scholarly journals Arrhenius plots of acetylcholinesterase activity in mammalian erythrocytes and in Torpedo electric organ. Effect of solubilization by proteinases and by a phosphatidylinositol-specific phospholipase C

1985 ◽  
Vol 231 (1) ◽  
pp. 237-240 ◽  
Author(s):  
P L Barton ◽  
A H Futerman ◽  
I Silman
Keyword(s):  
Phospholipase C ◽  
Electric Organ ◽  
Acetylcholinesterase Activity ◽  
Arrhenius Plots ◽  
Before And After ◽  
Break Points ◽  
Membrane Bound ◽  
Torpedo Electric Organ ◽  
Erythrocyte Ache ◽  
Lipid Environment

The temperature-dependence of the catalytic activity of acetylcholinesterase (AChE) from rat erythrocyte-ghost membranes and from Torpedo electric-organ membranes was examined. In the case of rat erythrocyte AChE, a non-linear Arrhenius plot was observed both before and after solubilization by a phosphatidylinositol-specific phospholipase C or by proteinase treatment. Similarly, no significant differences were observed in Arrhenius plots of Torpedo electric-organ AChE before or after solubilization. These results support our suggestion that the catalytic subunit of AChE does not penetrate deeply into the lipid bilayer of the plasma membrane and also suggest that care must be taken in ascribing break points in Arrhenius plots of membrane-bound enzymes to changes in their lipid environment.

scholarly journals Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ.

1985 ◽  
Vol 101 (5) ◽  
pp. 1757-1762 ◽  
Author(s):  
N Morel ◽  
J Marsal ◽  
R Manaranche ◽  
S Lazereg ◽  
J C Mazie ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Electric Organ ◽  
Acetylcholinesterase Activity ◽  
Cholinergic Nerve Terminals ◽  
Membrane Bound ◽  
Torpedo Electric Organ ◽  
Presynaptic Plasma Membrane ◽  
Glycera Convoluta

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.

scholarly journals Physicochemical behaviour and structural characteristics of membrane-bound acetylcholinesterase from Torpedo electric organ. Effect of phosphatidylinositol-specific phospholipase C

1985 ◽  
Vol 226 (2) ◽  
pp. 369-377 ◽  
Author(s):  
A H Futerman ◽  
R M Fiorini ◽  
E Roth ◽  
M G Low ◽  
I Silman
Keyword(s):  
Phospholipase C ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Electric Organ ◽  
Catalytic Subunit ◽  
Proteinase K ◽  
Short Amino Acid Sequence ◽  
Torpedo Electric Organ

Quantitative solubilization of the phospholipid-associated form of acetylcholinesterase (AChE) from Torpedo electric organ can be achieved in the absence of detergent by treatment with phosphatidylinositol-specific phospholipase C (PIPLC) from Staphylococcus aureus [Futerman, Low & Silman (1983) Neurosci. Lett. 40, 85-89]. The sedimentation coefficient on sucrose gradients of AChE solubilized in detergents (DSAChE) varies with the detergent employed. However, the coefficient of AChE directly solubilized by PIPLC is not changed by detergents. Furthermore, PIPLC can abolish the detergent-sensitivity of the sedimentation coefficient of DSAChE purified by affinity chromatography, suggesting that one or more molecules of phosphatidylinositol (PI) are co-solubilized with DSAChE and remain attached throughout purification. DSAChE binds to phospholipid liposomes, whereas PIPLC-solubilized AChE and DSAChE treated with PIPLC do not bind even to liposomes containing PI. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows that PIPLC-solubilized AChE, like unmodified DSAChE, is a catalytic subunit dimer; electrophoresis in the presence of reducing agent reveals no detectable difference in the Mr of the catalytic subunit of unmodified DSAChE, of AChE solubilized by PIPLC and of AChE solubilized by Proteinase K. The results presented suggest that DSAChE is anchored to the plasma membrane by one or more PI molecules which are tightly attached to a short amino acid sequence at one end of the catalytic subunit polypeptide.

scholarly journals Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides.

1982 ◽  
Vol 92 (2) ◽  
pp. 333-342 ◽  
Author(s):  
P A St John ◽  
S C Froehner ◽  
D A Goodenough ◽  
J B Cohen
Keyword(s):  
Binding Sites ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Relative Molecular Mass ◽  
Alkaline Extraction ◽  
Before And After ◽  
Torpedo Electric Organ ◽  
Extracellular Side ◽  
Alpha Bungarotoxin

