Abstract
In vitro synthesis of myelin proteolipid protein (PLP) was explored at different ages using rat brain total homogenates, incubated for 30 min with [3H]glycine. Total proteolipids, extracted from the incubated samples, were separated by SDSPAGE and the radioactivity was measured in the band corresponding to myelin PLP. The incorporation into PLP in relation to the incorporation into brain total proteins increased from 0.04% at 10 days of age to 0.63% at 20 days, and declined slowly thereafter. Time course experiments were carried out using brain homogenates obtained from rats of 20 days of age (i.e. at the period of maximal synthesis of PLP). Labeled PLP molecules were already found at 2.5 min of incubation and the incorporation of the label into this protein, relative to the incorporation into total proteins, did not vary throughout the entire incubation time (30 min). Pulsechase experiments using a similar system and adding cycloheximide at different incubation times showed that the appearance of label into mature PLP was immediately blocked by the inhibitor of protein synthesis. These data suggest that PLP is synthesized as such and not as a pre-protein which is subsequently processed to render mature PLP.
Similar content being viewed by others
References
Eng, L., Chao, R., Gerstl, B. Pratt, D., andTavastsjevna, M. 1968. The maturation of human white matter myelin: fractionation of myelin membrane proteins. Biochemistry 7:4455–4465.
Gonzalez-sastre, F. 1970. The protein composition of isolated myelin. J. Neurochem. 17:1049–1056.
Benjamins, J. A., Iwata, R., andHazlett, J. 1978. Kinetics of entry of proteins into myelin membrane. J. Neurochem. 31:1077–1085.
Konat, G. 1981. Intracellular translocation of newly synthesized myelin proteins in the rat brain stem slices. Exp. Neurol. 73:254–266.
Bizzozero, O. A., Pasquini, J. M., andSoto, E. F. 1982. Differential effect of colchicine upon the entry of proteins into myelin and myelin related membranes. Neurochem. Res. 7:1415–1425.
Pereyra, P. M., Braun, P. E., Greenfield, S., andHogan, E. L. 1983. Studies on subcellular fractions which are involved in myelin assembly: labeling of myelin proteins by a double radioisotope approach indicates developmental relationship. J. Neurochem. 41:974–988.
Agrawal, H. C., Randle, C. L., andAgrawal, D. 1982. In vivo acylation of rat brain myelin proteolipid proteins. J. Biol. Chem. 257:4588–4592.
Bizzozero, O. A., Soto, E. F., andPasquini, J. M., 1983. Myelin proteolipid protein is no esterified at the site of synthesis. Neurochem. International. 5:729–736.
Townsend, L. E., Agrawal, D., Benjamins, J. A., andAgrawal, H. C. 1982. In vitro acylation of rat brain myelin proteolipid protein. J. Biol. Chem. 257:9745–9750.
Campagnoni, C. W., Carey, G. D., andCampagnoni, A. T. 1978. Synthesis of myelin basic proteins in the developing mouse brain. Arch. Biochem. Biophys. 190:118–125.
Carey, G. D., andCampagnoni, A. T. 1979. In vitro synthesis of the myelin basic proteins in the developing mouse brain: properties of a homogenate system. J. Neurochem. 33:81–87.
Campagnoni, A. T., Carey, G., andYu, Y. 1980. In vitro synthesis of myelin basic proteins: subcellular site of synthesis. J. Neurochem. 34:677–686.
Yu, Y., andCampagnoni, A. T. 1982. In vitro synthesis of the four mouse myelin basic proteins: evidence for the lack of a metabolic relationship. J. Neurochem. 39:1559–1568.
Folch, J., andLees, M. B. 1951. Proteolipids, a new type of tissue lipoproteins. Their isolation from brain. J. Biol. Chem. 191:807–817.
Folch, J., Lees, M. B., andSloane-Stanley, G. H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:499–509.
Cavanna, R., andRapport, M. M. 1967. An improved preparation of bovine brain proteolipid. J. Lipid. Res. 8:65–68.
Morell, P., Wiggins, R., andGray, M. 1975. Polyacrylamide gel electrophoresis of myelin proteins: a caution. Analyt. Biochem. 68:148–154.
Neville, D. Jr. 1971. Molecular weight determination of protein-dodecyl sulphate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246:6328–6334.
Weber, K., andOsborne, K. 1969. The reliability of molecular weight determination by SDS-PAGE. J. Biol. Chem. 244:4406–4412.
Lowry, Q. H., Rosebrough, N. R., Farr, A. L., andRandall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.
Lees, M. B., andPaxman, S. 1972. Modification of the Lowry procedure for the analysis of proteolipid protein. Analyt. Biochem. 47:184–192.
Campagnoni, A. T., Campagnoni, C. W., Dutton, G. R., andCohen, J. 1976. A regional study of developing rat brain: the accumulation and distribution of proteolipid proteins. J. Neurobiol. 7:313–324.
Bizzozero, O. A., Besio Moreno, M., Pasquini, J. M., Soto, E. F., andGomez, C. J. 1982. Proteolipids from rat brain subcellular fractions: an electrophoretic analysis. Biochem. Biophys. Acta 691:281–292.
Ferguson, K. A. 1964. Starch-gel electrophoresis. Application to the classification of pituitary proteins and polypeptides. Metabolism. 13:985–1002.
Agrawal, H. C., Fujimoto, K., andBurton, R. M. 1976. Accumulation and turnover of the classical Folch-Lees proteolipid proteins in developing and adult rat brain. Biochem. J. 154:265–269.
Johnson, T. C. 1968. Cell-free protein synthesis by mouse brain during early development. J. Neurochem. 15:1189–1194.
Campagnoni, A. T., andHunkeler, M. J. 1980. Synthesis of the myelin proteolipid protein in the developing mouse brain. J. Neurobiol. 11:355–364.
Ramsey, J., andSteele, N. 1977. Quantitative isolation and properties of nearly homogeneous populations of undergraded free and bound polysomes from rat brain. J. Neurochem. 28:517–527.
Shapira, R., Chou, F. C., Wilhelmi, M. R., andKibler, R. F., 1978. “In vitro” incorporation of [3H]leucine into myelin basic protein. Fed. Proc. Am. Socs. Exp. Biol. 37:1784 (abstr.).
Colman, D., Kreibich, G., Frey, A., andSabatini, D. 1982. Biosynthesis of the myelin membrane proteins. Trans. Am. Soc. Neurochem. 13:152.
Dautigny, A., Alliel, P. M., Nussbaum, J. L., andJolles, P. 1983. Cell-free synthesis of rat brain myelin proteolipids and their identification by immunoprecipitation. Biochem. Biophys. Res. Commun. 110:432–437.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bizzozero, O.A., Pasquini, J.M. In vitro synthesis of myelin proteolipid protein from rat brain using a total homogenate system. Neurochem Res 9, 1339–1350 (1984). https://doi.org/10.1007/BF00973044
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00973044