Biochemical and Biophysical Research Communications
Interactions between α-conotoxin MI and the Torpedo marmorata receptor α–δ interface
Section snippets
Materials and methods
Soybean l-α-phosphatidylcholine, carbamylcholine, Chloramine-T, N-hydroxysuccinimidyl-4-azido salicylic acid, and V8 proteinase (EC 3.4.21.19) were from Sigma Chemical Co., Saint Louis, MO, USA; modified trypsin (EC 3.4.21.4), was from Promega, Madison, WI, USA; Affi-Gel 10 from Bio-Rad, Richmond, CA, USA, and the carrier-free 125INa from NEN Research Products, New England Nuclear-DuPont Co., Boston, MA, USA. Bromoacetylcholine was synthesized from choline bromide and bromoacetyl bromide from
V8-Proteolytic digestion of the photoaffinity-labeled α and δ subunits
The complex nAChR-ASA-[125I]CnTx MI was submitted to the method of Pedersen and Cohen [5]. Gel bands were electroblotted onto a PVDF membrane, subjected to autoradiography and then sequenced. The autoradiography detected 4 and 3 labeled fragments from modified α and δ subunits, respectively (Fig. 1). Table 1 summarizes N-terminal sequences. The position of the low Mr radiolabeled band in Fig. 1B is coincident with that of the tracking dye. This band also contains a peptide mixture.
Localization of V8 labeled fragments
It was
Acknowledgments
Grants from CONICET and University of Buenos Aires supported this work.
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Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors
2009, Biochemical PharmacologyCitation Excerpt :In earlier works the preparation and characterization of the peptide photoactivatable analogs in labeling of the whole subunits was achieved that first revealed the position of α-conotoxin-binding sites at the interfaces of the adjacent α1- and non-α1 nAChR subunits, as well as the involvement of the peptide N- and C-termini in the formation of complex with nAChR [76,77]. Later the employment of the phoactivatable analogs of α-conotoxins GI and MI resulted in detection of the crosslinked fragments of α1-, γ- and δ-subunits and partial mapping of the α1/γ[78] and α1/δ[79] sites from two Torpedo nAChRs, respectively. It is worth to mention, that goals of α-conotoxin analogs preparation are not limited to structural studies of nAChRs, but embrace numerous tasks (a fairly full list of synthesized α-conotoxin variants and derivatives is given in recent review [80]).
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