Biochemical characterization of postsynaptic membrane proteins

摘要

To comprehend the events that occur at the mammalian neuromuscular junction, one must correlate the release of neurotransmitter and its binding to receptors in the postsynaptic membrane, with the subcellular structures and molecular constituents localized to this region. The complexity of the specializations and structures involved in forming the mammalian neuromuscular junction has stimulated interest in identifying junction-associated proteins involved in its organization. Through the use of the Torpedo electric organ, membrane preparations highly enriched in AChRs have been obtained. In addition to the AChR, one might assume that junction specific proteins may also be present. Antibodies made against Torpedo AChR-enriched membrane preparations have in fact demonstrated that several non-AChR postsynaptic proteins are present. Three such antibodies, mabs 1403, 1351, and 1808 have been used to biochemically and immunologically characterize the M$sb{rm r}$ 55,000, 58,000 and 280,000 proteins respectively, from these Torpedo preparations.;The Torpedo 55K protein has an acidic pI, is insoluble in buffers containing 1% Triton X-100, and its distribution in the muscle is different from other known postsynaptic proteins. Further characterization of mab 1403 on mammalian tissues revealed that it specifically recognized the intermediate filament desmin. The 58K protein has been localized in Torpedo electrocytes to the cytoplasmic side of the postsynaptic membrane by immunogold labeling. It can be removed from the membranes through the use of chaotropic agents, and on two-dimensional gels behaves as a group of charge variants with isoelectric points of 6.4-6.7. The 58K protein is found concentrated at AChR-enriched sites in vertebrate muscle, but is also diffusely distributed throughout the extrasynaptic membrane. Amino terminal sequence analysis of the protein immunoaffinity-purified from Torpedo electric organ suggests that the 58K protein is unique. The 280K protein has a similar distribution in the muscle as that of the 58K protein. Although unlike the 43K and 58K proteins, the 280K protein is not removed from the membrane by low concentrations of chaoptopic agents or alkaline pH. Using a competitive ELISA assay, two mabs have been analyzed in their ability to bind to distinct epitopes of the 280K protein. Thus, my thesis has involved the characterization of three postsynaptic proteins from Torpedo electric organ and the examination of their mammalian counterparts.