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Molecular characterization of a non-glycinergic strychnine binding site in the rodent spinal cord

Holman, Hanqing Guo; (1996) Molecular characterization of a non-glycinergic strychnine binding site in the rodent spinal cord. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Treatment of rat or mouse spinal cord membranes with the arginine specific reagent 2,3-butanedione reveals strychnine binding sites which are not located on the inhibitory glycine receptor and which display a specificity for use-dependent cation channel blockers. The binding properties of these binding sites do not survive solubilisation in a variety of detergents and so cannot be purified by conventional affinity chromatography. However, as a first step towards investigating the possible function of this binding site as an ion channel, procedures have been developed for its affinity labelling and purification. Fractionation of rat spinal cord membranes on sucrose gradients showed the 2,3-butanedione induced binding of [3H]-strychnine to be enriched in the myelin rather than in the synaptosomal or mitochondrial fractions. Further subfractionation of myelin located this binding site to the peraxolemmal myelin, as opposed to the axolemmal or compact myelin fractions. Previous studies had shown quinacrine to be a high affinity ligand for the 2,3- butanedione induced [3H]-strychnine binding site and therefore quinacrine mustard has been employed as an irreversible affinity label. Incubation of crude spinal cord membranes or purified myelin with the dichloroethylamino derivative of quinacrine inactivated at least 50% of subsequent [3H]-strychnine binding. Subsequent solubilisation of such membranes in sodium dodecylsulphate followed by SDS polyacrylamide gel electrophoresis revealed a single fluorescent band at 23 kD. Both the inactivation of subsequent [3H]-strychnine binding and the fluorescent labelling of this peptide were reduced if membranes were treated with quinacrine mustard in the presence of strychnine. When employed with myelin membranes, affinity labelling with quinacrine mustard identifies a flourescent peptide on SDS-PAGE in sufficient quantities for sequencing, which reveals the 2,3-butanedione induced strychnine binding site to be located on a previously undescribed isoform of proteolipid protein.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Molecular characterization of a non-glycinergic strychnine binding site in the rodent spinal cord
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Spinal cord
URI: https://discovery.ucl.ac.uk/id/eprint/10103412
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