scholarly journals Interaction of Mg2+ with F0.F1 mitochondrial ATPase as related to its slow active/inactive transition

1991 ◽  
Vol 276 (1) ◽  
pp. 149-156 ◽  
Author(s):  
V V Bulygin ◽  
A D Vinogradov
Keyword(s):  
Atpase Activity ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Order Rate Constant ◽  
Mitochondrial Atpase ◽  
Sephadex Column ◽  
Specific Binding Sites ◽  
Mitochondrial Atp Synthase ◽  
Inhibitory Site ◽  
Activation Rates

Bovine heart submitochondrial particles incubated with a low concentration of ADP in the presence of Mg2+ and passed through a Sephadex column equilibrated with EDTA exhibit sensitivity of their initial ATPase activity to preincubation with Mg2+. By using particles thus prepared, several characteristics of a Mg(2+)-specific inhibitory site on F0.F1 ATPase were studied. The inhibition was shown to be both time- and Mg(2+)-concentration-dependent, with an equilibrium constant (at infinite time) of 2 x 10(-6) M (25 degrees C, pH 7.5). The dependence of the pseudo-first-order rate constant for the inhibition process on Mg2+ concentration suggests the presence of a single Mg(2+)-binding site with K8 = 1.1 x 10(-4) M. The data obtained are consistent with a two-step mechanism of Mg(2+)-F0.F1 interaction which results in a loss of the ATPase activity; it includes rapid pH-dependent binding of Mg2+ at the site with K8 = 1.1 x 10(-4) M, followed by a slow interconversion of the Mg(2+)-F1 complex into inactive ATPase (kin. = 0.65 min-1, kact. = 0.01 min-1). The Mg(2+)-inhibited ATPase is very slowly (t1/2 approximately 90 min) re-activated in the presence of EDTA. The rate of EDTA-induced re-activation is pH-independent and can be dramatically increased by added ATP, Pi and sulphite. The dissociation constants for free ATP and P1 (5 x 10(-7) M and 1 x 10(-3) M respectively) and the maximal activation rates were determined by measuring the hyperbolic dependencies of the EDTA-induced re-activation of Mg(2+)-de-activated ATPase on the concentrations of the accelerating ligands. Taken together, the data obtained show two functionally detectable free nucleotide-specific binding sites, one site for Pi and one Mg(2+)-specific ATPase-inhibitory site on the F0.F1 mitochondrial ATP synthase complex.

scholarly journals Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles

1980 ◽  
Vol 188 (3) ◽  
pp. 807-815 ◽  
Author(s):  
E A Vasilyeva ◽  
A F Fitin ◽  
I B Minkov ◽  
A D Vinogradov
Keyword(s):  
Atpase Activity ◽  
Rate Constant ◽  
Adenosine Diphosphate ◽  
Mitochondrial Atpase ◽  
Dependent Manner ◽  
Submitochondrial Particles ◽  
Alternative Pathways ◽  
Atpase Reaction ◽  
Inhibitory Site ◽  
Km Value

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.

Leukotriene D4 binding and signal transduction in rat glomerular mesangial cells

1989 ◽  
Vol 257 (2) ◽  
pp. F280-F287 ◽  
Author(s):  
K. F. Badr ◽  
S. Mong ◽  
R. L. Hoover ◽  
M. Schwartzberg ◽  
J. Ebert ◽  
...  
Keyword(s):  
Binding Sites ◽  
Radioligand Binding ◽  
Mesangial Cells ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Rank Order ◽  
Glomerular Mesangial Cells ◽  
Leukotriene D4 ◽  
Specific Binding Sites ◽  
Dose Dependent

