Reply from Lunt

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The insect neuronal nAChR: been tested in my laboratory a homo-oligomer or not? against SDS-patterns of Torpedo SIR:

In his article in the August issue of TINS 1, George G. Lunt discussed the suggestion of Heinz Breer and colleagues that the nACHR of Locusta migratoria (and possibly of other insect species also) is a homo-oligomer of a subunit of 65 kDa. In reading his article, I feel that Breer's case is much stronger than presented therein. A good starting point for a discussion of the issue is to remember that the SDS-gel pattern of Torpedo (and other) nAChRs overestimates the differences in molecular weight in Daltons of the subunits: (data from sequence in brackets): e, 40 kDa (50 kDa); 13, 50 kDa (54 kDa); y, 60 kDa (56 kDa); 6, 67 kDa (58 kDa). Thus, depending on structural considerations, including whether and to what extent the polypeptide chains are post-translationally modified, even hetero-oligomeric AChRs may show a single band or a 'smear' of bands in SDS-PAGE. This limiting fact should be kept in mind independent of the following points of consideration: (1) It is important to note that Breer's single SDS-band corresponds to a rather large polypeptide (65 kDa), whereas the single SDS-bands originally observed for electric eel and mammalian receptors have always corresponded to the smallest (o~) subunit. This proteolytic artifact can be avoided by using EDTA 2, as done by Breer. Interestingly, the SDS-gel pattern reported by Lunt's group for the Schistocerca receptor 3 shows only bands smaller than 65 kDa (60, 41 and 25 kDa). Although an additional (smaller) band was also observed by Breer's group 4, it is absent in the presence of appropriate protease inhibitors 5. In contrast, it is surprising that Lunt did not consider the possibility that the multiband patterns reported for AChR preparations from fly heads (never properly published and obtained with a preparation containing large amounts of nonneuronal tissue) could be the result of protease action. (2) The apparent molecular weight of Breer's 65 kDa band has TINS, Vol. 10, No. 3, 1987

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receptor and appears to be correct. When blotted, this band can be labelled with a monoclonal antibody that interferes with ligand binding and is specific for the e~-subunit of Torpedo AChR 5'6. This indicates that the Locusta 65 kDa band is different from the vertebrate b subunit and, thus, is not merely a proteolytic artifact. (3) Another of Breer's findings in favor of a single SDS-band of the Locusta nAChR should have been mentioned in Lunt's review: immune precipitation of radiolabelled nAChR expressed in Xenopus oocytes after injection of Locusta poly(A +) RNA resulted again in a single band of 65 kDa on SDS-PAGE. Together with his reconstitution and microsequencing studies, I believe that Breer has a strong case in favour of a single band SDS-gel pattern of the Locusta

Reply from Lunt SIR:

Maelicke raises some interesting points concerning the subunit composition of the insect nAChR. There is no doubt that the insect nAChR has a subunit of about 65 kDa; all of Breer's data demonstrate this most convincingly and other groups see this subunit in their preparations. What is still open to debate is whether the 65 kDa subunit is the only peptide in the insect nAChR. Additionally there is no direct evidence, e.g. from affinity labelling experiments, that it is analogous to the vertebrate o~ subunit. The immunological crossreactivity studies referred to by Maelicke provide further strong evidence in support of the 65 kDa subunit being part of the nAChR0 but the experiments described still leave open the question of whether it is the only subunit. I maintain that our knowledge of insect proteases is so meagre that we cannot be certain that proteolytic breakdown of other subunits occurs. Such cleavage of peptides would not necessarily be apparent until the oligomer was ©

1987, Elsevier Science Publishers B V . , A m s t e r d a m

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receptor. In view of the initially stated size range of nAChR subunits, and as is always emphasized in Breer's papers, this does not mean that a homo-oliogomeric Locusta nAChR is already proven. However, in these times of gene cloning, a final answer may not be far away. References

1 Lunt, G. G. (1986) Trends Neurosci. 9, 341-342 2 Schmiclt,T. J. and Raftery, M. A. (1973) Biochemistry 12, 852-857 3 Filbin,M. T., Lunt, G. G. and Donnellan, J.F. (1983) Eur. J. Biochem. 132, 151-156 4 Breer, H., Kleene, E. and Behnke, D. (1984) Neurosci. Lett. 46, 323-328 5 Breer,H., Kleene,E.and Hinz, G. (1985) J. Neurosci. 5, 3386-3392 6 Fels, G., Breer, H. and Maelicke, A. (1983) in Toxins as Tools in Neurochemistry (Hucho, F. and Ovchinnikov, Y. A., eds), pp. 127-140, de Gruyter

We welcome commentsfrom our readers.Short communicabons stand the bestchance ofpubh'cation,and the editorreservesthe A. Maelicke Max-Planck-lnstitut fiJr Ern~hrungsphysiologie, nght to takeextracts Rheinlanddamm201, D-4600Dortmund 1, FRG. from the longerones.

subjected to SDS treatment. Thus proteolysis in no way precludes any of the findings made with non-denatured receptor preparations. G. G. Lunt

Schoo/of Bib/oglca/Sciences,Umversityof Bath, C/averton Down, Bath BA2 7AY, UK.

The Biochemical Society Neurochemical Group Colloquium on

Neuronal Acceptors in Cell Targeting 9 April 1 9 8 7 Leicester, UK Speakers include G. B. Warren, B. Pearse, J. O. Dolly, S. Olsnes, J. Middlebrook, M. Lo, J. T. Kemshead, T. Reese and R. Levi-Montalcini. For information contact Dr J. O. Dolly, Dept of Biochemistry, Imperial College, South Kensington, London SW7 2AZ, UK.

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