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 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 The cerebral sodium-plus-potassium ion-stimulated adenosine triphosphatase of bovine brain and its microsomal matrix

1970 ◽  
Vol 119 (3) ◽  
pp. 367-376 ◽  
Author(s):  
I. Pull ◽  
H. McIlwain
Keyword(s):  
Amino Acids ◽  
Density Gradient ◽  
Adenosine Triphosphatase ◽  
Sodium Iodide ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sodium Thiocyanate ◽  
Adenosine Triphosphatase Activity ◽  
Ionic Detergent ◽  
High Concentrations

1. Adenosine triphosphatase activities of dispersions prepared from bovine cerebral cortex that had been frozen, were greater than those of dispersions prepared from fresh tissue. The subcellular distribution of components of the dispersion was not altered by freezing the tissue and a microsomal fraction enriched in Na++K+-stimulated adenosine triphosphatase activity was prepared. 2. The bovine cerebral microsomes were further treated with a 2m-sodium iodide reagent to obtain a particulate preparation with minimal Na++K+-independent adenosine triphosphatase activity. Na++K+-stimulated activity was increased by the sodium iodide treatment and this preparation was shown to be enriched in lipid constituents. 3. Density-gradient centrifugation of the sodium iodide treated preparation gave three main subfractions each containing approximately equal amounts of phospholipid and protein. Further exposure of the sodium iodide-treated preparation to the 2m-sodium iodide reagent altered the distribution of protein and phospholipid among the fractions obtained by density-gradient centrifugation. Dissociation of phospholipids from protein in the sodium iodide-treated preparation was brought about also by high concentrations of arginine. Concentrated solutions of arginine and sodium thiocyanate brought about dissociation of phospholipids from protein of the microsomal preparation. 4. Many amino acids were found to inhibit Na++K+-stimulated adenosine triphosphatase activity when present in high concentrations. The inhibition was complex but resulted, in part at least, from diminished affinity for ATP and Na+ in the presence of the amino acids. 5. A non-ionic detergent, Lubrol W, solubilized up to 40% of the enzyme activity of the sodium iodide-treated preparation together with 30% of the protein and phospholipid in the preparation. Protein was released from the sodium iodide-treated preparation by pancreatic elastase but Na++K+-stimulated adenosine triphosphatase activity of the residue was diminished. Ultrasonic treatment of the sodium iodide-treated preparation failed to release a significant proportion of Na++K+-stimulated adenosine triphosphatase activity into a form not deposited by ultracentrifugation.

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 Differential effects of temperature on a membrane adenosine triphosphatase and associated phosphatase

1975 ◽  
Vol 151 (2) ◽  
pp. 439-442 ◽  
Author(s):  
J A Walker ◽  
K P Wheeler
Keyword(s):  
Atpase Activity ◽  
Phosphatase Activity ◽  
Adenosine Triphosphatase ◽  
Arrhenius Plots ◽  
Differential Effects ◽  
Adenosine Triphosphatase Activity ◽  
Dependent Atpase ◽  
Effects Of Temperature

Arrhenius plots of a membrane (Na+ + K+)-dependent ATPase (adenosine triphosphatase) activity showed characteristic discontinuities, whereas those of the associated K+-dependent phosphatase activity did not. These findings support the contention that the phosphatase activity does not depend on phospholipid in the same way as does the ATPase activity.

scholarly journals LOCALIZATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN PRENEOPLASTIC AND CANCEROUS CELLS OF THE MOUSE SKIN INDUCED BY METHYLCHOLANTHRENE

1968 ◽  
Vol 16 (11) ◽  
pp. 678-687 ◽  
Author(s):  
JÁNOS SUGÁR ◽  
ORSOLYA CSUKA ◽  
JÓZSEF TÓTH
Keyword(s):  
Adenosine Triphosphatase ◽  
Cell Membranes ◽  
Light And Electron Microscopy ◽  
Mouse Skin ◽  
Intercellular Spaces ◽  
Adenosine Triphosphatase Activity ◽  
Dense Deposits ◽  
Membrane Bound ◽  
Cancerous Cells ◽  
Normal Epidermis

The localization of membrane-bound adenosine triphosphatase activity has been studied by both light and electron microscopy in the normal epidermis and in methylcholanthreneinduced preneoplastic alterations, i.e., hyperplasia, papilloma and also in similarly induced cancers of the skin of mice. In normal epidermis, the adenosine triphosphatase activity was seen to be located in contact with all of the cell membranes facing the intercellular spaces. In preneoplastic alterations, the enzyme activity was found along the cell membranes of the basal and suprabasal cells of the epithelial pegs and numerous dense deposits of lead phosphate could be seen on the surfaces of the cytoplasmic processes and in the enlarged intercellular spaces. The intensity of the reaction in preneoplastic conditions was greater than in the normal epidermis. No adenosine triphosphatase activity was observed in the cell membranes facing the basement membrane or in the desmosomes. In carcinoma a less intensive adenosine triphosphatase reaction was present and could be recognized on the surface of only few tumor cells.

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