scholarly journals The sodium-plus-potassium ion-activated adenosine triphosphatase of cerebral microsomal fractions: treatment with disrupting agents

1967 ◽  
Vol 102 (3) ◽  
pp. 675-683 ◽  
Author(s):  
J. R. Cooper ◽  
H. Mcilwain
Keyword(s):  
Atpase Activity ◽  
Protective Effect ◽  
Ammonium Sulphate ◽  
Ultrasonic Treatment ◽  
Adenosine Triphosphatase ◽  
Microsomal Fraction ◽  
Centrifugal Forces ◽  
Microsomal Preparation ◽  
Fold Enrichment ◽  
Low Concentrations

1. The Na(+)-plus-K(+)-stimulated adenosine triphosphatase [(Na(+),K(+))-ATPase] of microsomal preparations from ox brain was inactivated or diminished in activity by exposure to 2-8m-urea. Similar concentrations of urea diminished the turbidity of the suspensions. 2. Low concentrations (about 2.5mm) of NaATP with the urea gave partial or complete protection of the ATPase, without altering the concomitant change in turbidity. Some protection of the (Na(+),K(+))-ATPase was afforded by tris ATP, but the greatest protection was found with NaATP and in its presence the change in (Na(+),K(+))-ATPase with 3m-urea included a phase in which activity was enhanced by 40%. 3. The protective effect was specific to NaATP: KATP, NaADP, NaAMP and sodium pyrophosphate were without protective effect and in some cases they augmented the action of urea. 4. The turbidity of cerebral microsomal suspensions was diminished also by ultrasonic irradiation; NaATP did not alter this change. After ultrasonic treatment up to 55% of the protein and of the ATPase activity were no longer deposited by centrifugal forces of 4.5x10(6)g-min. 5. Ultrasonic treatment and centrifugation could be carried out with little or no loss of ATPase and ammonium sulphate flocculation of the supernatant then afforded in the first material precipitated a three- to five-fold enrichment of (Na(+),K(+))-ATPase activity. 6. Sodium borohydride and dimethyl sulphoxide also diminished the turbidity of the microsomal fraction but enrichment of the ATPase was not effected by these reagents; ten other compounds were without action on the ATPase. 7. Acetyl phosphate was hydrolysed by the microsomal preparation and this activity was increased by added K(+). Acetyl-phosphatase activity persisted in the ultrasonically treated and ammonium sulphate-fractionated preparations, which were more exacting in their requirements for K(+). 8. The findings are discussed in relation to the mechanism of the (Na(+),K(+))-ATPase.

scholarly journals The location of the thiol groups of myosin that are protected by pyrophosphate against reaction with 2,4-dinitrophenyl β-hydroxyethyl disulphide

1971 ◽  
Vol 125 (1) ◽  
pp. 261-266 ◽  
Author(s):  
Irena Kakol
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Adenosine Triphosphatase ◽  
Thiol Groups ◽  
Low Concentrations ◽  
Light Components

Myosin modified in the presence or in the absence of pyrophosphate by 2,4-dinitrophenyl β-hydroxyethyl disulphide was treated with iodo[1-14C]acetamide. The residual Ca2+-stimulated adenosine triphosphatase (ATPase) activity of the modified myosin was different depending on the presence or absence of PPi during modification and the number of 2,4-dinitrophenyl β-hydroxyethyl disulphide-modified thiol groups. The radioactivity incorporated into the light components of myosin correlated with the Ca2+-stimulated ATPase activity of the modified myosin and decreased with decreasing residual Ca2+-stimulated ATPase activity of the modified myosin. When native myosin was treated with low concentrations of iodo[1-14C]acetamide the residual Ca2+-stimulated ATPase activity of carboxyamidomethylated myosin was high and the radioactivity incorporated into the light components of myosin was negligible. The thiol groups of the light components of myosin are essential to preserve the ATPase activity of the protein and are close to the pyrophosphate-binding sites.

scholarly journals Inhibition of adenosine triphosphatase, 5-hydroxytryptamine transport and proton-translocation activities of resealed chromaffin-granule ‘ghosts’

1980 ◽  
Vol 190 (2) ◽  
pp. 273-282 ◽  
Author(s):  
David K. Apps ◽  
James G. Pryde ◽  
Raul Sutton ◽  
John H. Phillips
Keyword(s):  
Molecular Weight ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Proton Translocation ◽  
Low Molecular Weight ◽  
Chromaffin Granule ◽  
Low Concentrations ◽  
Tributyltin Chloride ◽  
Granule Membrane ◽  
Chromaffin Granule Ghosts

