Studies on subcellular fractions which are involved in myelin membrane assembly: isolation from developing mouse brain and characterization by enzyme markers, electron microscopy, and electrophoresis.
An extensive scheme for the subcellular fractionation of myelinating mouse brain is presented. Several centrifugation procedures for the separation of membranes involved in myelinogenesis are critically appraised, and guidelines for selection of centrifugation conditions are given. Characteristics of subcellular fractions are presented in the form of electron micrographs; also presented are distribution of RNA and protein; electrophoretic profiles of membrane proteins, and verification of the myelin-specific basic proteins, proteolipid protein, and glycoprotein by the immuno-electroblot technique; and the distribution of eight marker enzyme activities. Myelin-related membranes were found to differ both qualitatively and quantitatively in their complement of myelin-specific proteins. These myelin-containing fractions appear to represent different stages of myelination that coexist in developing mouse brain. These results provide the fundamental methodologies and background information for kinetic radioisotope analysis of intracellular events in the assembly of myelin presented in a companion article.
D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000032:analysis; D000818:Animals; D001921:Brain / Q000254:growth & development* / Q000648:ultrastructure; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods; D006056:Golgi Apparatus / Q000201:enzymology; D051379:Mice; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology; D009185:Myelin Proteins / Q000032:analysis; D009186:Myelin Sheath / Q000032:analysis / Q000648:ultrastructure*; D009419:Nerve Tissue Proteins / Q000032:analysis; D010727:Phosphoric Diester Hydrolases; D012313:RNA / Q000032:analysis
D009185:Myelin Proteins; D009419:Nerve Tissue Proteins; D012313:RNA; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases; D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; C571338:Cnp protein, mouse