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 Fluorescence Studies on Glyceraldehyde- 3-phosphate Dehydrogenase from Bovine Heart Muscle

2001 ◽  
Vol 56 (11-12) ◽  
pp. 1166-1168 ◽  
Author(s):  
Ewa Seweryn ◽  
Teresa Banaś ◽  
Izabela Berdowska ◽  
Bogdan Zieliński ◽  
Ireneusz Ceremuga
Keyword(s):  
Fluorescence Quenching ◽  
Heart Muscle ◽  
Inhibitory Effect ◽  
Cooperative Binding ◽  
Fluorescence Technique ◽  
Fluorescence Studies ◽  
Bovine Heart ◽  
The Hill

Abstract Glyceraldehyde-3-phosphate Dehydrogenase, ATP, Flu­ orescence Glyceraldehyde-3-phosphate dehydrogenase is a gly­colytic enzyme that catalyses conversion of glyceralde-hyde-3-phosphate to 1,3-diphosphoglycerate. ATP has been found to have an inhibitory effect on this enzyme. To establish the interaction between the enzyme and ATP, a fluorescence technique was used. Fluorescence quenching in the presence of ATP suggests cooperative binding of ATP to the enzyme (the Hill obtained coeffi­cient equals 2.78). The interaction between glyceralde-hyde-3-phosphate dehydrogenase and ATP may control not only glycolysis but other activities of this enzyme, such as binding to the cytoskeleton.

scholarly journals Effects of thyroidectomy and starvation on the activity and properties of hepatic carnitine palmitoyltransferase

1982 ◽  
Vol 208 (3) ◽  
pp. 667-672 ◽  
Author(s):  
E D Saggerson ◽  
C A Carpenter ◽  
B S Tselentis
Keyword(s):  
Inhibitory Effect ◽  
Carnitine Palmitoyltransferase ◽  
Hill Coefficient ◽  
Fed State ◽  
Malonyl Coa ◽  
Normal States ◽  
Carnitine Palmitoyltransferase Activity ◽  
The Hill ◽  
Made In

1. Hepatic carnitine palmitoyltransferase activity was measured over a range of concentrations of palmitoyl-CoA and in the presence of several concentrations of the inhibitor malonyl-CoA. These measurements were made in mitochondria obtained from the livers of fed and starved (24 h) normal rats and of fed and starved thyroidectomized rats. 2. In the fed state thyroidectomy substantially decreased overt carnitine palmitoyltransferase activity and also decreased both the Hill coefficient and the s0.5 when palmitoyl-CoA concentration was varied as substrate. Thyroidectomy did not appreciably alter the inhibitory effect of malonyl-CoA on the enzyme. 3. Starvation increased overt carnitine palmitoyltransferase activity in both the fed and the thyroidectomized state. In percentage terms this response to starvation was substantially greater after thyroidectomy. In both the hypothyroid and normal states starvation decreased sensitivity to inhibition by malonyl-CoA.

scholarly journals Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro

1986 ◽  
Vol 240 (2) ◽  
pp. 529-539 ◽  
Author(s):  
U H Lerner ◽  
B B Fredholm ◽  
M Ransjö
Keyword(s):  
Bone Resorption ◽  
Cyclic Amp ◽  
Phosphodiesterase Inhibitor ◽  
Inhibitory Effect ◽  
Cyclic Amp Accumulation ◽  
The Relationship ◽  
Dose Dependent ◽  
Old Mice ◽  
45Ca Release

The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible increase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 mumol/l), enhanced the cyclic AMP response to forskolin (50 mumol/l) from a net cyclic AMP response of 1234 +/- 154 pmol/bone to 2854 +/- 193 pmol/bone (mean +/- S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 mumol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 mumol/l, in the absence and the presence of rolipram (30 mumol/l), caused a dose-dependent cyclic AMP accumulation with an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 mumol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentration producing half-maximal inhibition) values at 1.6 and 0.6 mumol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 mumol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxycholecalciferol (0.1 mumol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 mumol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 mumol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of High Concentrations of KC1 and NaCl on Responses of Malate Dehydrogenase (Decarboxylating) to Malate and Various Inhibitors

