Partial purification and evidence for multiple molecular forms of the scrapie agent

Prusiner, Stanley B.; Hadlow, W. J.; Garfin, David E.; Cochran, S. Patricia; Baringer, J. Richard; Race, Richard E.; Eklund, Carl M.

doi:10.1021/bi00616a021

Cited by 95 publications

(52 citation statements)

References 31 publications

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“…We report a 20-fold and 200-fold increase of infectivity over RNA and DNA content in the final fraction, which again is comparable with earlier results [9,10]. These values represent the lowest limit of the purification.…”

supporting

confidence: 92%

Towards Purification of the Scrapie Agent

Diringer

Hilmert

Simon

et al. 1983

European Journal of Biochemistry

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A method for the partial purification of scrapie infectivity from hamster brain is described. About a 100–1000‐fold, 20‐fold, and 200‐fold enrichment in scrapie infectivity with respect to protein, RNA, and DNA content has been achieved using differential centrifugation, enzyme and detergent treatment. The inbred CLAC strain of hamsters used in our experiments contained about 10 times less infectivity in brain than has been found in randomly bred animals or other inbred strains.

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confidence: 92%

Towards Purification of the Scrapie Agent

Diringer

Hilmert

Simon

et al. 1983

European Journal of Biochemistry

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“…2E). This aggregation was presumably triggered by the extreme hydrophobicity of PrP 27-30 (2,9). In contrast, HDL particles were found all over the grid surface, without any apparent preference for PrP 27-30 ( Fig.…”

Section: Resultsmentioning

confidence: 96%

Human prions and plasma lipoproteins

Safar

Wille

Geschwind

et al. 2006

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

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Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrP Sc . The polyoxometalate phosphotungstic acid has been used to separate PrP Sc from its precursor PrP C by selective precipitation; notably, native PrP Sc has not been solubilized by using nondenaturing detergents. Because of the similarities between PrP Sc and lipoproteins with respect to hydrophobicity and formation of phosphotungstic acid complexes, we asked whether these molecules are bound to each other in blood. Here we report that prions from the brains of patients with sporadic Creutzfeldt-Jakob disease (CJD) bind to very low-density (VLDL) and low-density (LDL) lipoproteins but not to high-density lipoproteins (HDL) or other plasma components, as demonstrated both by affinity assay and electron microscopy. Immunoassays demonstrated that apolipoprotein B (apoB), which is the major protein component of VLDL and LDL, bound PrP Sc through a highly cooperative process. Approximately 50% of the PrP Sc bound to LDL particles was released after exposure to 4 M guanidine hydrochloride at 80°C for 20 min. The apparent binding constants of native human (Hu) PrP Sc or denatured recombinant HuPrP(90 -231) for apoB and LDL ranged from 28 to 212 pM. Whether detection of PrP Sc in VLDL and LDL particles can be adapted into an antemortem diagnostic test for prions in the blood of humans, livestock, and free-ranging cervids remains to be determined. prion protein ͉ apolipoprotein B ͉ blood ͉ Creutzfeldt-Jakob disease P rions are composed solely of an alternatively folded isoform of the prion protein, designated PrP Sc . Despite numerous efforts to solubilize native PrP Sc , no procedure has been developed except for incorporation of the protein into liposomes (1, 2). The hydrophobic properties of PrP Sc not only have impeded biological investigations but also have prevented structural studies at atomic resolution.During the development of an immunoassay for PrP Sc that does not depend upon limited proteolysis to hydrolyze the precursor protein designated PrP C , we found that the Na salt of phosphotungstic acid (PTA) selectively complexes with PrP Sc (3). PTA is a water-soluble salt featuring the nearly spherical trianion [PW 12 O 14 ] 3Ϫ that belongs to a broad class of polynuclear transition metal-oxo complexes known as polyoxometalates (POMs; see ref. 4). Recently, two of us (J.G.S. and S.B.P.) investigated the mechanism of selective complex formation between Keggin-type POMs and PrP Sc using a series of POM analogues (5). The ability of POMs to complex with PrP Sc as well as lipoproteins (6) raised the possibility that these two hydrophobic proteins might copurify.We began by examining the distribution of human (Hu) PrP Sc in human plasma fractions spiked with sporadic CreutzfeldtJakob disease (sCJD) prions from human brain. CJD prions were found with very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) but not with high-density lipoproteins (HDL) or any other plasma component. In addition to b...

