Identification and isolation of an axonal plasma membrane enriched fraction from cerebellar granule cell neurites

A procedure is described to isolate a fraction enriched in cerebellar granule cell neuritic membranes. Morphological markers that are specific for either the granule cell perikarya or neuritic membranes have been identified. Concanavalin A (Con A) has been shown to bind predominantly to the granule cell neurites whereas, the enzymes acetylcholinesterase (AChE) and 2',3',cyclic nucleotide-3'-phosphohydrolase (CNPase) are localized predominantly in the neuronal cell bodies. The membrane fraction enriched in Con A binding has been used to generate a monoclonal antibody which morphologically recognized the cerebellar granule cell neuritic membrane. Following fractionation of the granule cells, each marker was used to identify the cellular origin of the fractions. The neuritic markers Con A and the neuritic membrane antibody MR2 bound predominantly to membranes found in the 29.1% and 31.5% region of the sucrose gradient. The perikaryal markers, CNPase and AChE activity were most enriched in membrane fractions found at a sucrose concentration of 23% and 21%, respectively. Morphological examination of the neuritic enriched fraction shows that it contains predominantly membranous material with few subcellular organelles. The protein profiles of the cerebellar granule cell fractions are unique when compared with the protein profiles of other neuronal and non-neuronal fractions. The membrane fraction isolated from the cerebellar granule cells should prove useful in furthering our understanding of the axonal influence on glial development.