Membranes 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have been prepared to define more closely the target site of a-conotoxins on the acetylcholine receptor (R. Myers, unpublished data).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. 

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. 

Traylor and Singer (1967) have provided a detailed synthesis of the diazonium salts XIX and XX. Compound XIX has been used as an affinity label for acetylcholinesterase on erythrocyte membrane (Wofsy and Michaeli 1967) and for the cholinergic receptor from eel electroplax (Changeux et al. 1967). XX has been used for the modifica-... 

Because altered sodium channels have been implicated in kdr and kdr-like resistance phenomena in insects, basic research on the biochemistry and molecular biology of this molecule, which plays a central role in normal processes of nervous excitation in animals, is of immediate relevance. The results of recent investigations of the voltage-sensitive sodium channels of vertebrate nerves and muscles have provided unprecedented insight into the structure of this large and complex membrane macromolecule. Sodium channel components from electric eel electroplax, mammalian brain, and mammalian skeletal muscle have been solubilized and purified (for a recent review, see Ref. 19). A large a subunit (ca. 2 60 kDa) is a common feature of all purified channels in addition, there is evidence for two smaller subunits ( Jl and J2 37-39 kDa) associated with the mammalian brain sodium channel and for one or two smaller subunits of similar size associated with muscle sodium channels. Reconstitution experiments with rat brain channel components show that incorporation of the a and pi subunits into phospholipid membranes in the presence of brain lipids or brain phosphatidylethanolamine is sufficient to produce all of the functional properties of sodium channels in native membranes (AA). Similar results have been obtained with purified rabbit muscle (45) and eel electroplax (AS.) sodium channels. 

Several studies employing oocytes of the clawed frog, Xenopus laevis, for the in vitro translation of sodium channel encoding mRNAs (53-55) suggest that this experimental system may be particularly useful toward this end. The biophysical properties of sodium channels expressed in oocytes following injection of rat brain mRNA were similar to those of sodium channels in their native membrane environment, and were specifically inhibited by the sodium channel blockers tetrodotoxin and saxitoxin (i5.). Sodium channels encoded by mRNAs from rat skeletal muscle and eel electroplax have also been expressed in Xenopus oocytes (56-57). To date the expression of insect sodium channels in the Xenopus oocyte has not been reported, but the utility of this system for the translation and expression of insect acetylcholine receptor mRNA has recently been demonstrated (58). Successful application of this methodology to the expression of insect mRNAs encoding functional sodium channels offers a novel method to test some of the hypotheses for the molecular basis of the kdr mechanism. 

Duch DS, Levinson SR (1987) Neurotoxin-modulated uptake of sodium by highly purified preparations of the electroplax tetrodotoxin-binding glycopeptide reconstituted into lipid vesicles. J Membrane Biol 98 43-55 Duch DS, Recio-Pinto E, Frenkel C, Levinson SR, Urban BW (1989) Veratridine modification of the purified sodium channel a-polypeptide from eel electroplax. J Gen Physiol 94 813-831... 

W. S. Agnew, S. R. Levinson, J. S. Brabson, and M. A. Raftery, Purification of the Tetrodotoxin-Binding Component Associated with the Voltage-Sensitive Sodium Channel from Electrophorus electricus Electroplax Membranes, Proc. Natl Acad. Scl U A 75, 2606-2610 (1978). 

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. 

Occupaney of the nicotinic receptor (by A.) triggers a rapid response (1-2 ms) by direct activation of cation-selective ion channels, thereby causing depolarization of the postsynaptic membrane. The nicotinic cholinergic receptor has been isolated from the electroplax of Torpedo califomica (electric ray) and Electrophorus electricus (electric eel) and from vertebrate muscle. From all three tissues, it is a single membrane protein, M, 250,000, consisting of 4 glycoprotein subunits M, 40,000 (50,116) (a), 50,000... 

Direct use can be made of electroactive receptor species, such as the acetylcholine receptor extracted from the electroplax organ of fish. The idea would be to purify the material for reconstitution in a membrane which is immobilized on a sensor structure. Such a system could be used as a negative sensor for neurotoxins. Naturally, the military branches of a number of governments are particularly interested in this route. 

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. 

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...

Electroplax-exit-able membrane frog neuromuscular junction... 

The first report of a2,8-linked polySia on the a subunit of sodium channel proteins was by James and Agnew (1987), who identified such chains on the voltage-sensitive channel from the electric organ of the eel, Electrophorus elec-tricus. The sodium channel protein appears to be the only protein in electroplax membranes that is polysialylated, suggesting the presence of a specific a2,8-polysialyltransferase for the polysialylation of this protein (James and Agnew, 1989). 

Electroplax Torpedo membranes No effect on binding of a-bungarotoxin Un- published results... 

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