Sedimentation properties of the scrapie agent.

Abstract

The sedimentation behavior of the scrapie agent in homogenates of spleen from infected mice has been determined. Approximately 90% of the scrapie agent was sedimented at an w2t value of 3 X 1010 rad2/sec in a fixed-angle rotor. Sedimentation of the agent was not substantially affected by sonication or by treatment with the detergent sodium deoxycholate. The sedimentation profiles of the scrapie agent were similar to those observed for free polyribosomes, but differed from those exhibited by five other subcellular markers. Comparative studies showed that the sedimentation profiles of subcellular markers in spleen suspensions from mice infected with scrapie did not differ from uninoculated controls. These studies suggest that the scrapie agent is a discrete infectious particle which should be separable from cellular membranes. To date, the scrapie agent has defied isolation and identification. Studies on the transmission and neuropathology of scrapie suggest that it is a reasonable prototype for the other subacute spongiform encephalopathies, kuru, Creutzfeldt-Jakob disease, and transmissible mink encephalopathy (1)(2)(3)(4). The unusual physicochemical properties of the scrapie agent, its slow mode of replication, and its lack of detection by host defense mechanisms suggest that this causative agent is a novel infectious entity (5, 6). Unlike conventional viruses, the agent has not been seen by electron microscopy and has not been shown to be antigenic.The unique resistance of the scrapie agent to inactivation by heat, formalin, and ultraviolet radiation, and its extremely small ionizing radiation target size have led several investigators to speculate that the agent may not contain a nucleic acid, but may be composed only of carbohydrate, or possibly protein (5, 6). To date, attempts to purify the scrapie agent have been generally unsuccessful and a hypothesis that the agent is an integral part of cellular membranous structures has evolved (2, 6). This "membrane hypothesis" also suggests that separation of the scrapie agent from cellular membranes may not be possible.In part, the difficulties encountered by investigators attempting to purify the scrapie agent have been due to the inconvenient titration assay in mice. These titrations require care and observation of the mice over the 10-to 12-month period during which the disease develops (7). In order to develop a preparatory procedure for purification of the scrapie agent, we have studied the sedimentation characteristics of the agent in fixed angle rotors using the technique of analytical differential centrifugation (8). These studies indicate that the scrapie agent is probably a discrete infectious particle which can be sedimented at an w2t value of 3 X 1010 rad2/sec. The sedimentation behavior of the agent was not substantially altered by sonication or by treatment with the anionic detergent sodium deoxycholate (DOC), and the agent has been found to sediment with polyribosomes. The data suggest that it will be possible to isolate the scrapie agent. ...

Experimental Scrapie in the Mouse: Electrophoretic and Sedimentation Properties of the Partially Purified Agent

“…Sedimentation profiles were constructed by plotting the quantities of sedimenting particles remaining in the supernatant fluid as a function of d t , where o is the angular velocity of the centrifuge rotor in radians per second and t is the time of centrifugation in seconds (Anderson, 1968;Prusiner, 1978). Sedimentation coefficients were estimated as previously described (Prusiner et al, 1977;1978~).…”

“…Sub-cellular fractionation studies in some cases have led to the conclusion that the agent is distributed ubiquitously throughout the components of the cell (Semancik et al, 1976). Recent analytical centrifugation studies show that the agent in extracts of murine spleen is not attached to all subcellular organelles and that it sediments as a succession of particles within a discrete range of sizes (Prusiner et al, 1977 ; 1 9 7 8~) .…”

Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2

“…To perform such studies, Swiss mice were inoculated intracerebrally with the Chandler isolate of scrapie prions and the mice were sacrificed about 30 and 150 days later, when the titers in their spleens and brains, respectively, were expected to be at maximal levels. The two tissues were homogenized, extracted with detergent, and centrifuged for increasing times and speeds (47,52). The disappearance of scrapie infectivity was measured in supernatant fractions by endpoint titration, which required 1 year to score.…”

Creutzfeldt–Jakob Disease

“…6 In the late 1960s, the central dogma of molecular biology, which states that biological information is stored in DNA and transferred into proteins through a unidirectional [DNA → RNA → protein] flux was more than a sacred tablet, it governed any thought in biology. Proteins, polysaccharides, and even cellular membranes were incriminated, 5 but it is only a decade later, when Prusiner et al obtained by sedimentation highly purified preparation of infectious particles, 8 that an in-depth investigation of the chemical nature of the scrapie agent could be achieved. Francis Crick himself considered with skepticism the possibility that a protein could carry genetic information.…”