The polypeptides of isolated brain 10nm filaments and their association with polymerized tubulin

Thorpe, Robin; Delacourte, André; Ayers, Margaret; Bullock, Clive; Anderton, Brian H.

doi:10.1042/bj1810275

Cited by 57 publications

(29 citation statements)

References 39 publications

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“…We have tried unsuccessfully to resolve the 160-210K overlap fractions by preparative isoelectric focusing. The difference between the reported isoelectric points of these two proteins-5.1 for 160K and 5.6 for 210K (Thorpe et al, 1979;Brown et al, 1981)-is, theoretically, sufficient for separation with conventional, narrow-range isoelectric focusing. However, in the presence of 8 M urea the two proteins overlapped extensively in spite of a well-formed pH gra- Liem and Hutchison, 1982), our efforts to resolve the 160-210K fractions by this technique were unsuccessful.…”

Section: A F a S T G R E Ementioning

confidence: 87%

Bulk Preparation of CNS Cytoskeleton and the Separation of Individual Neurofilament Proteins by Gel Filtration: Dye‐Binding Characteristics and Amino Acid Compositions

Chiu

Norton

1982

Journal of Neurochemistry

154

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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.

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Section: A F a S T G R E Ementioning

confidence: 87%

Bulk Preparation of CNS Cytoskeleton and the Separation of Individual Neurofilament Proteins by Gel Filtration: Dye‐Binding Characteristics and Amino Acid Compositions

Chiu

Norton

1982

Journal of Neurochemistry

154

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“…It has been well documented in the literature that mammalian 10-nm neurofilaments contain a 68,000-dalton polypeptide as their major constituent (8,12,15,16 (27). Antibodies to this protein were shown by immunofluorescence to decorate cytoplasmic filamentous structures that resemble microtubules.…”

Section: Discussionmentioning

confidence: 99%

“…These three proteins have been assumed to be the subunit constituents of neurofilaments because they copurify with the filaments and they are not dissociated from them under a variety of chemical treatments. Recently it has been demonstrated that the 68,000-dalton polypeptide is present in conventionally purified microtubules from brain that are contaminated with 10-nm neurofilaments (14)(15)(16).…”

mentioning

confidence: 99%

The 68,000-dalton neurofilament-associated polypeptide is a component of nonneuronal cells and of skeletal myofibrils

Wang

Asai

Lazarides

1980

Proc. Natl. Acad. Sci. U.S.A.

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Purified preparations of 10-nm neurofilaments from rat spinal cord and bovine or porcine brain contain a predominant 68,000-dalton polypeptide. This polypeptide is also a major component of the neurofilaments that copurify with brain tubulin isolated by cycles of polymerization and depolymerization. A protein that has the same isoelectric point and molecular weight as the neurofilament-associated polypeptide has also been identified as a cytoskeletal protein in a variety of avian and mammalian cell types, including baby hamster kidney (BHK-21) mouse 3T3, Novikoff rat hepatoma, chicken fibroblast, and chicken muscle cells. This protein is also a component of isolated phicken skeletal myofibrils. One-dimensional peptide maps of the 68,000-dalton proteins purified by twodimensional isoelectric focusing/NaDodSO4/polyacrylamide gel electrophoresis from myofibrils, cycled tubulin, purified neurofilaments, and various cultured cell types were identical. In immunofluorescence this protein was associated with cytoplasmic intermediate filaments and myofibril Z discs. These results indicate that the neurofilament-associated polypeptide is a conserved protein that is present in many different cell types in addition to neuronal cells.

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“…Axoplasmic transport studies show the triplet to be present in vivo (36). Therefore, if there is a common precursor, processing must be done in the cell body (5 has revealed no homology among the neurofilament polypeptides (14)(15)(16)(17)(18)(19)(20), implying an independent origin for each. However, Julien and Mushynski (37) isolated a segment with a common amino acid sequence.…”

Section: Discussionmentioning

confidence: 99%

“…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)(15)(16)(17)(18)(19)(20) and point to an independent origin for each.…”

mentioning

confidence: 99%

Microheterogeneity ("neurotypy") of neurofilament proteins.

Goldstein¹,

Sternberger²,

Sternberger³

1983

Proc. Natl. Acad. Sci. U.S.A.

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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...

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The polypeptides of isolated brain 10nm filaments and their association with polymerized tubulin

Cited by 57 publications

References 39 publications

Bulk Preparation of CNS Cytoskeleton and the Separation of Individual Neurofilament Proteins by Gel Filtration: Dye‐Binding Characteristics and Amino Acid Compositions

Bulk Preparation of CNS Cytoskeleton and the Separation of Individual Neurofilament Proteins by Gel Filtration: Dye‐Binding Characteristics and Amino Acid Compositions

The 68,000-dalton neurofilament-associated polypeptide is a component of nonneuronal cells and of skeletal myofibrils

Microheterogeneity ("neurotypy") of neurofilament proteins.

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