Abstract
Rats were trained to drink alcohol solution by gradually increasing the ethanol content [2.5–15% (v/v)] in drinking water. After 11 months of alcohol (15% v/v) ingestion, animals were guillotined and the spinal cords were used for the preparation of neurofilaments (NF). NF triplet proteins were separated by SDS-PAGE and the phosphate contents of individual components were estimated. Results indicated a significant increase in phosphate content of 200 KD protein in alcohol fed rats (30.19±4.12 mol of phosphate/mole of protein: p<0.001) compared to control group (18.42 ±3.91 mol of phosphate/mole of protein). No significant change in the phosphate content of 150KD and 68KD components of NF were seen in experimental group. Further, the studies on NF associated protein phosphatase activity indicated a significant decrease in phosphatase activity among the alcohol fed rats (14.10±2.5 mU; p<0.001) against NF rich fraction as a substrate, as compared to control (20.15±2.15 mU). While the observed decrease in NF associated protein phosphatase would possibly explain the increase in phosphate content of NF proteins in alcohol fed rats, the precise mechanism of decrease in enzyme activity remains to be elucidated. Nevertheless, the change seen in phosphate content and NF associated protein phosphatase activity as a result of ethanol ingestion would possibly form the biochemical basis of some of the neuropathological changes seen in alcoholics.
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Baginski, E. S., Foa, P. P., and Zak, B. 1967. Determination of rat liver microsomal Glucose-6-phosphatase activity. Study of Citrate and G-6-Pase inhibition. Anal. Biochem. 21:201–207.
Carden, M. J., Schlaepfer, W. W., and Lee, V. M. Y. 1985. The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state. J. Biol. Chem. 260:9805–9817.
Charness, M. E. and Simon, R. P. 1989. Ethanol and the nervous system. N. Engl. J. Med. 321:442–454.
Chiu, E. C., and Norton, W. T. 1982. Bulk preparation of CNS cytoskeleton and the separation of individual neurofilament proteins by gel filtration: Dye-binding characteristics and amino acid compositions. J. Neurochem. 39:1252–1260.
Deitrich, R. A., Dunwiddie, T. V., Harris, R. A., and Erwin, V. G. 1989. Mechanisms of action of ethanol: Initial central nervous system actions. Pharmacol. Rev. 41:489–537.
Eyer, J., Mclean, W. G., and Leterrier, J. F. 1989. Effect of a single dose of B,B′-iminodipropionitrile invivo on the properties of neurofilaments invitro: Comparison with the effect of iminodipropionitrile added directly to neurofilaments in vitro. J. Neurochem. 52:1759–1765.
Goldman, J. E., and Yen, S. H. 1986. Cytoskeletal protein abnormalities in neurodegenerative diseases, Annal. Neurol. 19:209–223.
Harper, C., Kril, J., and Daly, J. 1987. Are We drinking our neurons away?. Br. Med. J. 294:534–536.
Howland, R. D., and Alli, P. 1986. Altered Phosphorylation of rat neuronal cytoskeletal proteins in acrylamide induced neuropathy. Brain. Res. 363:333–339.
Johnson, G. V. W., and Jope, R. S. 1988. Phosphorylation of rat brain cytoskeletal proteins increased after orally administered aluminium. Brain. Res. 456:95–103.
Jones, S. M. and Williams Jr. R. C. 1982. Phosphate content of mammalian neurofilaments. J. Biol. Chem. 257:9902–9905.
Kawahara, H., Cadrin, M., and French, S. W. 1990. Ethanol-induced phosphorylation of cytokeratin in cultured Hepatocytes. Life. Sci. 47:859–863.
Laemmli, E. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680–685.
Lieber, C. S. 1988. Biochemical and molecular basis of alcohol induced injury to liver and other tissues. N. Engl. J. Med. 319:1639–1650.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265–275.
