scholarly journals The phosphorylation potential generated by respiring bovine heart submitochondrial particles

1977 ◽  
Vol 168 (2) ◽  
pp. 299-303 ◽  
Author(s):  
S J Ferguson ◽  
M C Sorgato
Keyword(s):  
Adenosine Triphosphatase ◽  
Membrane Vesicles ◽  
Submitochondrial Particles ◽  
Phosphorylation Potential ◽  
Adenosine Triphosphatase Activity ◽  
Bovine Heart

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.

scholarly journals A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles

1975 ◽  
Vol 148 (3) ◽  
pp. 533-537 ◽  
Author(s):  
R B Beechey ◽  
S A Hubbard ◽  
P E Linnett ◽  
A D Mitchell ◽  
E A Munn
Keyword(s):  
Electron Microscopy ◽  
Rapid Method ◽  
Adenosine Triphosphatase ◽  
Pure Form ◽  
Heart Mitochondria ◽  
Polyacrylamide Gels ◽  
Submitochondrial Particles ◽  
Bovine Heart

An almost pure form of the bovine heart mitochondrial adenosine triphosphatase (ATPase) is released from the membrane by shaking submitochondrial particles with chloroform. Analyses on polyacrylamide gels and by electron microscopy, and also sensitivity to inhibitors, show that the chloroform-released enzyme is similar to other ATPase preparations from bovine heart mitochondria.

scholarly journals The adenosine triphosphatase-inhibitor content of bovine heart submitochondrial particles. Influence of the inhibitor on adenosine triphosphate-dependent reactions

1977 ◽  
Vol 162 (2) ◽  
pp. 351-357 ◽  
Author(s):  
S J Ferguson ◽  
D A Harris ◽  
G K Radda
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Type I ◽  
Type Ii ◽  
Inhibitor Protein ◽  
Isolation Method ◽  
Atpase Inhibitor ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Tightly Bound Nucleotides

1. The activity of the ATPase (adenosine triphosphatase) of phosphorylating particles prepared by sonication of bovine heart mitochondria in the presence of MgCl2 and ATP is influenced by the isolation method for the mitochondria used in the preparation of particles. Type-I particles, made from mitochondria isolated in a medium lacking succinate, have a lower ATPase activity than to Type-II particles, which are prepared from mitochondria isolated in a medium containing succinate. 2. Centrifugation under appropriate energized conditions increases the ATPase activity of Type-I particles almost to that of the Type-II particles. The ATPase activity of Type-II particles was only slightly stimulated by this procedure. These data are interpreted as indicating a higher content of the ATPase-inhibitor protein in the Type-I particles. 3. A comparison was made of the ATP-driven enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and the exchange of the endogenous tightly bound nucleotides of the ATPase in Type-I and Type-II particles. The effect of exogenous inhibitor protein on both these reactions was also studied. 4. The time-scale on which the inhibitor protein can exchange between ATPase molecules is discussed.

scholarly journals The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential

1978 ◽  
Vol 174 (1) ◽  
pp. 237-256 ◽  
Author(s):  
M C Sorgato ◽  
S J Ferguson ◽  
D B Kell ◽  
P John
Keyword(s):  
Membrane Potential ◽  
Reaction Medium ◽  
Atp Synthesis ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Submitochondrial Particles ◽  
Electron Mediator ◽  
Phosphorylation Potential ◽  
Bovine Heart ◽  
Gradient Component

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0′ + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.

scholarly journals A method for determining the adenosine triphosphatase content of energy-transducing membranes. reaction of 4-chloro-7-nitrobenzofurazan with the adenosine triphosphatase of bovine heart submitochondrial particles

1976 ◽  
Vol 159 (2) ◽  
pp. 347-353 ◽  
Author(s):  
S J Ferguson ◽  
W J Lloyd ◽  
G K Radda
Keyword(s):  
Mitochondrial Membrane ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Single Amino Acid ◽  
Mitochondrial Atpase ◽  
Inner Mitochondrial Membrane ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Single Amino Acid Residue ◽  
Membrane Bound

1. Modification of a single amino acid residue by introduction of the nitrobenzofurazan group inactivates mitochondrial ATPase (adenosine triphosphatase) when membrane-bound in submitochondrial particles. The similarity between the reactions of both membrane-bound and isolated ATPase with 4-chloro-7-nitrobenzofurazan indicates that the single essential tryosine residue identified in the isolated enzyme [Ferguson, Loyd, Lyons & Radda (1975) Eur. J. Biochem. 54, 117-126] Is also a feature of the membrane-bound ATPase. 2. A procedure is presented for estimating the ATPase content of the inner mitochondrial membrane. It is based on the specificity of the incorporation of the nitrobenzofurazan group, and the ready removal of this group by compounds that contain a thiol group. This method indicates that 8.5% of the membrane protein is ATPase. The procedure should be applicable to the titration of the energy-transducing ATPases of bacterial plasma membranes and of the thylakoid membranes of chloroplasts. 3. Combination of the data obtained on the ATPase content of the bovine heart inner mitochondrial membrane with a titration of the cytochrome bc1 complex with antimycin indicates that these two components of the membrane are present in approximately equal amounts.

scholarly journals Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms

1989 ◽  
Vol 84 (3) ◽  
pp. 315-323 ◽  
Author(s):  
Italo M. Cesari ◽  
Elis Aldana ◽  
Francisco Gil ◽  
Madalina Condrescu
Keyword(s):  
Basement Membrane ◽  
Atpase Activity ◽  
Schistosoma Mansoni ◽  
Adenosine Triphosphatase ◽  
Surface Membrane ◽  
Membrane Vesicles ◽  
Alkaline Ph ◽  
Double Membrane ◽  
Similar Activity ◽  
Adenosine Triphosphatase Activity

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized whithin the tegument of fixed worms on the cytoplasmic leaflet of both the doubel surface membrane and the basement membrane; this reaction was inhibited by 1 µM chloropromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosimes are discussed.

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