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“…After elution of a peak absorbing at 260 and 280 nm, the electrophoresis was terminated. The scrapie agent was removed by electroelution for 16 hr or by pulverizing the gel with a Brinkmann Polytron (9). These fractions (designated E6) were eluted into 60 mM Tris-HOAc (pH 8.3)/1 mM EDTA/0.2% Sarkosyl.…”

Section: Methodsmentioning

confidence: 99%

Scrapie agent contains a hydrophobic protein.

Prusiner¹,

McKinley²,

Groth³

et al. 1981

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

178

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The scrapie agent causes a degenerative nervous system disorder of sheep and goats. Considerable evidence indicates that the scrapie agent contains a protein that is necessary for infectivity [Prusiner, S. B., Groth, D. F., Cochran, S. P., Masiarz, F. R., McKinley, M. P. & Martinez, H. M. (1980) Biochemistry 19, 4883-4891], but direct demonstration of a protein moiety has been hampered by lack ofsufficiently purified preparations. Employing preparations of the scrapie agent enriched 100-to 1000-fold with respect to protein, we found that digestion by proteinase K destroyed more than 99.9% of the infectivity. Diethylpyrocarbonate, which chemically modifies amino acid residues in proteins with high efficiency, also inactivated the scrapie agent in these purified preparations. Reductions of infectivity by proteinase K and diethylpyrocarbonate were not observed with less purified preparations. The agent bound to phenyl-Sepharose could not be eluted with 8.5 M ethylene glycol; however, a combination of ethylene glycol and detergents did release the agent. These observations provide good evidence for a protein and for hydrophobic domains within the scrapie agent. Whether the protein required for infectivity is the same protein responsible for the hydrophobic properties of the scrapie agent remains to be established.The scrapie agent causes a degenerative nervous system disorder in sheep and goats and is considered a prototype for two similar disorders of humans: kuru and Creutzfeldt-Jakob disease (1). Many months or even years pass from the time of exposure or inoculation until onset of neurological dysfunction; hence the term "slow virus" disease. Because the unusual properties of the scrapie agent readily distinguish it from conventional viruses, we prefer to use the term "unusual slow viruslike agent". Studies on the structure of unusual slow virus-like agents are of great interest because they may provide new approaches to the investigation of many common degenerative diseases of unknown etiology.In this communication we show that the scrapie agent possesses a protein that is required for infectivity. The titer of the scrapie agent in a purified fraction was reduced by proteolytic digestion and by chemical modification. Hydrophobic chromatography provides further evidence for the hydrophobicity of the agent (2).Our findings that the scrapie agent contains a protein, to our knowledge, represent the most convincing identification so far of a macromolecule within the agent. The requirement. for a protein to maintain infectivity clearly separates the scrapie agent from infectious "naked" nucleic acids such as plant viroids (3). To date we have been unable to identify a nucleic acid within the agent. These results raise the possibility that the genome coding for the scrapie agent protein may not reside within the infectious particle itself. MATERIALS AND METHODSChemicals and Reagents. Crystalline proteinase K was purchased from Merck; micrococcal nuclease, phenylmethylsulfonyl fluoride (PhMeSO2F), and diethyl...

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Partial purification and evidence for multiple molecular forms of the scrapie agent

Cited by 95 publications

References 31 publications

Towards Purification of the Scrapie Agent

Towards Purification of the Scrapie Agent

Human prions and plasma lipoproteins

Scrapie agent contains a hydrophobic protein.

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