1887

Abstract

SUMMARY

Plasma membrane-enriched preparations from scrapie-infected and healthy hamster brains, as well as preparations of neural retina, were sonicated, then separated by rate-zonal sedimentation in 10 to 25% Nycodenz® gradients. Gradient fractions were extracted with 0.5% Triton X-100 and re-fractionated by equilibrium density centrifugation in linear 25 to 40% CsCl gradients. Infectivity was highest in a fraction having a density of 1.280 g/ml and which contained a visible band of material. Digestion of the Nycodenz fractions with proteinase K before detergent extraction and CsCl fractionation resulted in a shift in the visible band to a density of 1.235 g/ml with most of the scrapie infectivity remaining at 1.280 g/ml. When labelled with I after 40-fold concentration, this 1.280 g/ml CsCl fraction from the proteinase K-treated gradients contained only a single band of protein(s) having a mol. wt. near 30000. No differences were seen between proteins in healthy or scrapie-infected preparations.

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1984-02-01
2024-04-19
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References

  1. Bartlett G. R. 1959; Phosphorus assay in column chromatography. Journal of Biological Chemistry 234:466–469
    [Google Scholar]
  2. Bligh E. G., Dryer W. J. 1959; A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 55:297–302
    [Google Scholar]
  3. Bolton D. C., McKinley M. P., Prusiner S. B. 1982; Identification of a protein that purifies with the scrapie prion. Science 218:1309–1310
    [Google Scholar]
  4. Bradford M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
    [Google Scholar]
  5. Burton K. 1956; A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemistry 5:315–324
    [Google Scholar]
  6. Dubois M., Giles K. A., Hamilton J. A., Rebers P. A., Smith A. 1956; Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28:350–356
    [Google Scholar]
  7. Greenwood F. C., Hunter W. M., Glover J. S. 1963; The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochemical Journal 89:114–123
    [Google Scholar]
  8. Jorgenson P. L. 1982; Mechanism of the Na+ K+ pump. Protein structure and conformations of the pure (Na+, K +)-ATPase. Biochimica et biophysica acta 694:27–68
    [Google Scholar]
  9. Kimberlin R. H., Marsh R. F. 1975; Comparison of scrapie and transmissible encephalopathy in hamsters. I. Biochemical studies of brain during development of disease. Journal of Infectious Diseases 131:97–103
    [Google Scholar]
  10. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  11. Lax A. J., Millson G. C., Manning E. J. 1983; Can scrapie titres be calculated accurately from incubation periods?. Journal of General Virology 64:971–973
    [Google Scholar]
  12. Millson G. C., Hunter G. D., Kimberlin R. H. 1971; An experimental examination of the scrapie agent in cell membrane mixtures. II. Association of scrapie activity with membrane fractions. Journal of Comparative Pathology 81:255–265
    [Google Scholar]
  13. Millson G. C., Hunter G. D., Kimberlin R. H. 1976; The physico-chemical nature of the scrapie agent. In Slow Virus Diseases of Animals and Man pp 243–266 Edited by Kimberlin R. H. Amsterdam: North-Holland;
    [Google Scholar]
  14. Munro N. H., Fleck A. 1966; The determination of nucleic acids. In Methods of Biochemical Analysis vol 14 pp 113–176 Edited by Glick D. New York: Interscience;
    [Google Scholar]
  15. Prusiner S. B., Cochran S. P., Groth D. F., Downey D. E., Bowman K. A., Martinez H. M. 1981; Measurement of the scrapie agent using an incubation time interval assay. Annals of Neurology 11:353–358
    [Google Scholar]
  16. Prusiner S. B., Bolton D. C., Groth D. F., Bowman K. A., Cochran S. P., McKinley M. P. 1982; Further purification and characterization of scrapie prions. Biochemistry 21:6942–6950
    [Google Scholar]
  17. Semancik J. S., Marsh R. F., Geelen J. L. M. C., Hanson R. P. 1976; Properties of the scrapie agent-endomembrane complex from hamster brain. Journal of Virology 18:693–700
    [Google Scholar]
  18. Wray W., Boulikas T., Wray V. P., Hancock R. 1981; Silver staining of proteins in polyacrylamide gels. Analytical Biochemistry 118:197–203
    [Google Scholar]
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