Experimental scrapie in the mouse: electrophoretic and sedimentation properties of the partially purified agent
| Autor: | Eklund Cm, Stanley B. Prusiner, Richard E. Race, W. J. Hadlow, Cochran Sp, Darlene Groth, Garfin De, Michael P. McKinley |
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| Rok vydání: | 1980 |
| Předmět: |
Differential centrifugation
Gel electrophoresis Chromatography Hot Temperature Chemistry Prions Sodium Detergents chemistry.chemical_element Biochemistry Sedimentation coefficient Cellular and Molecular Neuroscience chemistry.chemical_compound Mice Viral Proteins DNA Viral Centrifugation Density Gradient Animals RNA Viral Centrifugation Electrophoresis Polyacrylamide Gel Female Sodium dodecyl sulfate Polyacrylamide gel electrophoresis Percoll |
| Zdroj: | Journal of neurochemistry. 35(3) |
| ISSN: | 0022-3042 |
| Popis: | Some biochemical and biophysical properties of the scrapie agent in a partially purified fraction P5 from murine spleen are described in this communication. The agent was stable in the nonionic detergents Triton X-100 and Nonidet P40 and stable in the nondenaturing, anionic detergents sodium cholate and sodium N-lauroyl sarcosinate. In contrast, sodium dodecyl sulfate (SDS) inactivated the agent at high concentrations (1% or >) when the detergent-to-protein ration approached 1.5 g SDS/g protein. The agent was resistant to inactivation by nucleases and proteases, even in the presence of 0.1% SDS. A broad peak of infectivity was exhibited in modified colloidal silica (Percoll) density gradients. Maximal titers were found at a Percoll density of 1.10 g/cm3 in the presence and absence of 0.05% SDS. Gel electrophoresis of the agent in the presence of 0.1% SDS resulted in inactivation of > 95% of the agent loaded onto the gel. Free-flow electrophoresis showed that > 99% of the agent in fraction P5 migrated toward the anode, but not as a discrete species. Sedimentation analysis of the agent in fraction P5 in the presence of 1% lysolecithin showed that the agent has a sedimentation coefficient of 30S. Heating P5 preparations caused the agent to associate with cellular elements and form aggregates with sedimentation coefficients > 10,000S. Removal by differential centrifugation of the large forms of the agent produced upon heating permitted characterization of a discrete subpopulation of scrapie agent particles. Rate-zonal sucrose gradient studies showed that > 95% of the infectivity in this subpopulation sedimented as uniform particles with a sedimentation coefficient of 240S. |
| Databáze: | OpenAIRE |
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