BIOCHEMICAL STUDIES OF MUSCARINE RECEPTORS

本研究计划专门用于纯化和表征

This research proposal is specifically designed to purify and characterize the muscarine receptor from two separate brain regions, forebrain and brainstem. Preliminary work has demonstrated that the muscarine receptor is amenable to purification by affinity chromatography on columns of Sepharose-centrine eluted with carbachol. The current application is a proposal to scale-up the purification procedures, after perfection in rat brain tissue, to work with 60 g. lots of bovine brain tissue, which have about 1. mg of pure receptor. Characterization of the purified receptor will include: measurement of molecular weight by sedimentation equilibrium or sedimentation velocity; determination of the active subunit and the subunit composition of the receptor by SDS gel electrophoresis; and determination of amino acid composition by automated microanalysis and the N-terminal amino acid by microdansylation. Comparison of receptor isolated from these brain regions will enable us to answer the question of whether differences in agonist and antagonist binding in these two regions are due to structurally different receptors. This question is significant because current knowledge, of how the aging process and the related Alzheimer's disease affects brain regions, is limited. It is apparent, however, that there is a significant impairment in cholinergic neurotransmission, confined to only the forebrain and hippocampus, and that the postsynaptic receptor may be a site of the dysfunction. The results of these studies can be directly applied to the design of therapies to specifically target receptors, in a particular brain region, that are defective.