Cholinergic Proteolipid Research Articles

Absence of triphosphoinositide in the chloroform-methanol extract of cerebral cortex containing the cholinergic proteolipid.

Absence of triphosphoinositide in the chloroform-methanol extract of cerebral cortex containing the cholinergic proteolipid.

Lipid bilayers and liposomes in reconstitution experiments with cholinergic proteolipid from torpedo electroplax

The cholinergic proteolipid from Torpedo electroplax was used in reconstitution experiments in artificial membranes being incorporated directly into the membrane-forming solution or into liposomes (proteoliposomes) which interacted with lecithin bilayers. In both cases the membrane became reactive to acetylcholine by a decrease in resistance and an increase in the frequency and amplitude of minute current fluctuations of 3·10 −11 to 4·10 −10mho. The injection of d-tubocurarine prod u ced an increase in membrane resistance and a decrease in the amplitude of the current fluctuations. These results suggest that the cholinergic proteolipid is reconstituted in an active form in the bilayer.

Differences in the cholinergic proteolipid isolated by affinity chromatography from normal and denervated rat leg muscle

The cholinergic proteolipid from rat leg muscle, isolated by Sephadex LH-20 column chromatography, was purified about 5 fold further by affinity chromatography in organic solvents. The affinity column consisted of p-phenyltrimethylammonium as the active group and a pulse of acetylcholine or acid was used to desorb the specific fraction. From normal controls 36 μg of specific protein per g fresh tissue were obtained. In the denervated muscle there was no increase in this protein after three days, but it increased by 120% after six days. Binding studies carried out with 14C-acetylcholine, 3H-α-bungarotoxin and 14C-d-tubocurarine showed that only the specific fraction was able to bind the ligands. Three days after denervation the specific activity (nmoles/mg protein) for 14C-acetylcholine increased 400% and for 3H-α-bungarotoxin 100% over the control; on the contrary, there was no change in the binding of 14C-d-tubocurarine. These results are discussed in relation to the different pharmacological properties of the functional and extrajunctional receptors in skeletal muscle.

Isolation by affinity chromatography of a cholinergic proteolipid from [formula omitted

A cholinergic proteolipid fraction (i.e. a hydrophobic lipoprotein) was separated from the n. caudatus of the cow, using affinity chromatography with the lipophilic gel Sephadex LH-20 and p-phenyltrimethylamonium as the active group. High affinity binding studies showed that only the specific fraction, desorbed after an acetylcholine (or acid) pulse, and corresponding to 0,72% of the proteolipids, is the one that binds the cholinergic ligands. The binding of ( 3H)atropine and ( 14C)d-tubocurarine demonstrated that there are 814 picomoles/g fresh tissue of muscarinic sites and only 76 picomoles/g of nicotinic sites. The specific radioactivity for ( 3H)atropine is 10,000 nmoles/g protein, suggesting a high degree of purification of the specific cholinergic proteolipid.

Letter: Similarities between "cholinergic proteolipid" and detergent-extracted cholinergic proteins.

Letter: Similarities between "cholinergic proteolipid" and detergent-extracted cholinergic proteins.

Similarities between “cholinergic proteolipid” and detergent-extracted cholinergic proteins (reply)

Similarities between “cholinergic proteolipid” and detergent-extracted cholinergic proteins (reply)

Differences between detergent-extracted acetylcholine receptor and "cholinergic proteolipid.".

SNAKE venom α-toxins1 in combination with reversible2,3 or alkylating4,5 cholinergic ligands give a selective labelling of the cholinergic (nicotinic) receptor protein (AChR)6–8. Since this protein is membrane-bound and therefore expected to possess hydrophobic properties, two different methods have been used for its dissolution and purification: (1) chloroform–methanol extraction9 and (2) non-denaturing detergent extraction in aqueous media2,3. The former solubilises the so-called “cholinergic proteolipid” which, still in organic media, binds charged cholinergic ligands9. The latter procedure has made possible the purification of the AChR by affinity chromatography and the study of the binding of the above-mentioned ligands in physiological media (for reviews see refs 6–8). Furthermore, the serum of rabbits immunised against the purified AChR10–12 blocks in vivo the physiological response to cholinergic agonists10,11, thereby confirming that the detergent-extracted AChR is indeed involved in the permeability change caused by acetylcholine. On the other hand, the physiological importance of the “cholinergic proteolipid” remains undetermined, though the challenge to the actual purification13 has recently been refuted14. It is therefore the aim of the present work to compare the “cholinergic proteolipid” and the detergent-extracted AChR from the same sources: the electric organs of Electrophorus electricus and Torpedo marmorata using the selective labels α-toxin15 and the covalent affinity reagent 4-(N-maleimido)-phenyltrimethylammonium iodide4 in combination with immunodiffusion tests11.