1. Highly purified resealed chromaffin-granule ‘ghosts’ were assayed for ATPase and ATP-driven H+-translocation and 5-hydroxytryptamine-uptake activities, and for 5-hydroxytryptamine uptake driven by an imposed transmembrane H+-gradient. The effects of several inhibitors on these activities were studied. 2. Dicyclohexylcarbodi-imide inhibits all of these activities, but not in parallel; at low concentrations it decreases the permeability of the membrane to protons. 3. 4-Chloro-7-nitrobenzofuran (Nbf-Cl) and silicotungstate inhibit ATP-dependent activities, without effect on 5-hydroxytryptamine uptake driven by an imposed H+-gradient. 4. Tributyltin chloride inhibits all of the activities. 5. Treatment of the ‘ghosts’ with low concentrations of urea inhibits 5-hydroxytryptamine uptake and ATP-dependent generation of a transmembrane H+-gradient, without inhibiting ATPase activity. 6. Nbf-Cl and silicotungstate are without effect on the rate of leakage of 5-hydroxytryptamine from preloaded ‘ghosts’, whereas dicyclohexylcarbodi-imide and tributyltin chloride accelerate the rate of leakage. 7. Treatment of the membranes with 14C-labelled Nbf-Cl labels several proteins; membranes treated with dicyclohexyl[14C]carbodi-imide are labelled predominantly in a protein of low molecular weight, which may be analogous to the mitochondrial H+-conducting proteolipid. 8. It is concluded that Nbf-Cl and silicotungstate inhibit the H+-translocating ATPase of the granule membrane; that dicyclohexylcarbodi-imide inhibits the ATPase, and inhibits 5-hydroxytryptamine accumulation by accelerating leakage of the amine; and that the effects of tributyltin chloride are due to inhibition of the ATPase, and collapse of the transmembrane H+-gradient through OH−-anion exchange.

scholarly journals Comparison of some minor activities accompanying a preparation of sodium-plus-potassium ion-stimulated adenosine triphosphatase from pig brain

1968 ◽  
Vol 106 (1) ◽  
pp. 113-121 ◽  
Author(s):  
M. Fujita ◽  
K. Nagano ◽  
N. Mizuno ◽  
Y. Tashima ◽  
T. Nakao ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Atpase Activity ◽  
Ultrasonic Treatment ◽  
Adenosine Triphosphatase ◽  
Enzyme Preparation ◽  
Sodium Iodide ◽  
Low Ph ◽  
Pig Brain ◽  
Brain Microsomes ◽  
Decreasing Ph

1. An ATPase (adenosine triphosphatase) preparation obtained from pig brain microsomes by treatment with sodium iodide showed four apparently different ouabain-sensitive activities under various conditions. They were (a) ouabain-sensitive Mg2+-stimulated ATPase, (b) K+-stimulated ATPase, (c) (Na+,K+)-stimulated ATPase and (d) Na+-stimulated ATPase activities. 2. These activities showed the same substrate specificity, ATP being preferentially hydrolysed and CTP slightly. AMP was not hydrolysed. 3. These activities were inhibited by low concentration of ouabain. The concentration producing 50% inhibition was 0·1μm for ouabain-sensitive Mg2+-stimulated ATPase, 0·2μm for K+-stimulated ATPase, 0·1μm for (Na+,K+)-stimulated ATPase and 0·003μm for Na+-stimulated ATPase activity. 4. The ouabain-sensitive ATPase activities were inactivated by N-ethylmaleimide but the insensitive ATPase activity was not. 5. The three ouabain-sensitive ATPase activities were inhibited about 50% by 1mm-Ca2+, whereas the ouabain-sensitive Mg2+-stimulated ATPase activity was activated by the same concentration of Ca2+. The preparation was treated with ultrasonics at 20kcyc./sec. The 2min. ultrasonic treatment inactivated the ATPase activities by 50%. 7. The temperature coefficient Q10 was 6·6 for K+-stimulated ATPase activity, 3·7 for (Na+,K+)-stimulated ATPase and 2·6 for Na+-stimulated ATPase. 8. Organic solvents inactivated the ATPase activities, to which treatment the K+-stimulated ATPase was the most resistant. 9. The phosphorylation of the enzyme preparation became less dependent on Na+ with decreasing pH. This Na+-independent phosphorylation at low pH was sensitive to K+ and hydroxylamine as well as the Na+-dependent phosphorylation at neutral pH.

scholarly journals Isolation and partial characterization of magnesium ion-and calcium ion-dependent adenosine triphosphatase activity from bovine brain microsomal fraction

1979 ◽  
Vol 181 (2) ◽  
pp. 321-330 ◽  
Author(s):  
Torben Særmark ◽  
Hans Vilhardt
Keyword(s):  
Atpase Activity ◽  
Kinetic Analysis ◽  
Adenosine Triphosphatase ◽  
Microsomal Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Ph Gradients ◽  
Adenosine Triphosphatase Activity ◽  
Km Value