Experiments were conducted to examine the topographic arrangement of the polypeptides of the acetylcholine receptor (AcChR) and the nonreceptor Mr 43,000 protein in postsynaptic membranes isolated from Torpedo electric organ. When examined by electron microscopy, greater than 85% of vesicles were not permeable to ferritin or lactoperoxidase (LPO). Exposure to saponin was identified as a suitable procedure to permeabilize the vesicles to macromolecules with minimal alteration of vesicle size or ultrastructure. The sidedness of vesicles was examined morphologically and biochemically. Comparison of the distribution of intramembrane particles on freeze-fractured vesicles and the distribution found in situ indicated that greater than 85% of the vesicles were extracellular-side out. Vesicles labeled with alpha-bungarotoxin (alpha-Bgtx) were reacted with antibodies against alpha-BgTx or against purified AcChR of Torpedo. Bound antibodies were detected by the use of ferritin-conjugated goat anti-rabbit antibody and were located on the outside of greater than 99% of labeled vesicles. Similar results were obtained for normal vesicles or vesicles exposed to saponin. Quantification of the amount of [3H]-alpha-BgTx bound to vesicles before and after they were made permeable with saponin indicated that less than 5% of alpha-BgTx binding sites were cryptic in normal vesicles. It was concluded that greater than 95% of postsynaptic membranes were oriented extracellular-side out. LPO-catalyzed radioiodinations were performed on normal and saponin-treated vesicles and on vesicles from which the Mr (relative molecular mass) 43,000 protein had been removed by alkaline extraction. In normal vesicles, polypeptides of the AcChR were iodinated while the Mr 43,000 protein was not. In vesicles made permeable with saponin, the pattern of labeling of AcChR polypeptides was unchanged, but the Mr 43,000 protein was heavily iodinated. The relative iodination of AcChR polypeptides was unchanged in membranes equilibrated with agonist or with alpha-BgTx or after alkaline-extraction. It was concluded that the Mr 43,000 protein is present on the intracellular surface of the postsynaptic membrane and that AcChR polypeptides are exposed on the extracellular surface.

scholarly journals Soluble low-Km 5′-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage

1992 ◽  
Vol 284 (3) ◽  
pp. 621-624 ◽  
Author(s):  
M Vogel ◽  
H Kowalewski ◽  
H Zimmermann ◽  
N M Hooper ◽  
A J Turner
Keyword(s):  
Cerebral Cortex ◽  
Phospholipase C ◽  
High Speed ◽  
Electric Organ ◽  
Bovine Brain ◽  
Soluble Enzyme ◽  
Torpedo Marmorata ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Membrane Bound

Soluble and membrane-bound low-Km 5′-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6%) than in bovine cerebral cortex (27.9%). Membrane-bound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5′-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5′-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5′-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPI-anchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

scholarly journals Immunological identification of a new 14X10(3) Mr membrane-bound protein in Torpedo electric organ

1991 ◽  
Vol 98 (3) ◽  
pp. 351-361
Author(s):  
N. Morel ◽  
G. Brochier ◽  
M. Synguelakis ◽  
G. Le Gal La Salle
Keyword(s):  
External Surface ◽  
Electric Organ ◽  
Cell Types ◽  
Immunogold Labelling ◽  
Electron Microscope Level ◽  
Capillary Endothelial Cells ◽  
Membrane Bound ◽  
Torpedo Electric Organ ◽  
Immunological Identification

A series of monoclonal antibodies binding to different epitopes shared by a 14 × 10(3)Mr membrane-bound polypeptide has been obtained. By indirect immuno-fluorescence, it was shown that the 14 × 10(3)Mr antigen is present in various cell types in Torpedo electric organ and muscle, especially fibroblasts, capillary endothelial cells, axonal cuff cells and, to a lesser extent, Schwann cells. At the electron-microscope level, after immunogold labelling, the antigen was found associated with the external surface of the plasma membrane of these cells, with the exception of the axonal cuff cells where part of the labelling was intracellular. The possible biological role of this 14 × 10(3)Mr protein is unknown but preliminary experiments suggest that this antigen has affinity for other Torpedo electric organ membrane proteins.

scholarly journals The Profound Influence of Lipid Composition on the Catalysis of the Drug Target NADH Type II Oxidoreductase

Membranes ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 363
Author(s):  
Albert Godoy-Hernandez ◽  
Duncan G. G. McMillan
Keyword(s):  
Acyl Chain ◽  
Cellular Respiration ◽  
Type Ii ◽  
Nadh:Quinone Oxidoreductase ◽  
Chain Composition ◽  
Membrane Bound ◽  
Lipid Environment ◽  
Lipid Specificity ◽  
Mediated Catalysis ◽  
Quinone Pool

Lipids play a pivotal role in cellular respiration, providing the natural environment in which an oxidoreductase interacts with the quinone pool. To date, it is generally accepted that negatively charged lipids play a major role in the activity of quinone oxidoreductases. By changing lipid compositions when assaying a type II NADH:quinone oxidoreductase, we demonstrate that phosphatidylethanolamine has an essential role in substrate binding and catalysis. We also reveal the importance of acyl chain composition, specifically c14:0, on membrane-bound quinone-mediated catalysis. This demonstrates that oxidoreductase lipid specificity is more diverse than originally thought and that the lipid environment plays an important role in the physiological catalysis of membrane-bound oxidoreductases.

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