McLane, J. A. 1987. Decreased axonal transport in Rat nerve following acute and chronic exposure. Alcohol. 4:385–389.
Nestler, E. J. and Greengard, P. 1984. eds.) Protein phosphorylation in the nervous system. pp. 81–94. John Wiley & Sons. (Pub). New York
Nhamburo, P. T., Salafsky, B. P., Tabakoff, B., and Hoffman, P. L. 1987. Effect of ethanol on ouabain inhibition of mouse brain (Na+K+) ATP ase activity. Biochem. Pharmacol. 36:2027–2033.
Nixon, R. A., and Lewis, S. E. 1986. Differential turnover of phosphate groups on neurofilament subunits in mammalian neuroncs in vivo. J. Biol. Chem. 26:16298–16301.
Pant, H. C., Virmani, M., and Majchrowicz, E. 1982. Changes in erythrocyte ghost phosphorylation associated with physical dependence upon ethanol. Sub. Alcohol Actions/Missuse, 3:343–351.
Robin, R. A. 1988. Differential response of Adenylate cyclase and ATPase activities after chronic ethanol exposure of PC12 Cells. J. Neurochem. 51:1148–1155.
Saito, T., Lee, J. M., Hoffman, P. L., and Tabakoff, B. 1987. Effect of chronic ethanol treatment on the B-adrenergic receptor-coupled adenylate cyclase system of mouse cerebral cortex, J. Neurochem. 48:1817–1822.
Sapru, M. K., Geetha, H., and Shetty, K. T. 1987. A single reagent method of phosphate estimation for phosphatases assay, Int. J. Biochem. Biophys. 24:340–343.
Shetty, K. T., Veeranna, and Guru, S. C. Neurofilament associated protein phosphatase: Effect of aluminium and neuropsy-choactive drugs, (Communicated).
Sternberger, N. H., Sternberger, L. A., and Ulrich, J. 1985. Aberrant neurofilament phosphorylation in Alzheimer disease, Proc. Natl. Acad. Sci. USA. 83:4274–4276.
Tokutake, S., Hutchison, S. B., Pachter, J. S., and Liem, R. K. H. 1983. A batch wise purification procedure of neurofilament proteins. Anal. Biochem, 135:102–105.
Toru-Delbauffe, D., and Pierre, M. 1983. A Rat brain protein kinase phosphorylating specifically neurofilaments. FEBS. 162:230–234.
Toru-Delbauffe, D., Pierre, M., Osty, J., Chantoux, F., and Francon, J. 1986. Properties of neurofilament kinase, Biochem. J. 235:283–289.
Ulrich, J., Haugh, M., Anderton, B. H., Probst, A., Lautenschlager, C., and His, B. 1987. Alzheimer dementia and Pick's disease: Neurofibrillary tangles and pick bodies are associated with identical phosphorylated neurofilament epitopes. Acta Neuropathol. 73:240–246.
Urbano-Marqueza, A., Estruch, R., Navarro-Lopez, F., Grau, J. M., Mont, L., and Rubin, E. 1989. The effects of alcoholism on skeletal and cardiac muscle. N. Engl. J. Med. 320:409–415.
Veeranna, and Shetty, K. T. 1990. Phosphoserine phosphatase of human brain: partial purification, characterization, regional distribution and effects of modulators including psychoactive drugs. Neurochem. Res. 15:1203–1210.
Wible, B. A., Smith, K. E., and Angelides, K. J. 1989. Resolution and purification of neurofilament-specific kinase. Proc. Natl. Acad. Sci. USA. 86:720–724.
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Guru, S.C., Taranath Shetty, K. & Shankar, S.K. Effect of chronic ethanol ingestion on phosphate content of neurofilament proteins and neurofilament associated protein phosphatase in rat spinal cord. Neurochem Res 16, 1193–1197 (1991). https://doi.org/10.1007/BF00966695
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DOI: https://doi.org/10.1007/BF00966695