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TIiI: COUNCIL FdR ToBAcco RESEARCA-U.S.1A., INC. ME[NORANDUM TO: W. U. Gardner and Staff FROM: D. H. Ford and D. Stone March 27, 1979 SUBJECT: Grant 1230. Donald J. Nelson, Ph.D., Clark University, Worcester, Mass. "Spectroscopic Studies of the Interaction of Cholinergic Ligands with Nicotinic Receptor Proteins." _ _ Site Visit on March 20, 1979. NMR Spectroscopy. Has made good progress in developing the optimum conditions under which one can use 3H as a probe. Has succeeded in developing a tritium probe in which a narrow resolution line of high peak occurs with a. 37/1 signal to noise ratio. He is now ready to start using nicotine labeled with 3H in different positions as well as analogues of nicotine to determine how the configuration changes the signal obtained from tritium label. The first tritium labeled molecules he plans to study will be nicotine and acetylcholine. Receptor Studies. He has begun to purify the venom from Naja naja, which binds reversibly to the nicotine ACh receptor, using cation exchange columns „ These will be followed by molecular sieving gel columns. An affinity column will be used. Then in the next step a sepharose column will be activated with cy anogen bromide so the toxin will bind to it. Once this model is functioning predictably, he will obtain the electric rays and obtain electric tissue from them to start separating out the nACh receptor on the columns. Receptor will finally be ei uted from the civlumns with carbamyl choline. Will then use 3H-acetylcholine or 3H-bungarotoxin for estimation of receptor purity. At this point he doesn't foresee anyr problems with any aspect of the study. From what he has accompl ished so farr it would appear that he and his group form a well-organized team with a clear understanding of all the details (and problems) of their procedures. Other studies discussed (not suPported by CPR) but of interest to CTR. a l -AntitrMsin Dr. Nelson uses a nitroxide spin label in the antiprotease, i.e., N I 0 . Single elect ron signal. By employing purified a 1-AT and trypsin, he has shown that the spectrum shifts from broad peaks to 3 narrow pe aks. This is also seen with elastase and is due to the location of attachment of the probe due to conformational changes with 0~,1-AT and protease interactions. over . .