Microsomal fraction was prepared by ultracentrifugation of homogenates of cortical tissue from bovine brains. The preparation displayed ATPase (adenosine triphosphatase) activity in the presence of Mg2+ (6.4μmol of Pi/h per mg of protein) and Ca2+ (3.4μmol of Pi/h per mg of protein). Kinetic analysis of the activation of the enzyme preparation by Ca2+ resulted in the demonstration of two apparent Km values for Ca2+ (6.0×10−8m and 1.2×10−6m). Treatment of the microsomal membranes with Triton X-100 resulted in solubilization of the ATPase, though with some loss of activity. The solubilized microsomal proteins were incorporated into liposomes. By incubation of the liposomes in media containing 45Ca2+ an ATP-dependent uptake of Ca2+ was demonstrated. The solubilized preparation was subjected to preparative isoelectric focusing in granulated gel beds. Two distinct peaks of Mg2+- and Ca2+-dependent ATPase activity were observed at pH4.8 (peak 4.8) and at pH6.3 (peak 6.3). The material isolated in peaks 4.8 and 6.3 was focused in polyacrylamide gel with pH gradients. The material corresponding to peak 4.8 consisted of a single protein, whereas peak 6.3 contained one major and at least one minor protein. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis confirmed these results and indicated that the major component of peak 4.8 and the protein of peak 6.3 both had a molecular weight of 105000. The material in peaks 4.8 and 6.3 was assayed for ATPase activity in the presence of various concentrations of Ca2+. Kinetic analysis of the results for peak 4.8 demonstrated an apparent Km value for Ca2+ of 4.1×10−8m. The enzyme isolated at pH6.3 had an apparent Km value of 3.8×10−6m. However, when the material from peak 4.8 was incubated in the presence of 1mm-Mg2+ the ATPase could not be activated by Ca2+.

Localization of Adenosine Triphosphatase Activity in the Phleom of Nicotiana Tabacum

1972 ◽  
Vol 30 ◽  
pp. 230-231
Author(s):  
James Cronshaw ◽  
Jamison E. Gilder
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Higher Plants ◽  
High Surface ◽  
Root Tip ◽  
Animal Cells ◽  
Physiological Processes ◽  
Tip Cells ◽  
Atpase Localization

Adenosine triphosphatase (ATPase) activity has been shown to be associated with numerous physiological processes in both plants and animal cells. Biochemical studies have shown that in higher plants ATPase activity is high in cell wall preparations and is associated with the plasma membrane, nuclei, mitochondria, chloroplasts and lysosomes. However, there have been only a few ATPase localization studies of higher plants at the electron microscope level. Poux (1967) demonstrated ATPase activity associated with most cellular organelles in the protoderm cells of Cucumis roots. Hall (1971) has demonstrated ATPase activity in root tip cells of Zea mays. There was high surface activity largely associated with the plasma membrane and plasmodesmata. ATPase activity was also demonstrated in mitochondria, dictyosomes, endoplasmic reticulum and plastids.

scholarly journals Effect of Pseudomonas moorei KB4 Cells’ Immobilisation on Their Degradation Potential and Tolerance towards Paracetamol

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 820
Author(s):  
Robert Surma ◽  
Danuta Wojcieszyńska ◽  
Jagna Karcz ◽  
Urszula Guzik
Keyword(s):  
Protective Effect ◽  
Kinetic Parameters ◽  
Toxicity Assessment ◽  
Bacterial Cells ◽  
Low Concentrations ◽  
Toxicity Analysis ◽  
Immobilised Cells ◽  
Degradation Activity ◽  
High Concentrations ◽  
The Hill

Pseudomonas moorei KB4 is capable of degrading paracetamol, but high concentrations of this drug may cause an accumulation of toxic metabolites. It is known that immobilisation can have a protective effect on bacterial cells; therefore, the toxicity and degradation rate of paracetamol by the immobilised strain KB4 were assessed. Strain KB4 was immobilised on a plant sponge. A toxicity assessment was performed by measuring the concentration of ATP using the colony-forming unit (CFU) method. The kinetic parameters of paracetamol degradation were estimated using the Hill equation. Toxicity analysis showed a protective effect of the carrier at low concentrations of paracetamol. Moreover, a pronounced phenomenon of hormesis was observed in the immobilised systems. The obtained kinetic parameters and the course of the kinetic curves clearly indicate a decrease in the degradation activity of cells after their immobilisation. There was a delay in degradation in the systems with free cells without glucose and immobilised cells with glucose. However, it was demonstrated that the immobilised systems can degrade at least ten succeeding cycles of 20 mg/L paracetamol degradation. The obtained results indicate that the immobilised strain may become a useful tool in the process of paracetamol degradation.

