Purification of the putative alpha-latrotoxin receptor from bovine synaptosomal membranes in an active binding form.
alpha-Latrotoxin (alpha-LTx, apparent mol. wt. 130 000) is a presynaptically active neurotoxin purified from the venom of the black widow spider that causes massive exocytotic release of neurotransmitters, presumably via binding to presynaptic membrane protein(s). Solubilization and purification experiments were undertaken to identify and characterize this membrane component. An immunoaffinity matrix was prepared by sequentially binding anti-alpha-LTx antibodies and alpha-LTx to Protein A-Sepharose CL-4B. Beads were irreversibly cross-linked with dimethyl pimelimidate. These beads were capable of extracting alpha-LTx binding activity from Triton X-100 solubilized bovine synaptosomal membranes. Following extensive washing, bound material was eluted with 6 M urea. Analysis of silver stained and radiolabel-containing gels revealed one major band (apparent mol. wt. 200 000) under non-reducing conditions and two major bands (apparent mol. wts. 66 000 and 54 000) under reducing conditions. The purified material was still capable of specifically binding alpha-LTx as determined by solid phase assays on microtiter plates. The affinity for alpha-LTx of the purified preparation was similar to that of the native membrane (KA approximately 10(10) M). It is concluded that a putative alpha-LTx receptor protein can be purified from synaptosomal membranes using an immunoaffinity matrix in a form that retains its defined biological property (alpha-LTx binding).
Scheer
H
H
Meldolesi
J
J
D000818:Animals; D001180:Arthropod Venoms / Q000378:metabolism*; D001923:Brain Chemistry; D002417:Cattle; D002540:Cerebral Cortex / Q000032:analysis; D008970:Molecular Weight; D018377:Neurotransmitter Agents / Q000378:metabolism; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D018000:Receptors, Peptide; D012995:Solubility; D013111:Spider Venoms / Q000378:metabolism*; D013574:Synaptosomes / Q000032:analysis*
D001180:Arthropod Venoms; D018377:Neurotransmitter Agents; D011950:Receptors, Cholinergic; D018000:Receptors, Peptide; D013111:Spider Venoms; C034800:alpha-latrotoxin receptor