Factors Affecting the Inhibitor Sensitivity of the Mitochondrial Membrane-Bound Adenosine Triphosphatase Activity

1974 ◽  
Vol 2 (2) ◽  
pp. 205-207 ◽  
Author(s):  
M. PARTIS ◽  
A. D. MITCHELL ◽  
J. WHITEHEAD ◽  
J. F. DONNELLAN ◽  
P. E. LINNETT ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Adenosine Triphosphatase ◽  
Factors Affecting ◽  
Adenosine Triphosphatase Activity ◽  
Membrane Bound

scholarly journals PROBLEMS ASSOCIATED WITH THE DEMONSTRATION BY LEAD METHODS OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN RESIDENT PERITONEAL MACROPHAGES AND EXUDATE MONOCYTES OF THE GUINEA PIG

1973 ◽  
Vol 21 (5) ◽  
pp. 488-498 ◽  
Author(s):  
R. E. POELMANN ◽  
W. T. DAEMS ◽  
E. J. VAN LOHUIZEN
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Microscopic Study ◽  
Heterogeneous Nucleation ◽  
Adenosine Triphosphatase ◽  
Peritoneal Macrophages ◽  
Electron Microscopic ◽  
Adenosine Triphosphatase Activity ◽  
Eosinophilic Granulocytes ◽  
Membrane Bound

This cytochemical and electron microscopic study on peritoneal macrophages of the guinea pig has raised doubts concerning the validity of lead methods for the demonstration of plasma membrane-bound adenosine triphosphatase activity. The problems encountered are inherent in the use of lead ions as a capture reagent. The nonenzymatically formed precipitates reflect sites of heterogeneous nucleation specific for certain kinds of cells, e.g., resident peritoneal macrophages, eosinophilic granulocytes and, to a lesser degree, exudate monocytes. This type of precipitation is also catalyzed on the surface of nonbiologic matrices such as latex particles. Enzymatic processes may well occur, but they cannot be distinguished from nonenzymatic processes.

scholarly journals Factors affecting the translocation of oxaloacetate and l-malate into rat liver mitochondria

1968 ◽  
Vol 109 (5) ◽  
pp. 921-928 ◽  
Author(s):  
J. M. Haslam ◽  
D. E. Griffiths
Keyword(s):  
Rat Liver ◽  
Adenosine Triphosphatase ◽  
Liver Mitochondria ◽  
Spectrophotometric Assay ◽  
Rat Liver Mitochondria ◽  
Anaerobic Conditions ◽  
Factors Affecting ◽  
Adenosine Triphosphatase Activity ◽  
Energy Dependent ◽  
Stimulation Of

1. The rates of translocation of oxaloacetate and l-malate into rat liver mitochondria were measured by a direct spectrophotometric assay. 2. Penetration obeyed Michaelis–Menten kinetics, and apparent Km values were 40μm for oxaloacetate and 0·13mm for l-malate. 3. Arrhenius plots of the temperature-dependence of rates of penetration gave activation energies of +10kcal./mole for oxaloacetate and +8kcal./mole for l-malate. 4. The translocation of both oxaloacetate and l-malate was competitively inhibited by d-malate, succinate, malonate, meso-tartrate, maleate and citraconate. The Ki values of these inhibitors were similar for the penetration of both oxaloacetate and l-malate. 5. Rates of penetration were stimulated by NNN′N′-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate under aerobic conditions or by ATP under anaerobic conditions. 6. The energy-dependent stimulation of translocation was abolished by uncouplers of oxidative phosphorylation. Oligomycin A, aurovertin, octyl-guanidine and atractyloside prevented the stimulation by ATP, but did not inhibit the stimulation by NNN′N′-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate. 7. Mitochondria prepared in the presence of ethylene-dioxybis(ethyleneamino)tetra-acetic acid did not exhibit the energy-dependent translocation, but this could be restored by the addition of 50μm-calcium chloride. 8. Valinomycin or gramicidin plus potassium chloride enhanced the energy-dependent translocation of oxaloacetate and l-malate. 9. Addition of oxaloacetate stimulated the adenosine triphosphatase activity of the mitochondria, and the ratio of ‘extra’ oxaloacetate translocation to ‘extra’ adenosine triphosphatase activity was 1·6:1. 10. Possible mechanisms for the energy-dependent entry of oxaloacetate and l-malate into mitochondria are discussed in relation to the above results.

scholarly journals LOCALIZATION OF MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE ACTIVITY IN CULTURED HUMAN CELLS

1965 ◽  
Vol 13 (8) ◽  
pp. 647-656 ◽  
Author(s):  
EDWARD ESSNER ◽  
JØRGEN FOGH ◽  
PATRICIA FABRIZIO
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Membrane ◽  
Adenosine Triphosphatase ◽  
Cultured Cells ◽  
Enzyme Reaction ◽  
Outer Mitochondrial Membrane ◽  
Adenosine Triphosphatase Activity ◽  
Different Types ◽  
Cultured Human Cells ◽  
Membrane Reaction

Adenosine triphosphatase activity has been localized by light microscopy in the mitochondria of four different types of cultured cells using the Wachstein-Meisel lead method after brief fixation in formol-calcium. The resolutions of the method permits the study of mitochondrial size, form, number and distributions in these cultured cells. Electron microscopy shows enzyme reaction product within the mitochondrion but not on the outer mitochondrial membrane. Reaction product is also localized to the plasma membrane and its infoldings.

scholarly journals A simple and rapid method for the reversible removal of lipids from a membrane-bound enzyme

1978 ◽  
Vol 169 (2) ◽  
pp. 305-311 ◽  
Author(s):  
S L Goodman ◽  
M Isern de Caldentey ◽  
K P Wheeler
Keyword(s):  
Phosphatase Activity ◽  
Rapid Method ◽  
Adenosine Triphosphatase ◽  
Complete Loss ◽  
Initial Activity ◽  
Experimental Conditions ◽  
Adenosine Triphosphatase Activity ◽  
Ionic Detergent ◽  
Reproducible Method ◽  
Membrane Bound

A simple, rapid and reproducible method for the reversible removal of lipids from a membrane-bound enzyme is described. Essentially, a membrane preparation containing (Na+ + K+)-dependent adenosine triphosphatase was extracted with the non-ionic detergent Lubrol WX in the presence of glycerol, and partial separation of protein from lipid was achieved with the use of only two centrifugations. About 74% of the endogenous phospholipid and 79% of the cholesterol were removed, concomitant with a virtually complete loss of ouabain-sensitive adenosine triphosphatase activity, but with retention of 60-100% of the K+-dependent phosphatase activity. The addition of pure phosphatidylserine re-activated the enzyme to more than 80% of the initial activity, and up to 30% of the protein was recovered. Excess of phosphatidylserine could be washed off the enzyme to give a stable ‘reconstituted’ preparation. The effects of variation in the experimental conditions were examined, and the results are discussed with respect to the possibility of adapting the method to the study of other lipid-dependent enzymes bound to membranes.

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.

Sign in / Sign up

Export Citation Format

Share Document