Physicochemical and immunological studies of the N-terminal domain of the Torpedo acetylcholine receptor alpha-subunit expressed in Escherichia coli.

The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of alpha2 beta gamma delta subunits. Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail. In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N-terminal fragment 1-209 of the Torpedo californica AChR alpha-subunit in Escherichia coli. Two vectors coding for a (His)6 tag, either preceding or following the 1-209 sequence, were used and the recombinant proteins obtained (designated alpha1-209pET and alpha1-209pQE, respectively) were purified by affinity chromatography on a Ni2+-agarose column. The chemical structure of both proteins was verified by Edman degradation and mass spectrometry. The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents. The two proteins bound [125I]alpha-bungarotoxin with equal potency (KD approximately 130 nm, Bmax approximately 10 nmol.mg-1). Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR. The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly beta-structure (50-56%), the fraction of alpha-helices amounting to 32-35%. Nonionic (beta-octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of beta-structure or increased the alpha-helicity, respectively. The predominance of beta-structure is in accord with recent data on the N-terminal domain of the mouse muscle AChR alpha-subunit expressed in the mammalian cells [West et al. (1997) J. Biol. Chem. 272, 25 468]. Thus, expression in E. coli provides milligram amounts of the protein that retains several structural characteristics of the N-terminal domain of the Torpedo AChR alpha-subunit and appears to share with the latter a similar secondary structure. The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis.

Prevalencia de leishmaniasis visceral Humana y canina en la ciudad de Lambaré, Paraguay / Humana and canine visceral leishmaniasis in Lambaré city, Paraguay

Estudio de una muestra representiva y aleatoria de la población de perros de toda la ciudad de Lambaré, para determinar la prevalencia de los casos de LV canina y a la búsqueda activa de personas de la misma ciudad, consideradas con riesgo de contagio para LV, a fin de determinar la ocurrencia de casos de LV en humanos, un total de 348 muestras de sangre canina y 930 personas fueron analizadas analizadas

Labeling of Torpedo californica nicotinic acetylcholine receptor subunits by cobratoxin derivatives with photoactivatable groups of different chemical nature at Lys23.

Different photoactivatable derivatives of toxin 3 (CTX) Naja naja siamensis were obtained after CTX reaction with N-hydroxysuccinimide esters of p-azidobenzoic, p-azidotetraflourobenzoic, p-benzoylbenzoic and p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acids. The ion-exchange HPLC profiles for the reaction products were very similar in four cases, with one predominant peak corresponding to the derivative containing the label at Lys23. After [125I]iodination, CTX photoactivatable derivatives were cross-linked to the nicotinic acetylcholine receptor from Torpedo californica under optimized conditions. The highest cross-linking yield (up to 16% of the bound toxin) was observed for azidobenzoyl-Lys23-CTX. Different receptor subunits were found to be labelled depending on the nature of the photoactivatable group: the azido derivatives labelled the gamma and delta subunits, benzoylbenzoyl derivative labelled the alpha and delta subunits, while p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl derivative reacted with alpha, gamma and delta subunits. The cross-linking experiments in the presence of varying concentrations of (+)-tubocurarine demonstrated that the Lys23-attached diazirinyl group contacts the delta and alpha subunits in one ligand-binding site, whereas at the other site, for another CTX molecule, the contacts of the Lys23-diazirinyl are with gamma and alpha subunits. This means that the central loop in the two CTX molecules binds at the alpha/gamma and alpha/delta interfaces. Calculation of the sterically possible displacement of diazirinyl nitrogen, basing on the known X-ray structure of CTX, showed that this value does not exceed 13 A. The results obtained favor the disposition of the ligand-binding sites at the subunit interfaces, with the distance between alpha and delta, or alpha and gamma subunits at these sites being not more than 13 A.