Heterodimeric Associations between Neuronal Intermediate Filament Proteins*
- From the Department of Biochemistry, McGill University, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada
Abstract
Formation of protein dimers involving α-internexin, peripherin, and the neurofilament (NF) proteins NFH, NFM, and NFL was investigated by partial renaturation of various combinations of individually purified subunits in buffered 2m urea. Oligomers that were formed were resolved by “blue” native electrophoresis (Schägger, H., Cramer, W. A., and von Jagow, G. (1994) Anal. Biochem. 217, 220–230) modified to include urea in the polyacrylamide gels. Combining this method with Western blot analysis, disulfide cross-linking, and SDS-polyacrylamide gel electrophoresis in the second dimension showed that NFL readily forms significant amounts of heterodimer with NFH, NFM, α-internexin, or peripherin in the presence of 2 murea. α-Internexin and peripherin also formed heterodimers with NFH or NFM under these conditions. The modified version of blue native gel electrophoresis described here may be useful in monitoring the impact of post-translational modifications and mutations on the dimerization of intermediate filament proteins.
Footnotes
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↵* This work was supported in part by a grant from the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Recipient of a studentship from the Fonds de la Recherche en Santé du Québec.
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↵§ To whom correspondence and reprint requests should be addressed. Tel.: 514-398-7286; Fax: 514-398-7384.
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↵1 The abbreviations used are: IF, intermediate filament; nIF, neuronal intermediate filament; CuP2, copper phenanthroline; DRG, dorsal root ganglion; NF, neurofilament; NFH, NFM, and NFL, heavy, mid-sized, and low molecular mass neurofilament subunits, respectively; BN-PAGE, blue native polyacrylamide gel electrophoresis; DTT, dithiothreitol; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PVDF, polyvinylidene fluoride; mAb, monoclonal antibody.
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- Received July 14, 1997.
- Revision received September 18, 1997.
