Torpedo electroplax

Radioiodinated derivatives have been prepared to define more closely the target site of a-conotoxins on the acetylcholine receptor (R. Myers, unpublished data). In membrane preparations from Torpedo electroplax, photoactivatable azidosalicylate derivatives of a-conotoxin GIA preferentially label the p and 7 subunits of the acetylcholine receptor. However, when the photoactivatable derivative is cross-linked to detergent solubilized acetylcholine receptor (AChR), only the 7 subunit is labeled. Since snake a-neurotoxins mainly bind to the a subunits of AChR and a-conotoxins compete directly with a-bungarotoxin, the cross-linking results above are both intriguing and problematic. 

In the following the Cl -channels will be subdivided into those of the central nervous system, of muscle and Torpedo electroplax, of apolar non-excitable cells and of epithelia. 

The acetylcholine receptor (AChR) of Torpedo electric organ is also a PCP "receptor." However, this nicotinic AChR has about one-tenth the affinity for PCP than that of the rat brain PCP receptor [K0.5 = 0.3 pM, versus = 4-6 pM for Torpedo (Heidmann et al. 1983 flaring et al. 1984)]. Moreover, the nicotinic AChR has subunits of MR<66 kD, and these are the subunits that are specifically labelled with 3H-Az-PCP in the Torpedo electroplax membranes (Heidmann et al. 1983 Haring and Kloog 1984 Haring et al. 1984). These data indicate that the nicotinic AChR-PCP receptor differs from the rat brain PCP receptor. Furthermore, our findings are... 

Most of the above studies have been made possible because of the high density of nAChRs in the Torpedo electroplax membrane. However, with recent technological developments, it may be hoped that similar approaches will prove useful when applied to receptors of much lower abundance. 

Eldefrawi and Eldefrawi [98] reported the purification of the acetylcholine of Torpedo electroplax on an affinity column consisting of cobra (Naja naja siamensis) toxin coupled to Sepharose 4B. Desorption with 10 mM benzoquinonium produced a protein that bound [ I]a-bungarotoxin but not [ H]acetyl-choline. However, desorption with 1 mM carbamylcholine gave a receptor protein that bound pH]acetylcholine decamethonium, [ H]nicotine [ C]dimethyl-(-l-)-tubocuranrine, and [ I]a bungarotoxin. Schmidt and Raftery [99] also purified acetylcholine receptor, from Narcine, on a N-(e-aminohexanoyl)-3-aminopropyltrimethyl-ammonium bromide-HBr-agarose column. 

Returning to the topic of ACh receptors, preliminary three-dimensional models are available for this receptor from Torpedo electroplax (S8,j5 ). Figure 5 shows the possible structure of the ACh binding site and the residues likely to be involved in ACh binding. Considerable progress can be expected in developing the structure of the comparable receptor from the locust CNS (60). 

Torpedo electroplax membranes Percentage of cholinergic receptor... 

M sucrose, 0.05 mM Na2EDTA, and 1 mM ethanol. Purification of torpedo electroplax membranes were also carried out, and the separation of various bacterial cells were also described, including the purification of different strains of Escherichia coli and the separation of Salmonella typhirum cells, using PEG-dextran ATPS systems. Moreover, these CCC devices were also applied to larger cells, such as the separation of various species of red blood cells. 

THE RELATIONSHIP BETWEEN CHOLINERGIC PROTEOLIPID AND PROTEODETERGENT IN TORPEDO ELECTROPLAX MEMBRANES... 

In our experiments of affinity labeling of Torpedo electroplax membranes we confirmed the results of Karlin (1974) and Barrantes et al., (1975a) as far as that, after chloroform-methanol extraction, most of the label remained in the residue. As shown in Table 2 in two experiments done with different concentrations of 3h -MPTA the radioactivity extracted from affinity labeled membranes only... 

Membranes of Torpedo electroplax enriched with cholinergic receptor (Changeux et al., 1970) were labeled with pH -MPTA (Karlin and Cowburn 1973). In experiment 1, 0.3 nmoles and in 2, 6 nmoles of ]3h -MPTA were used for labeling the membranes. 

It is interesting that by affinity chromatography Meunier et al., (1974) reported the extraction of 2.9 mg/Kg of cholinergic receptor from the Electropborus. In the original paper of La Torre et al., (1970) is already stressed the higher amount of proteolipid present in the Torpedo electroplax as compared with the Electropborus. 

Fig. 2. Experiment in which Torpedo electroplax membranes were labeled with j Hj-MPTA and then submitted to extraction with 1% Triton X-100. The reextraction of the proteodetergent by chloroform-methanol(2 1) is also indicated (see the description in the text).

To investigate further the problem of the relationship between the cholinergic proteolipid and the proteodetergent, receptor-enr ched membranes from Torpedo electroplax were submitted to SDS polyacrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (1970) as modified for slab gels by Studier (1973). 

Fig. 3. Polyacrylamide slab gel of the proteins present in Torpedo electroplax membranes. C, control E, membranes extracted with chloroform-methanol (2 1) 0, origin F, front. The extraction...

A radioactive perhydro-derivative of histrionicotoxin (pH]H,2-HTX (10, 92) has proved a very useful tool for investigation of the binding sites for histrionicotoxin in membranes and microsacs from Torpedo electroplax preparations (see Table 12 for references). Its use has not been extended to muscle preparations where the density of nicotinic receptor channel complexes are many fold lower than in Torpedo electroplax. Indeed, no specific binding could be detected in the electroplax of the Egyptian electric fish (Malapterurus electricus) where the density of nicotinic receptors also appear very low (97). In membranes from Torpedo electroplax pH]Hi2-HTX binds to a site with an affinity constant (Kd) of about 0.4 pM. The density of the binding sites for pH]Hi2 HTX is apparently about twice that of the acetylcholine-binding sites in electroplax membranes. The rate of... 

Eldefrawi, M. E., D. S. Copio, C. S. Hudson, J. Rash, N. A. Mansour, A. T. Eldefrawi, and E. X. Albuquerque Effects of antibodies to Torpedo acetylcholine receptor on the acetylcholine receptor-ionic channel complex of Torpedo electroplax and rabbit intercostal muscle. Exp. Neurol. 64, 428—444 (1979). 

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