scholarly journals No major thermogenic role for (Na+ + K+)-dependent adenosine triphosphatase apparent in hepatocytes from hyperthyroid rats

1982 ◽  
Vol 202 (3) ◽  
pp. 661-665 ◽  
Author(s):  
D G Clark ◽  
M Brinkman ◽  
O H Filsell ◽  
S J Lewis ◽  
M N Berry
Keyword(s):  
Oxygen Consumption ◽  
Oxygen Uptake ◽  
Atpase Activity ◽  
Heat Production ◽  
Adenosine Triphosphatase ◽  
Heat Output ◽  
Liver Parenchymal ◽  
Dependent Atpase ◽  
Parenchymal Cells ◽  
Isolated Liver

(Na+ + K+)-dependent ATPase activity, heat production and oxygen consumption were increased by 59%, 62% and 75% respectively in hepatocytes from tri-iodothyronine-treated rats. Ouabain at concentrations of 1 and 10 mM decreased oxygen uptake by 2-8% in hepatocytes from euthyroid rats and by 5-15% in hepatocytes from hyperthyroid animals. Heat output was decreased by 4-9% with the glycoside in isolated liver parenchymal cells from the control animals and by 11% in the cells from the tri-iodothyronine-treated animals. These results do not support the hypothesis that hepatic (Na+ + K+)-ATPase plays a major role in increased heat production in hepatocytes from hyperthyroid rats.

scholarly journals The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1970 ◽  
Vol 120 (1) ◽  
pp. 15-24 ◽  
Author(s):  
P. S. G. Goldfarb ◽  
R. Rodnight
Keyword(s):  
Potassium Chloride ◽  
Magnesium Chloride ◽  
Adenosine Triphosphatase ◽  
Time Course ◽  
Atp Hydrolysis ◽  
Protein Ratio ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Emission Flame

1. The intrinsic Na+, K+, Mg2+ and Ca2+ contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na+ from 90±20 to 24±12, the bound K+ from 27±3 to 7±2, the bound Mg2+ from 20±2 to 3±1 and the bound calcium from 8±1 to <1nmol/mg of protein. 3. The activities of the Na++K++Mg2+-stimulated adenosine triphosphatase and the Na+-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5μm (ATP/protein ratio 12.5pmol/μg). 4. The Na+-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5μm-magnesium chloride and 2μm-potassium chloride. Addition of 2.5μm-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na+-dependent ATP hydrolysis was partly restored with 2.5μm-magnesium chloride; addition of K+ in the range 2–10μm-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0°C with 0.5nmol of K+/mg of protein so that the final added K+ in the reaction mixture was 0.1μm restored the Na+-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [42K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K+/mg of protein was linear over a period of 20min and was inhibited by Na+. Half-maximal inhibition of 42K+-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na+-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K+ and Mg2+ of the Na++K++Mg2+-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K+ from a solution of 0.5μm-potassium chloride.

Effect of high salt intake on sodium, potassium-dependent adenosine triphosphatase activity in the erythrocytes of normotensive men

1988 ◽  
Vol 75 (2) ◽  
pp. 167-170 ◽  
Author(s):  
Antonio P. Quintanilla ◽  
Maria I. Weffer ◽  
Haengil Koh ◽  
Mohammed Rahman ◽  
Agostino Molteni ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Inorganic Phosphate ◽  
Adenosine Triphosphatase ◽  
Salt Intake ◽  
Control Period ◽  
Plasma Renin ◽  
Normal Diet ◽  
High Salt ◽  
Sodium Potassium ◽  
High Salt Intake

1. We measured ouabain-insensitive adenosine triphosphatase (ATPase), sodium, potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) and intracellular Na+ and K+ in the erythrocytes of 19 healthy volunteers, before and after supplementation of their normal diet with 6.0–8.9 g of salt (102–137 mmol of NaCl) per day, for 5 days. 2. The subjects had a small but significant gain in weight. Mean plasma renin activity decreased from 1.57 to 0.73 pmol of angiotensin I h−1 ml−1 and plasma aldosterone from 0.46 to 0.24 nmol/l. 3. Total ATPase activity fell from 197.9 nmol of inorganic phosphate h−1 mg−1 during the control period to 173.5 during the high-salt period (P < 0.0125). Na+,K+-ATPase activity fell from 162.2 to 141.4 nmol of inorganic phosphate h−1 mg−1 (P < 0.05). Intracellular Na + and intracellular K+ did not change. 4. These results are consistent with the hypothesis that salt-induced volume expansion causes the release of a factor inhibitory to the Na+ pump.

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