Experimental scrapie in the mouse: electrophoretic and sedimentation properties of the partially purified agent

• Published in: Journal of neurochemistry Vol. 35; no. 3; p. 574
• Main Authors: Prusiner, S B, Garfin, D E, Cochran, S P, McKinley, M P, Groth, D F, Hadlow, W J, Race, R E, Eklund, C M
• Format: Journal Article
• Language: English
• Published: England 01.09.1980
• Subjects: Animals; Centrifugation, Density Gradient; Detergents - pharmacology; DNA, Viral - analysis; Electrophoresis, Polyacrylamide Gel; Female; Hot Temperature; Mice; Prions - analysis; Prions - drug effects; RNA, Viral - analysis; Viral Proteins - analysis
• ISSN: 0022-3042
• DOI: 10.1111/j.1471-4159.1980.tb03693.x

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 < 300S but > 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.