Neurofilaments were isolated from desheathed and minced segments of rat peripheral nerve by osmotic shock into 0.01 M Tris-HCI buffer, pH 7.2. Freshly isolated neurofilaments were observed to undergo disassembly by progressive fragmentation upon exposure of dilute tissue extracts to this buffer. Low- and high-speed centrifugations of these tissue extracts separated membranous and particulate constituents and produced a progressive enrichment of 68,000-dalton polypeptide band in successive supernates, as determined by analyses of soluble proteins by SDS-polyacrylamide electrophoresis. The final high-speed supernatant fractions (S3) of nerve extracts, which were predominantly composed of 68,000-dalton polypeptide, were used to raise a specific experimental antisera in rabbits.
Utilizing techniques of immune electron microscopy, experimental rabbit antisear was shown to contain antibodies against neurofilaments. Intact neurofilaments isolated from rat nerves and attached to carbon-coated grids became decorated when exposed to experimental rabbit antisera or purified gamma globulin (IgG) derivatives. The decoration of neurofilaments closely resembled the IgG coating seen in immune electron microscopy. Antibody absorption techniques were used to identify the biochemical constituency of neurofilamentous antigenic determinants. The decoration of neurofilament by experimental IgG was not altered by additions of tubulin or bovine serum albumin, but was prevented by additions of S3 fractions as well as the 68,000-dalton polypeptide of this fraction which was eluted and recovered from polyacrylamide gels.
These findings are indicative that a 68,000-dalton polypeptide is a constituent subunit of rat peripheral nerve neurofilaments.