RESUMEN
Biospecific sorbents for isolation of the latrotoxin receptor have been obtained and studied. Binding of the receptor components solubilized from the bovine cerebral cortex membrane to the immobilized toxin critically depends on the presence of calcium ions and the solution ionic strength. The procedure for semipreparative isolation of the highly active receptor preparation with Kd = 9 x 10(-10) M and Bmax = 0.9 nmol/mg has been developed. Binding activity of the isolated receptor is inhibited by heating as well as by proteases and denaturing agents. According to electrophoretic analysis in the presence of SDS the receptor complex contains protein components of molecular mass 200, 160, 79, 43 kDa, the former two being glycoproteins.
Asunto(s)
Venenos de Artrópodos/metabolismo , Química Encefálica , Neurotoxinas/metabolismo , Receptores Colinérgicos/aislamiento & purificación , Receptores de Péptidos , Venenos de Araña/metabolismo , Animales , Bovinos , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Cinética , Membranas/metabolismo , Peso Molecular , Receptores Colinérgicos/metabolismoRESUMEN
A presynaptic neurotoxin isolated from the venom of the Central Asia spider karakurt (Black Widow Spider, Latrodectus mactans tredecimguttatus) is shown to consist of two identical subunits of mol. weight about 118 kDa. The iodinated neurotoxin binds to the rat brain synaptosomal plasma membranes with Kd 0.1 nM (Bmax 0.1 pmol/mg of protein) at 37 degrees C, and with Kd 0.35 nM (Bmax 0.2 pmol/mg of protein) at 5 degrees C. At intermediate temperatures both types of receptors are detectable. It is supposed that the dimeric form of the toxin interacts with a single class of receptors possessing lateral mobility in the membrane. By the use of different bifunctional reagents it is revealed that the neurotoxin interacts with a presynaptic membrane protein of mol. weight 95 kDa. A protein of the same size accompanied by a 71 kDa protein was isolated by the affinity chromatography of solubilized synaptosomal membranes on the absorbent, containing immobilized neurotoxin.
Asunto(s)
Venenos de Artrópodos/metabolismo , Encéfalo/metabolismo , Neurotoxinas/metabolismo , Receptores de Neurotransmisores/metabolismo , Venenos de Araña/metabolismo , Animales , Araña Viuda Negra , Cloruro de Calcio/farmacología , Cromatografía en Gel , Concanavalina A/farmacología , Electroforesis Discontinua , Técnicas In Vitro , Cinética , Lectinas/farmacología , Modelos Biológicos , Neurotoxinas/aislamiento & purificación , Ratas , Receptores de Neurotransmisores/efectos de los fármacos , Venenos de Araña/aislamiento & purificación , Aglutininas del Germen de TrigoRESUMEN
Black widow spider toxin (latrotoxin) was studied for its effect on bilayer phospholipid membrane conductance. Under the toxin action membranes became selective with respect to divalent cations. The selectivity decreased in the following order: Ba2+, Sr2+, Ca2+, Mg2+. The ion channels formed by toxin in a planar membrane were effectively blocked by Cd2+. Current-voltage characteristics of the membranes permit concluding that toxin molecules are incorporated into a plane bilayer in the oriented way and the formed channels are potential-dependent.