Abstract
Neurofilament proteins from brain and spinal cord of immature rat (20–35 days of age) and rabbit (15–17 days of age) were prepared by an axonal flotation technique. Examination of rat filament preparations by electron microscopy revealed a preponderance of 10 nm diameter filaments that were usually loosely aggregated although some bundles of more tightly packed filaments were present as well. The neurofilament triplet proteins of the rat and rabbit central nervous system were found to be phosphorylated 24 hr after the intracerebral injection of [32P]orthophosphate when examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography. Examination of each eluted neurofilament protein from both species showed that [32P]phosphate was retained after reelectrophoresis and fluorography. Evidence is presented that the [32P] phosphate is covalently linked to the purified neurofilament proteins by phosphoester bonds.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Schlaepfer, W. W., andFreeman, L. A. 1978. Neurofilament proteins of rat peripheral nerve and spinal cord. J. Cell Biol. 78:653–662.
Norton, W. T., andGoldman, J. E. 1980. Neurofilaments. Pages 301–329in Bradshaw, R. A. andSchneider, D. M. (eds.), Proteins of the Nervous System, 2nd edition, Raven Press, New York.
Willard, M., andSimon, C. (1981). Antibody decoration of neurofilaments. J. Cell Biol. 89:198–205.
Eagles, P. A. M., Gilbert, D. S., Hopkins, J. M., Maggs, A., andWais, C. (1980). Neurofilament structure and enzymatic modification. Cell Biol. Int. Rep. 4:731 (Abs.).
Pant, H. C., Shecket, G., Gainer, H., andLasek, R. J. (1978). Neurofilament protein is phosphorylated in the squid giant axon. J. Cell Biol. 78:R23-R27.
Pant, H. C., Yoshioka, T., Tasaki, I., andGainer, H. (1979). Divalent cation dependent phosphorylation of proteins in squid giant axon. Brain Res. 162:303–313.
Shecket, G., andLasek, R. (1978). Phosphorylation of 10 nm neurofilaments. Page 48,in Goldman, R. D. andOlmsted, J. B. (eds.), The Cytoskeletal and Contractile Networks of Nonmuscle Cells, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Shecket, G., andLasek, R. J. (1979). Phosphorylation of neurofilaments from mammalian peripheral nerve. Trans. Am. Soc. Neurochem. 10:140.
Eagles, P., Bell, C., Maggs, A., Wais, C., andGilbert, D. (1978). Neurofilament structure and its enzymatic modification. Page 47,in Goldman, R. D. andOlmsted, J. B. (eds.), The Cytoskeletal and Contractile Networks of Nonmuscle Cells, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Runge, M. S., El-Maghrabi, M. R., Claus, T. H., Pilkis, S. J., andWilliams, R. C., Jr. 1981. A MAP-2-stimulated protein kinase activity associated with neurofilaments. Biochemistry 20:175–180.
Chiu, F.-C., Korey, B., andNorton, W. T. 1980. Intermediate filaments from bovine, rat and human CNS: Mapping analysis of the major proteins. J. Neurochem. 34:1149–1159.
Bunge, M. B. (1977). Initial endocytosis of peroxidase or ferritin by growth cones of cultured nerve cells. J. Neurocytol. 6:407–439.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., andRandall, R. J. 1951, Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.
Neville, D. M., Jr. 1971. Molecular weight determinations of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246:6328–6334.
Agrawal, H. C., Burton, R. M., Fishman, M. A., Mitchell, R. F., andPrensky, A. L. 1972. Partial characterization of a new myelin protein band. J. Neurochem. 19:2083–2089.
Bonner, W. M., andLaskey, R. A. 1974. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83–88.
Fishman, M. A., Hagen, S., Trotter, J. L., O'Connell, K., andAgrawal, H. C. 1979. Use of a stable fluorescent reagent, 2-methoxy-2,4-diphenyl-3(2H)-furanone, for the visualization and purification of myelin proteins. J. Neurochem. 32:1077–1083.
Willard, M. B. 1973. Genetically determined protein polymorphism in the rabbit nervous system. Proc. Natl. Acad. Sci. 73:3614–3645.
Selkoe, D. J., Liem, R. K. H., Yen, S.-H. andShelanski, M. L. 1979. Biochemical and immunological characterization of neurofilaments in experimental neurofibrillary degeneration induced by aluminum. Brain Res. 163:235–252.
Czosnek, H., andSoifer, D. 1980. Comparison of the proteins of 10 nm filaments from rabbit sciatic nerve and spinal cord by electrophoresis in two dimensions. FEBS Lett. 117:175–178.
Levine, J., andWillard, M. 1981. Fodrin: Axonally transported polypeptides associated with the internal periphery of many cells. J. Cell. Biol. 90:631–643.
Johnson, E. M., Maeno, H., andGreengard, P. 1971. Phosphorylation of endogenous protein of rat brain by cyclic adenosine 3′, 5′-monophosphate-dependent protein kinase. J. Biol. Chem. 246:7731–7739.
Julien, J.-P., andMushynski, W. E. (1981). The in vitro and in vivo phosphorylation of rat neurofilaments. Trans. Am. Soc. Neurochem. 12:132 (Abs.).
Nixon, R. A., Brown, B. A., andMarotta, C. A. 1981. Modification of neurofilament proteins in the mouse retinal ganglion cell. Trans. Am. Soc. Neurochem. 12:172 (Abs.).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Honchar, M.P., Bunge, M.B. & Agrawal, H.C. In vivo phosphorylation of neurofilament proteins in the central nervous system of immature rat and rabbit. Neurochem Res 7, 365–372 (1982). https://doi.org/10.1007/BF00965490
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00965490