1974 ◽  
Vol 1 (1) ◽  
pp. 15 ◽  
Author(s):  
H Greenway ◽  
AP Sims
Keyword(s):  
Malate Dehydrogenase ◽  
Active Sites ◽  
Substrate Inhibition ◽  
Maximum Velocity ◽  
Inhibitory Effect ◽  
The Other ◽  
Appreciable Change ◽  
Other Hand ◽  
The Hill ◽  
Hill Number

The effects of KC1 on catalytic and allosteric properties of malate dehydrogenase (decarboxylating) were studied. Chloride salts (50 mM and higher) strongly inhibited enzyme activity at low malate concentrations. At high malate (4 mM) , on the other hand, the degree of inhibition induced by chloride was very small. The s0.5 value (i.e. the malate concentration required for half maximum velocity) was about 0.4 mM in the absence of KC1 and increased to 0.75 and 1.5 mM at 50 and 100 mM KC1 respectively. High chloride concentrations also removed a small degree of substrate inhibition, which in the absence of KC1 occurred at 8 mM malate. At low malate concentrations (< 0 . 5 mM) 50 mM KC1 increased the Hill number from 1.3 to 1.9. Thus chloride treatment revealed a strong degree of cooperativity for the enzyme. This potential for homotropic effects was much less realized in MES buffer (potassium salt), presumably because affinity of malate for individual active sites was already very high. These effects of KC1 were readily reversible and the enzyme showed no appreciable change in molecular weight in the presence of 200 mM KCl. At malate concentrations between 1 and 12 mM, inhibitions of malate dehydrogenase (decarboxylating) activity induced by oxaloacetate, D-malate, and phosphoglycerate were reduced by KCl. At low malate concentrations, on the other hand, the inhibitory effect induced by oxaloacetate changed to a strong stimulatory effect in the presence of KCl. The inhibitory effect due to oxaloacetate was greatly diminished in malate dehydrogenase (decarboxylating) which was eluted from a DE52 cellulose column, but the inhibitory effect due to KC1 was retained. Elution from DE52 cellulose also reduced the Hill number to 1 and increased the s0.5 value. The above results suggest that KC1 reduced the affinity of both substrate and some allosteric inhibitors of malate dehydrogenase (decarboxylating).

scholarly journals Modification of the acetylcholine-induced current of the snail Helix pomatia L. by fast temperature changes

2005 ◽  
Vol 57 (3) ◽  
pp. 181-187
Author(s):  
M. Nedeljkovic ◽  
Gordana Kartelija ◽  
Lidija Radenovic
Keyword(s):  
Helix Pomatia ◽  
Hill Coefficient ◽  
Temperature Changes ◽  
Inward Current ◽  
Electrode Voltage ◽  
Fast Cooling ◽  
Snail Helix Pomatia ◽  
The Hill ◽  
Dose Dependent ◽  
Font Color

Using the single electrode voltage clamp method, we found that acetylcholine (aCh) induces transient inward dose-dependent current on the membrane of the identified Helix pomatia Br neuron. We analyzed the effects of fast cooling and heating as well as thermal acclimation on the aCh inward current. the experiments were conducted on active and dormant snails acclimated to either 20 or 7?C for at least four weeks. the Hill coefficient remained approximately 1 in all cases, which means that there is a single aCh binding site on the membrane. Fast temperature alternations induce binding affinity changes. in the work presented, we analyzed the effects of cooling on the aCh-induced inward current. the amplitude of aCh-induced inward current was markedly reduced after cooling, and the speed of decay of the aCh response was lower. <br><br><font color="red"><b> This article has been retracted. Link to the retraction <u><a href="http://dx.doi.org/10.2298/ABS1501341E">10.2298/ABS1501341E</a><u></b></font>

scholarly journals The Effect of Hydrophobic Monoamines on Acid-Sensing Ion Channels ASIC1B

Acta Naturae ◽  
2015 ◽  
Vol 7 (2) ◽  
pp. 95-101 ◽  
Author(s):  
E. I. Nagaeva ◽  
N. N. Potapieva ◽  
D. B. Tikhonov
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Patch Clamp Technique ◽  
Recombinant Receptors ◽  
Whole Cell Patch Clamp ◽  
Acid Sensing Ion Channels ◽  
The Hill ◽  
Cooperative Activation

