Studies on the Biosynthesis of Intermediate Filament Proteins in the Rat CNS

Abstract

The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isolated by immunoaffinity chromatography and then analyzed by 2-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population synthesized 2 neurofilament proteins (MW 145K [kilodaltons], pI [isoelectric point] 5.4 and MW 70K, pI 5.3) and 3 isomers of GFA (.alpha., .beta. and .gamma.) that differ in pI. Wheat germ homogenate containing mRNA extracted from free cord polysomes synthesized 2 proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the .alpha., .beta. and .gamma. isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Synthesis of neurofilament proteins requires multiple mRNA. Synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthesized by the rough microsomes.