Conductance properties of artificial lipidic membranes containing a proteolipid from Electrophorus. Response to cholinergic agents.

Previous studies have shown that a special proteolipid extract from the electric organ of Electrophorus showed high affinity binding for acetylcholine and other cholinergic agents. This proteolipid has now been incorporated into ultrathin lipidic membranes, and the membrane resistance was studied. The resistance decreased from 7.27 +/- 0.82 x 10(5) ohm cm(2) in the control membrane to 1.83 x 10(5) ohm cm(2) with addition of 72 microg/ml proteolipid. The decrease in resistance followed a potential function of order four with the proteolipid concentration in the membrane-forming solution. The presence of this proteolipid determined some type of cationic selectivity which was not observed in the control. At a critical point of proteolipid concentration the conductance spontaneously fluctuated between two levels. The membrane current jumped from one state to another by way of single discrete steps, reminiscent of those obtained with the excitatory inducing material or the macrocyclic antibiotics. In membranes containing another proteolipid having no cholinergic binding properties, the increase in conductance was smaller, and had a linear function with the concentration. In this case the "flip flop" fluctuation and the cationic selectivity were not observed. The membranes containing the cholinergic proteolipid reacted to the addition of acetylcholine by a rapid and transient increase in conductance that was considerably reduced or abolished by a previous application of d-tubocurarine. These membranes also interacted with other cholinergic agents, such as gallamine triethiodide, hexamethonium, and alpha-bungarotoxin. These results suggest that this special proteolipid, when added to the artificial membranes, induces a "chemical excitability" toward cholinergic ligands.

Binding of α-bungarotoxin to the cholinergic receptor proteolipid from Electrophorus electroplax

1. 1. This investigation was initiated to study the similarities or differences between the cholinergic proteolipid first isolated in our laboratory from Electrophorus electroplax and the protein-detergent-α-bungarotoxin complex isolated by others. 2. 2. The binding of α-[ 131I]bungarotoxin to the proteolipid extracted from electric tissue or electroplax membranes was studied using column chromatography and a partition method. 3. 3. The saturation curve obtained revealed a single site of binding per molecule of proteolipid of molecular weight 37000. 4. 4. The binding of α-bungarotoxin could not be displaced with acetylcholine or decamethonium. 5. 5. It is concluded that the cholinergic proteolipid extracted with organic solvents is similar to the protein-α-bungarotoxin complex separated by the use of strong detergents.

Fine structure of ultrathin artificial membranes

A special technique for the electron-microscope study of the fine structure of ultrathin artificial membranes is described. Membranes made of total phospholipids of the cerebral cortex and cholesterol showed globular elements of 40 Å embedded in a denser and diffuse matrix. These same elements were also seen organized in a periodic banded pattern. Identical patterns were observed with and without supporting films. Lipidic membranes containing small amounts of proteolipid fromElectrophorus showed a lower electron density, a finer and smoother texture and a decrease in electrical resistance.Lipidic membranes containing the cholinergic receptor proteolipid fromElectrophorus, upon addition of acetylcholine, showed a rapid and transient rise in conductance which was accompanied by changes in fine structure, consisting in a more uneven corrugated appearance of the membrane and the presence of dense spots of 20 Å. These results are discussed in relation to the channel hypothesis of ion permeability. It is postulated that the binding of acetylcholine by the receptor proteolipid results in conformational changes in this protein that facilitate the translocation of ions through the membrane.

Isolation of a Cholinergic Proteolipid Receptor from Electric Tissue

A proteolipid protein having a high affinity for methyl (14)C-hexamethonium, (3)H-p-(trimethylammonium)-benzene diazonium fluoroborate, and (acetyl-1-(14)C)choline chloride was isolated and purified from the electric tissue of Torpedo marmorata and Electrophorus electricus. At variance with the "receptor proteolipid" from the brain the one from electric tissue apparently does not bind atropine sulfate.

"Trophic" influences of nerve on muscle.

"Trophic" influences of nerve on muscle.