scholarly journals The allosteric properties of rat liver fructose-1,6-bisphosphatase

1984 ◽  
Vol 222 (1) ◽  
pp. 131-138 ◽  
Author(s):  
D W Meek ◽  
H G Nimmo
Keyword(s):  
Rat Liver ◽  
Opposite Effect ◽  
Catalytic Site ◽  
Hill Coefficient ◽  
Allosteric Site ◽  
Fructose 1,6 Bisphosphatase ◽  
Three State Model ◽  
Fructose 1,6 Bisphosphate ◽  
The Hill ◽  
Allosteric Properties

Inhibition of rat liver fructose-1,6-bisphosphatase by AMP was uncompetitive with respect to fructose 1,6-bisphosphate in the absence of fructose 2,6-bisphosphate, but non-competitive in its presence. AMP was unable to bind to the enzyme except in the presence of one of the fructose bisphosphates; the binding stoicheiometry was 2 molecules/tetramer. Increasing concentrations of Mg2+ increased the Hill coefficient h and the apparent Ki for AMP, whereas fructose 2,6-bisphosphate had the opposite effect. Increasing concentrations of both AMP and fructose 2,6-bisphosphate decreased h and increased the apparent Ka for Mg2+. AMP slightly decreased, and Mg2+ slightly increased, the apparent Ki for fructose 2,6-bisphosphate, but each had only small effects on h. These results are interpreted in terms of a new three-state model for the allosteric properties of the enzyme, in which fructose 2,6-bisphosphate can bind both to the catalytic site and to an allosteric site and AMP can bind to the enzyme only when the catalytic site is occupied.

scholarly journals Effects of phosphorylation on the kinetic properties of rat liver fructose-1,6-bisphosphatase

1984 ◽  
Vol 222 (1) ◽  
pp. 125-130 ◽  
Author(s):  
D W Meek ◽  
H G Nimmo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Kinetic Properties ◽  
Purification Procedure ◽  
Dependent Protein Kinase ◽  
Fructose 1,6 Bisphosphatase ◽  
Dependent Protein ◽  
A Subunit ◽  
Fructose 1,6 Bisphosphate

A new purification procedure for rat liver fructose-1,6-bisphosphatase that involves use of Procion Red-Sepharose is described. The purified enzyme was homogeneous, had a subunit Mr of 40 000-41 000 and seemed to be undegraded. The enzyme could be phosphorylated by cyclic AMP-dependent protein kinase with a stoicheiometry of one per subunit. Phosphorylation caused a 2-fold decrease in the Km of the enzyme for fructose 1,6-bisphosphate, but did not affect its allosteric responses to AMP, Mg2+ and fructose 2,6-bisphosphate.

Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

1989 ◽  
Vol 67 (1) ◽  
pp. 16-24 ◽  
Author(s):  
George Deliconstantinos ◽  
Lioudmila Kopeikina ◽  
Vassiliki Villiotou
Keyword(s):  
Fluorescence Polarization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Break Point ◽  
Hill Coefficient ◽  
Dog Brain ◽  
Integral Proteins ◽  
The Hill ◽  
Synaptosomal Plasma Membranes ◽  
Allosteric Properties

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5–5 μM) follows an exponential curve described by the general formula y = a∙ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 μM cholesterol glucoside, with a Hill coefficient of h = 2.98 ± 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 μM) or its glucoside (up to 50 μM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 μM) incorporation. Cholesterol (5 μM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 ± 1.1 °C in control SPM, which was elevated to 29.5 ± 1.4 °C in SPM treated with cholesterol glucoside (50 μM) and abolished in SPM treated with cholesterol (5 μM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F− and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 μM) produced an increased packing of the bulk lipids, while cholesterol (5 μM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.Key words: cholesterol, cholesterol glucoside, (NA+/K+)ATPase, Ca2+-ATPase, membrane fluidity, fluorescence polarization.

scholarly journals A subunit interface mutant of yeast pyruvate kinase requires the allosteric activator fructose 1,6-bisphosphate for activity

1995 ◽  
Vol 310 (1) ◽  
pp. 117-123 ◽  
Author(s):  
R A Collins ◽  
T McNally ◽  
L A Fothergill-Gilmore ◽  
H Muirhead
Keyword(s):  
Pyruvate Kinase ◽  
Mutant Enzyme ◽  
Hill Coefficient ◽  
Kinetic Properties ◽  
Variant Form ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Kinase Gene ◽  
Fructose 1,6 Bisphosphate ◽  
Allosteric Properties

A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mutated to proline has been engineered in order to study the allosteric properties of this enzyme. Both the mutant and wild-type enzymes were overexpressed in a strain of yeast in which the genomic copy of the pyruvate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effect fructose 1,6-bisphosphate with concomitant reduction in co-operativity. In contrast, the mutant was found to be dependent on the presence of the effector for catalytic activity and was inactive in its absence. The fully activated mutant enzyme had a kcat. 1.6 times greater than that of the wild-type enzyme. The mutation introduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although kcat. is also affected. The S0.5 for PEP and S0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic parameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0.07 in the presence of the effector, compared with a value of 0.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectroscopy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiary and quaternary structures such as intersubunit and interdomain interactions may be modified. An improved purification procedure has been devised that allows large quantities of enzyme to be rapidly prepared.

scholarly journals Inosine 5′-monophosphate dehydrogenase of Escherichia coli K12: the nature of the inhibition by guanosine 5′-monophosphate (Short Communication)

1973 ◽  
Vol 133 (3) ◽  
pp. 607-608 ◽  
Author(s):  
Paul R. Lambden ◽  
William T. Drabble
Keyword(s):  
Escherichia Coli ◽  
Short Communication ◽  
Hill Coefficient ◽  
Imp Dehydrogenase ◽  
Escherichia Coli K12 ◽  
Non Linear ◽  
The Hill ◽  
Variable Substrate ◽  
Allosteric Properties ◽  
Monophosphate Dehydrogenase

When IMP is the variable substrate, IMP dehydrogenase (EC 1.2.1.14) gives non-linear Lineweaver–Burk plots in the presence of GMP. At 0.1mm-GMP the Hill coefficient n=1.15 and at 0.2mm-GMP n=1.34. In the absence of GMP n=1.0. The fact that n exceeds 1.0 in the presence of GMP indicates that the enzyme possesses regulatory (allosteric) properties. The inhibition by GMP is competitive with respect to IMP and non-competitive with respect to NAD+.

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).

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.

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