Characterization and Comparison of Neurofilament Proteins from Rat and Mouse CNS

Abstract

Rat and mouse CNS neurofilament proteins (NFPs) were characterized and compared, in terms of electrophoretic properties on polyacrylamide gels and by peptide mapping, with one another and with other co-purifying lower-molecular-weight CNS proteins, including alpha and beta tubulin. NFPs were partially purified by modification of the axon flotation procedure of Norton and co-workers and were demyelinated with Triton X-100. On one-dimensional SDS polyacrylamide gels the molecular weights of the triad of NFPs from both rat and mouse were approximately 200,000, 140,000, and 70,000. Prominent lower-molecular-weight proteins (63,000-16,000) as well as minor amounts of tubulin and actin were observed after gel electrophoresis. On two-dimensional gels (isoelectric focusing followed by SDS gel electrophoresis) each of the NFPs appeared to be composed of more than one component and the corresponding NFPs from rat and mouse had similar isoelectric points. Gel electrophoresis peptide mapping using Staphylococcus aureus V8 protease indicated the following: (1) the triad of NFPs of different sizes have different peptide maps; (2) alpha and beta tubulin have nonidentical digestion products, which are dissimilar to those of the NFPs; (3) other proteins that co-purify by the axon flotation procedure also have nonidentical peptide maps; and(4) the corresponding NFPs from rat and mouse have similar peptide maps. The co-purifying proteins examined in detail (63,000-49,000) do not appear to be derived by proteolytic cleavage of NFPs and may represent other cytoskeletal constituents.

Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons

“…The myelinated axons were then disrupted by treatment with Triton X-100 (21) and the neurofilaments were purified as previously described (6). Samples were stored at -70°C until used.…”

“…The protein samples used were either homogenized individual gel bands (6,8) or were intact segments of gel lanes using the general procedure of Bordier and Crettol-Jarvinen (4). When intact gel segments were to be digested the procedure was modified by embedding the gel segment directly in the stacking gel (29).…”

“…One-dimensional SDS PAGE was performed by the procedure of Laemmli (20) using 5-15% linear polyacrylamide gradients (6). Long slab gels were used in all experiments (dimensions, 140 × 260 x 1.5 mm3).…”

“…Two-dimensional gels were prepared using the general procedures of O'Farrell (39), modified as previously described (6,29). Gels were stained and fluorographed as described for one-dimensional gels.…”

“…Neurofilaments are believed to be major components of the neuronal cytoskeleton (14,23,46), although their precise structural or functional role has not been clearly established. Pufffled mammalian neurofdaments contain at least three major protein species in the molecular weight classes 68,000-72,000, 140,000-170,000, and 200,000-220,000 (1,6,7,25,57). The association of these proteins in a single organelle was initially suggested by their movement within one phase of axoplasmic transport (-0.2 mm/d) (3,18).…”