Acid-sensing ion channels (ASICs) are widely distributed in both the central and peripheral nervous systems of vertebrates. The pharmacology of these receptors remains poorly investigated, while the search for new ASIC modulators is very important. Recently, we found that some monoamines, which are blockers of NMDA receptors, inhibit and/or potentiate acid-sensing ion channels, depending on the subunit composition of the channels. The effect of 9-aminoacridine, IEM-1921, IEM-2117, and memantine both on native receptors and on recombinant ASIC1a, ASIC2a, and ASIC3 homomers was studied. In the present study, we have investigated the effect of these four compounds on homomeric ASIC1b channels. Experiments were performed on recombinant receptors expressed in CHO cells using the whole-cell patch clamp technique. Only two compounds, 9-aminoacridine and memantine, inhibited ASIC1b channels. IEM-1921 and IEM-2117 were inactive even at a 1000 M concentration. In most aspects, the effect of the compounds on ASIC1b was similar to their effect on ASIC1a. The distinguishing feature of homomeric ASIC1b channels is a steep activation-dependence, indicating cooperative activation by protons. In our experiments, the curve of the concentration dependence of ASIC1b inhibition by 9-aminoacridine also had a slope (Hill coefficient) of 3.8, unlike ASIC1a homomers, for which the Hill coefficient was close to 1. This finding indicates that the inhibitory effect of 9-aminoacridine is associated with changes in the activation properties of acid-sensing ion channels.

scholarly journals Sodium/calcium exchange in smooth-muscle microsomal fractions

1984 ◽  
Vol 218 (2) ◽  
pp. 421-427 ◽  
Author(s):  
N Morel ◽  
T Godfraind
Keyword(s):  
Smooth Muscle ◽  
Microsomal Fraction ◽  
Rat Aorta ◽  
Maximum Activity ◽  
Hill Coefficient ◽  
Ionophore A23187 ◽  
Half Maximum ◽  
Maximum Inhibition ◽  
Nacl Concentration ◽  
The Hill

The existence of Na+ -dependent Ca2+ transport was investigated in microsomal fractions from the longitudinal smooth muscle of the guinea-pig ileum and from the rat aorta, and its activity was compared with that of the plasmalemmal ATP-dependent Ca2+ pump previously identified in these preparations. The rate of Ca2+ release from plasmalemmal vesicles previously loaded with Ca2+ through the ATP-dependent Ca2+ pump was transiently faster in the presence of 150 mM-NaCl in the medium than in the presence of 150 mM-KCl or -LiCl or 300 mM-sucrose. Na+-loaded vesicles took up Ca2+ when an outwardly directed Na+ gradient was formed across the membrane. The Ca ionophore A23187 induced a rapid release of 85% of the sequestered Ca2+, whereas only 15% was displaced by La3+. Ca2+ accumulated by the Na+-induced Ca2+ transport was released by the addition of NaCl, but not KCl, to the medium. Ca2+ uptake in Na+-loaded vesicles was inhibited in the presence of increasing NaCl concentration in the medium. Half-maximum inhibition was observed with 28 mM-NaCl. Data fitted the Hill equation, with a Hill coefficient (h) of 1.9. Na+-induced Ca2+ uptake was a saturable function of Ca2+ concentration in the medium. Half-maximum activity was obtained with 18 microM-Ca2+ in intestinal-smooth-muscle microsomal fraction and with 50 microM-Ca2+ in aortic microsomal fraction. The results suggest that in these membrane preparations a transmembrane movement of Ca2+ can be driven by a Na+ gradient. However, the Na+-induced Ca2+ transport had a lower capacity, a lower affinity and a slower rate than the ATP-dependent Ca2+ pump.

