkaline Bohr effect. The effect of Zn2+ on 02 affinity of haemoglobin can also be observed for other haemoglobins as diverse as those of cow and chicken. This paper presents new data on the haemoglobin-zinc interaction for normal haemoglobin, des-His146beta-haemoglobin and N-ethylsuccinimide-haemoglobin of humans. For normal haemoglobin (0.05 mM in tetramers), at 20 degrees C in buffer containing 0.1 M-Cl-, 02-dissociation-curve experiments showed that the addition of 0.4-0.5 mM-ZnS04 did not change the Bohr effect between pH 6.71 and 7.29. Similar experiments, with “zinc-ion buffers”, showed that the value of the Hill coefficient, h, decreased only slightly if the concentration of free Zn2+ was held constant. For N-ethylsuccinimide-haemoglobin, Zn2+ caused less increase in O2 affinity than for normal haemoglobin. These studies, together with data on the equilibrium binding of Zn2+ to oxy-, deoxy- and des-His146beta-haemoglobins, suggest that zinc is chelated in oxyhaemoglobin by at least three amino acids, two of which are histidine-146beta and cysteine-93beta.

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.

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.

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