Bulk preparation of CNS cytoskeleton and the separation of individual neurofilament proteins by gel filtration: dye-binding characteristics and amino acid compositions.
The three major proteins of mammalian neurofilaments, of molecular weight 70,000, 160,000, and 210,000, have been resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and more recently, by ion-exchange chromatography in urea solution. We describe here a method to separate the neurofilament proteins by gel filtration without the use of SDS. A bulk preparation of cytoskeleton from rat spinal cord was first characterized. This preparation was then solubilized in a buffer containing 8 M urea and subjected to gel filtration. Individual neurofilament proteins, in milligram quantities, were harvested following the pooling of appropriate fractions. Gel electrophoresis showed a high degree of homogeneity in each of the three pooled fractions. Dye binding studies demonstrated that the protein of molecular weight 210,000 was relatively underrepresented when stained with Coomassie Blue, while all three neurofilament proteins showed similar dye binding properties with Fast Green. Amino acid analysis indicated that (1) all three neurofilament proteins contained a high content of acidic residues; (2) the molecular weight 210,000 protein contained greater than 8 mol% proline; and (3) no simple oligomeric relationship existed among the neurofilament triplets.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation; D002850:Chromatography, Gel; D004396:Coloring Agents; D003599:Cytoskeleton / Q000032:analysis*; D007381:Intermediate Filament Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D051381:Rats; D012394:Rosaniline Dyes; D013116:Spinal Cord / Q000648:ultrastructure*
D000596:Amino Acids; D004396:Coloring Agents; D007381:Intermediate Filament Proteins; D012394:Rosaniline Dyes; C024757:Coomassie brilliant blue R; C035906:Fast Green