CELLULAR AND MOLECULAR APPROACHES TO NEUROTOXICOLOGY

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The calcium-requlated activity of chromaffin cells provide a well-studied system for investigating molecular and cell-surface mediated mechanisms of neurotoxin action. The storage granules of these cells, chromaffin granules, accumulate large concentrations of catecholamines and ATP which are eventually released by exocytosis. Isolated chromaffin granules will aggregate and fuse in the presence of calcium. We have been exploring the molecular basis of these activities. Fluorescent-labelled lipid probes have been successfully inserted into chromaffin granule membranes in vitro without altering the storage properties of the particles. Resonance energy transfer studies of calcium-promoted fusion of these membranes show that, unlike artificial phospholipid vesicles, fusion runs 5-10 fold slower than aggregation. These results support the previous findings that substantial rearrangement of the protein and lipid components of the membrane is required for fusion to occur. This in vitro fusion is inhibited by both organic and inorganic monovalent anions and cations and is insensitive to the presence of Mg-ATP. It is abolished by lysis and resealing the granules. Neurotoxins are known to disrupt the structure of myelin. Myelin basic protein (MBP), which accounts for 30 percent of CNS meylin proteins, has no known physiological function, although injection of purified MBP will cause experimental Ascending Encephalomyelities (EAE), considered by some as a model for Multiple Sclerosis. A molecular model for the structure of MBP generates a series of testable predictions. We have been examining the structural properties of MBP using fluorescence and optical spectroscopy. Contrary to many reports, we find evidence for extensive long range structural specificity of myelin basic protein in agreement with the model. These studies may lead to a rapid, more precise functional assay for MBP than induction of EAE.