Comparative effects of a low-carbohydrate diet and exercise plus a low-carbohydrate diet on muscle sarcoplasmic reticulum responses in males

2006 ◽  
Vol 291 (4) ◽  
pp. C607-C617 ◽  
Author(s):  
T. A. Duhamel ◽  
H. J. Green ◽  
J. G. Perco ◽  
J. Ouyang
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Phase 1 ◽  
Hill Coefficient ◽  
Carbohydrate Diet ◽  
Low Carbohydrate Diet ◽  
Diet And Exercise ◽  
Low Carbohydrate ◽  
The Hill ◽  
The Relationship

We employed a glycogen-depleting session of exercise followed by a low-carbohydrate (CHO) diet to investigate modifications that occur in muscle sarcoplasmic reticulum (SR) Ca2+-cycling properties compared with low-CHO diet alone. SR properties were assessed in nine untrained males [peak aerobic power (V̇o2 peak) = 43.6 ± 2.6 (SE) ml·kg−1·min−1] during prolonged cycle exercise to fatigue performed at ∼58% V̇o2 peak after 4 days of low-CHO diet (Lo CHO) and after glycogen-depleting exercise plus 4 days of low-CHO (Ex+Lo CHO). Compared with Lo CHO, Ex+Lo CHO resulted in 12% lower ( P < 0.05) resting maximal Ca2+-ATPase activity ( Vmax = 174 ± 12 vs. 153 ± 10 μmol·g protein−1·min−1) and smaller reduction in Vmax induced during exercise. A similar effect was observed for Ca2+ uptake. The Hill coefficient, defined as slope of the relationship between cytosolic free Ca2+ concentration and Ca2+-ATPase activity, was higher ( P < 0.05) at rest (2.07 ± 0.15 vs. 1.90 ± 0.10) with Ex+Lo CHO, an effect that persisted throughout the exercise. The coupling ratio, defined as the ratio of Ca2+ uptake to Vmax, was 23–30% elevated ( P < 0.05) at rest and during the first 60 min of exercise with Ex+Lo CHO. The ∼27 and 34% reductions ( P < 0.05) in phase 1 and phase 2 Ca2+ release, respectively, observed during exercise with Lo CHO were not altered by Ex+Lo CHO. These results indicate that when prolonged exercise precedes a short-term Lo CHO diet, Ca2+ sequestration properties and efficiency are improved compared with those during Lo CHO alone.

scholarly journals The Relationship Between Regulation of Prostaglandin Metabolism and Platelet Aggregation

1977 ◽  
Author(s):  
T. Tohjima ◽  
Y. Takahashi ◽  
H. Funayama ◽  
Y. Shiokawa
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Dextran Sulfate ◽  
Qualitative Difference ◽  
Inhibitory Effect ◽  
Platelet Suspension ◽  
The Relationship ◽  
Dose Dependent ◽  
Incubation Method

Platelet aggregation is mediated by prostaglandin (PG) endoperoxide and cAMP. But exogenous PGs might modify platelet function and might play a major role on the PG metabolism in platelet as well as endogenous PGs do. We studied the relationship between quantitative and qualitative regulation of PGs and cAMP and platelet aggregation, by incubation method adding exogenous PGs (PGE1, etc) and antiaggregating agents such as heparin, dextran sulfate, carbocromen, α-tocopherol, persein human platelet suspension. Platelet aggregation was studied photometrically adding thrombin. PGE1, E2, F1α and F2α were assayed by radioimmunoassay (RIA). cAMP was assayed by RIA. PG formation after incubation with the agents in platelet suspension was as follows; 1) PGF system was stimulated by PGE1 and PGE2. 2) PGE system was depressed by PGF2α. 3) PGF system was depressed by PGA1. 4) PGE1, stimulated cAMP and PGF system. 5)Aspirin, heparin, dextran sulfate and carbocromen had dose dependent inhibitory effect on thrombin induced platelet aggregation and had reducing effect on formation before adding thrombin. 6) Plenylamine and CDP-choline had inhibitory effect in vitro on platelet aggregation at higher concentrations than that of clinical use. CDP-choline reduced PGE levels but did not influence PGF levels. Prenylamine reduced PGF levels and stimulated cAMP formation at higher concentrations. 7)Dipyridamole and α-topcopherol did not reduce PGs levels. This approach provided the results that platelet function was regulated by PG metabolism which is modified by exogenous PGs or some drugs. Antiaggregating agents can be classified by the quantitative and qualitative difference of PG formation.

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.

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