Partial purification of the scrapie agent from mouse brain by pressure disruption and zonal centrifugation in sucrose-sodium chloride gradients

Siakotos, A. N.; Gajdusek, D. Carleton; Gibbs, Clarence J.; Traub, Roger D.; Bucana, Corazon D.

doi:10.1016/0042-6822(76)90261-0

Cited by 40 publications

(20 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There are differences in the infectivity density values previously published [52], [53], [54] and ours, which are likely explained by the use of different starting material, distinct gradient medium and the degree of solubilization achieved. Our density values found for caveolin, -a protein recovered in fractions nearby PrP Sc and infectivity-, are consistent with those published [55].…”

Section: Discussioncontrasting

confidence: 71%

Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

et al. 2013

View full text Add to dashboard Cite

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrPSc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). Stable variations in PrPSc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrPSc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrPSc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrPSc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrPSc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrPSc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrPSc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.

show abstract

Section: Discussioncontrasting

confidence: 71%

Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

et al. 2013

View full text Add to dashboard Cite

show abstract

“…For many years, investigators searched for a detergent that would solubilize scrapie prion infectivity (32,33). Attempts at purification, as well as characterization of the scrapie agent, were plagued by the smearing of scrapie infectivity across centrifugation gradients, electrophoretic fractions, and chromatography profiles (34,35). The experimental protocols described here could not have been done without functional solubilization of scrapie prions into DLPC (12,18).…”

Section: Methodsmentioning

confidence: 99%

Immunoaffinity purification and neutralization of scrapie prion infectivity.

Gabizon

McKinley

Groth

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

183

View full text Add to dashboard Cite

show abstract

“…The 400 S value for the scrapie agent is consistent with available filtration data, which indicate that the agent passes through a filter containing pores 50 nm in diameter, but is retained by filters having pore sizes less than 30 nm in diameter (32)(33)(34). In addition, scrapie agent infectivity was found to appear in the void volume of an agarose column that excluded the particles of molecular weight of 5 X 107 or greater (28). Ionizing radiation target size data suggesting that the scrapie agent has a molecular weight of about 105 are not unreasonable if it is assumed that the core needed for infectivity is quite small relative to the overall physical size of the particle (35 It will be of interest to see whether fractions of brain suspensions from mice infected with scrapie will exhibit sedimentation profiles similar to those observed with spleen.…”

Section: Resultsmentioning

confidence: 99%

Sedimentation properties of the scrapie agent.

Prusiner¹,

Hadlow²,

Eklund³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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. ...

show abstract

Partial purification of the scrapie agent from mouse brain by pressure disruption and zonal centrifugation in sucrose-sodium chloride gradients

Cited by 40 publications

References 8 publications

Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

Immunoaffinity purification and neutralization of scrapie prion infectivity.

Sedimentation properties of the scrapie agent.

Contact Info

Product

Resources

About