Microheterogeneity ("neurotypy") of neurofilament proteins.

Abstract

Neurofilaments purified from adult rat brainstem by two methods were electrophoresed on NaDodSO4/polyacrylamide gels to separate the triplet proteins (approximate Mrs of 200,000, 155,000, and 68,000) which, in turn, were electroblotted onto nitrocellulose paper. On Coomassie blue-stained gels that were not electroblotted, the same banding pattern was seen with both methods of preparation. Immunocytochemical staining of the electroblots with each of five monoclonal antibodies revealed that three of the monoclonal antibodies were specific for the Mr 200,000 neurofilament protein and two, for both the Mrs 200,000 and 155,000 neurofilament proteins. None of the antibodies reacted with the Mr 68,000 band. The Mr 200,000 band could be resolved into doublet bands. Individual monoclonal antibodies reacted with either one or both of the Mr 200,000 doublets. The immunocytochemical staining of the neurofilament triplets on electroblots was compared to that of adult rat cerebellar paraffin sections. Each monoclonal antibody had a unique pattern of staining, reacting only with certain subpopulations of neurons or their processes. Correlation of the staining patterns in cerebellar tissue sections with those of neurofilament polypeptides on electroblots suggested that different neurofilament polypeptides can be localized to different structures and subpopulations of neurons and that molecular heterogeneity ("neurotypy") may be revealed within the MrS 200,000 and 155,000 neurofilament polypeptides.Neurofilaments are neuron-specific intermediate filaments with a diameter of 10 nm and consist of the neurofilament triplet, three major polypeptides with Mrs of 200,000, 150,000, and 68,000 (1-5). The origin of the triplet is uncertain. Immunological crossreactivity between the neurofilament polypeptides with polyclonal antibodies suggests common antigenic sites (1,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and implies that the neurofilament proteins are derived from a common precursor (15). On the other hand, peptide maps reveal little homology among the neurofilament polypeptides (14-20) and point to an independent origin for each.Monoclonal antibodies to a single neurofilament polypeptide would be useful to explore the origin, structure, and organization of neurofilaments and their polypeptides. Monoclonal antibodies that recognize unshared (21) and shared (21, 22) determinants on the three polypeptides have been reported.In the present study, we have produced five monoclonal antibodies, each of which recognizes one or more of the neurofilament polypeptides. The staining, by each monoclonal antibody, of neurofilament proteins separated by gel electrophoresis and electroblot transfer onto nitrocellulose paper has provided additional evidence for the existence of one or more common antigenic sites. Comparison of these electroblots to the staining of rat cerebellar tissue sections has allowed for closer examination of the structure and distribution of neurofilaments and their polypeptides in the brain. The different staining patter...

Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.

“…Thus, all structures stained by antibodies of the anti-neurofibrillar group were not stained by antibodies from the anti-perikaryonal-neurofibrillar group, or vice versa. Nevertheless, analysis of electroblots of brain homogenates, cytoskeletal preparations (3,4), and isolated neurofilaments (5) showed that antibodies of both groups reacted exclusively with doublet Mr 200,000 bands and one Mr 150,000 band of the Mr neurofilament triplet (three major polypeptides of Mrs 200,000, 150,000, and 68,000) (6). These data and differences in immunocytochemical reaction of each antibody in each group suggested two levels of heterogeneity of neurofilaments, one detectable by difference of reaction between the anti-neurofibrillar and anti-perikaryonal-neurofibrillar group and the other by differences with antibodies within each of these groups.…”

“…The incubated electroblots and the paraffin sections were stained immunocytochemically (9, 10) with mouse peroxidaseanti-peroxidase prepared from monoclonal anti-peroxidase (Clono PAP) (2,6,11) tibodies 06-17, 04-7, and 07-5 (Fig. 2).…”

“…§1734 solely to indicate this fact. (4) were applied in amounts ranging from 2 to 10 ,ug on gels containing 8% acrylamide, 0.375 M Tris-HCl (pH 8.8), and 0.1% NaDodSO4 and were electrophoresed and electroblotted along with molecular weight standards by the method of Towbin et al (8) as detailed (6). One strip containing M, standards and one containing cytoskeletal preparations in each blot were stained by Coomassie blue, and the rest of the strips containing cytoskeletal preparations were incubated at 320C for 2.5 hr either in 0.1 M Tris HCl, pH 8.0/0.01 M phenylmethylsulfonyl fluoride containing 43 Ag of calf intestinal alkaline phosphatase (type VII, Sigma) per ml or in the same solution devoid of phosphatase.…”

The entorhinal cortex of the Megachiroptera: a comparative study of Wahlberg’s epauletted fruit bat and the straw-coloured fruit bat

“…This may imply that the 2 bat species may not be as phylogenetically close as we presume. It could also be that the EC is a less adept model for this hypothesis, and a lack of immunoreactivity in the EC of Strawcoloured fruit bat, may indicate that the function subserved by SMI-32 immunoreactive cytoskeletal components may either not be required or is assumed by another protein (Campbell and Morrison 1989;Goldstein et al 1983). There are similar reports of a lack of SMI-32 immunoreactivity in the EC that include an Australian echidna (Tachyglossus aculeatus) and the Tamar wallaby (Macropus eugenii) Hassiotis et al 2004;Hassiotis et al 2005).…”

<b>Perikaryal proteins that react with an antibody against the 220 K component of axonal </b><b>neurofilaments </b>

“…Proteins separated in two-dimensional gels were blotted onto a nitrocellulose paper and allowed to react with the anti-220 K antibody. Bound antibody was stained with the peroxidase-antiperoxidase system (2,14). 170 K and 220 K components (Fig.…”