We examined the characteristics of [3H]leukotriene D4 (LTD4) binding to mesangial cells in culture. Binding is stereoselective, specific, saturable, and rapidly reversible. Two binding sites are recognized with dissociation constants and binding site densities at equilibrium of 2.2 and 16.8 nM and 1.1 x 10(4) and 3 x 10(4) binding sites per cell. LTD4, LTE4, (5R,6S)LTD4, LTB4, and the LTD4-receptor antagonist, SKF 104353, competitively inhibit radioligand binding in the following rank order of potency: LTD4 greater than LTE4 = SKF 104353 greater than (5R,6S)LTD4 greater than LTB4. LTD4 also induces time- and concentration-dependent phosphoinositide hydrolysis in mesangial cells. Formation of inositol 1,4,5-trisphosphate (IP3) is maximal at 5 s, followed by a time-dependent increase in inositol monophosphate generation, and inhibited by 100-fold excess concentration of SKF 104353. Addition of LTD4 to mesangial cells is associated with an increase in intracellular pH and dose-dependent stimulation of [3H]thymidine incorporation and mitogenesis. Thus rat mesangial cells possess specific binding sites for LTD4, the activation of which stimulates IP3 formation and induces cellular alkalinization and mitogenic responses. These studies provide insight into the cellular basis for LTD4-mesangial cell interactions, which are of potential pathophysiological relevance during acute glomerular inflammatory injury.

scholarly journals Allosteric Modulation of Haemocyanin Oxygen-affinity by L-Lactate And Urate in the Lobster Homarus Vulgaris: II. Characterization of Specific Effector Binding Sites

1992 ◽  
Vol 168 (1) ◽  
pp. 111-124 ◽  
Author(s):  
A. NIES ◽  
B. ZEIS ◽  
C. R. BRIDGES ◽  
M. K. GRIESHABER
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Oxygen Affinity ◽  
Allosteric Modulators ◽  
Specific Binding Sites ◽  
Ligand Binding Sites ◽  
Specific Effector ◽  
Purine Ring

The haemocyanin of Homarus vulgaris possesses specific binding sites for L-lactate and urate, two allosteric modulators of haemocyanin oxygen-affinity. The affinities for both ligands have been determined. The dissociation constants, KD, are 0.87±0.26mmoll−1 for L-lactate and 0.03±0.01mmoll−1 for urate at 15°C and pH 8.0. The affinity of the haemocyanin is about 40 times larger for urate than for L-lactate. The stoichiometry of the binding is two ligands per dodecamer in both cases. l-Lactate does not compete with urate for its binding site and vice versa, indicating that the ligand binding sites are independent of each other. The specificity of urate binding to haemocyanin was investigated in competition experiments with allantoin, caffeine and hypoxanthine. The purine derivatives caffeine and hypoxanthine reduce the binding of urate to haemocyanin, whereas allantoin has no effect. Thus, the purine ring system seems to be essential for the binding of urate to haemocyanin. Note: To whom reprint requests should be addressed.

Relationship between potency of L-isoprenaline and β-adrenoceptor density estimated in single cells from tracheal smooth muscles of guinea pigs of different ages

1992 ◽  
Vol 70 (4) ◽  
pp. 458-461 ◽  
Author(s):  
Issei Takayanagi ◽  
Mitsutoshi Satoh ◽  
Noriko Kokubu ◽  
Teruko Kato
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Single Cells ◽  
Regression Line ◽  
Smooth Muscles ◽  
Receptor Density ◽  
Age Related ◽  
Β Receptor

An age-related change in potency of L-isoprenaline in the presence of ascorbic acid, desmethylimipramine, corticosterone, pargyline, and phentolamine was obtained in tracheal strips from guinea pigs of differing ages between 6 and 40 weeks. The potency in the strips from 100-week-old guinea pigs did not significantly differ from that in strips from 40-week-old animals. Single cells were prepared from the tracheal muscles of 6-, 10-, 40-, and 100-week-old guinea pigs. The specific binding of [3H]dihydroalprenolol to the single cells was saturable. The dissociation constants of [3H]dihydroalprenolol were in good agreement with those of the membrane fractions from the guinea-pig tracheal muscles, and did not change with age. An excellent relationship between the potency of L-isoprenaline and the maximum binding of [3H]dihydroalprenolol estimated in the preparations from 6- to 40-week-old guinea pigs was found, suggesting that the increase in the potency of L-isoprenaline is due to the increase in the maximum binding or receptor density. The value in the preparations from 100-week-old guinea pigs deviated significantly from the regression line. This suggests the possibility that the decrease in potency in the strips from 100-week-old animals is due to a change in post β-receptor processes in responsiveness.Key words: guinea-pig trachea, single cells, β-receptor density, ageing, dissociation constant.

