SYNTHESIS OF NITRODIAZIRINYL DERIVATIVES OF NEUROTOXIN-II FROM NAJA-NAJA-OXIANA AND THEIR INTERACTION WITH THE TORPEDO-CALIFORNICA NICOTINIC ACETYLCHOLINE-RECEPTOR

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-I I) from Naja naja oxiana were obtained after NT-II reaction with N-hyd roxysuccinimide ester of 3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy}a cetic acid followed by chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis . Tryptic digests were separated by reversed phase-HPLC, the labeled p eptides being identified by mass spectrometry. The derivatives contain ing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 w ere [I-125]iodinated by the chloramine T procedure. Each iodinated der ivative was found to form photoinduced cross-links with the membrane-b ound nicotinic acetylcholine receptor (AChR) from Torpedo californica. The pattern of labeling the receptor's alpha, beta, gamma, or delta s ubunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series of p -azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AC hR binding, and (ii) fragments of the different AChR subunits are loca ted close together at the neurotoxin-binding sites.