scholarly journals Specific Binding of Rubidium in Chlorella

1962 ◽  
Vol 45 (5) ◽  
pp. 959-977 ◽  
Author(s):  
Dan Cohen
Keyword(s):  
Active Transport ◽  
Binding Sites ◽  
Specific Binding ◽  
High Concentration ◽  
External Concentration ◽  
Specific Binding Sites ◽  
Dense Suspension ◽  
Concentration Of Ions ◽  
The Individual ◽  
A Site

Specific binding sites for potassium, which may be components of the carriers for active transport for K in Chlorella, were characterized by their capacity to bind rubidium. A dense suspension was allowed to take up Rb86 from a low concentration of Rb86 and a high concentration of ions which saturate non-specific sites. The amount bound was derived from the increase in the external concentration of Rb86 following addition of excess potassium. The sites were heterogeneous. The average affinity of Rb and various other ions for the sites was determined by plotting the degree of displacement of Rb86 against log molar concentration of the individual ions. Interpolation gave the concentration for 50 per cent displacement of Rb, which is inversely related to affinity. The order of affinity was not changed when the cells were frozen, or boiled either in water or in 70 per cent ethanol. The affinity is maximal for ions with a crystalline radius of 1.3 to 1.5 A and a high polarizability, and is not related to the hydrated radius or valency. It is suggested that binding groups in a site are rigidly arranged, the irregular space between them being 2.6 to 3.0 A across, so that affinity is high for ions of this diameter and high polarizability.

Inhibition of muscle actomyosin ATPase activity by mitochondrial ATPase inhibitor

1977 ◽  
Vol 75 (4) ◽  
pp. 1104-1110 ◽  
Author(s):  
Shojiro Yamazaki ◽  
Hiroshi Hasebe ◽  
Haruhiko Takisawa ◽  
Yutaka Tamaura ◽  
Yuji Inada
Keyword(s):  
Atpase Activity ◽  
Mitochondrial Atpase ◽  
Atpase Inhibitor ◽  
Actomyosin Atpase

Studies of the uptake of macromolecules by cells. I. Uptake of proteins by cells of the Ehrlich–Lettré ascites carcinoma

1968 ◽  
Vol 46 (12) ◽  
pp. 1443-1450 ◽  
Author(s):  
Y. C. Choi ◽  
E. R. M. Kay
Keyword(s):  
Nucleic Acid ◽  
Cell Membrane ◽  
Binding Sites ◽  
Specific Binding ◽  
Protein Uptake ◽  
Specific Binding Sites ◽  
Model Protein ◽  
Uptake Process ◽  
Kinetics Of ◽  
Protein Treatment

The uptake of protein by cells of the Ehrlich–Lettré ascites carcinoma was characterized kinetically by using hemoglobin as a model protein. An attempt was made to show that the process is not an artefact due to nonspecific adsorption of protein to the cell membrane. The kinetics of the uptake process suggested that an interaction exists between the exogenous protein and specific binding sites on the membrane. Acetylation of hemoglobin enhanced the rate of uptake of this protein. Treatment of cells with neuraminidase, phospholipase A, and Pronase resulted in an inhibition of protein uptake. The experimental evidence for the uptake of hemoglobin was supported by evidence that L-serine-U-14C-labelled hemoglobin is transported into the cytoplasm and utilized subsequently, resulting in labelling of the nucleic acid nucleotides.

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