NR AJSU
AU Prusiner,S.B.; McKinley,M.P.; Groth,D.F.; Bowman,K.A.; Mock,N.I.; Cochran,S.P.; Masiarz,F.R.
TI Scrapie agent contains a hydrophobic protein
QU Proceedings of the National Academy of Sciences of the United States of America 1981 Nov; 78(11): 6675-9
PT journal article
AB 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 of sufficiently 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.
IN Die Infektiosität von vorgereinigtem und angereichertem Scrapie-Agens in 60 mM Tris / 1 mm EDTA / 0,2%Sarkosyl kann durch Proteinase K und das Aminosäuren modifizierende Diethylpyrocarbonat konzentrationsabhängig und zeitabhängig um mehr als 99,9% reduziert werden. Nicht vorbehandeltes Scrapie-Agens ist dagegen unempfindlich gegen diese Behandlungen. Proteinase K kann die Infektiosität bei 60° nur in Anwesenheit von 1 M KCl abbauen. Auch Trypsin kann das Scrapie-Agens inaktivieren. Die Wirkung des nur Aminosäuren und nicht DNA chemisch modifizierenden Diethylpyrocarbonats kann durch NH2OH per nukleophiler Substitution wieder rückgängig gemacht werden. Ribonuklease A und Desoxyribonuklease I können die Infektiosität weder bei unbehandeltem, noch bei angereichertem Scrapie-Agens reduzieren. Das Bindungsverhalten des Scrapie-Agens an Säulen zeigt, dass es hydrophobe Domänen besitzen muß.
MH Animal; Deoxyribonuclease I; Deoxyribonucleases/metabolism; Diethyl Pyrocarbonate/pharmacology; Endonucleases/metabolism; Endopeptidase K; Endopeptidases/metabolism; Female; Hamsters; Oxidation-Reduction; Prions/*analysis; Ribonuclease, Pancreatic; Ribonucleases/metabolism; Support, Non-U.S. Gov't; Support, U.S. Gov't, Non-P.H.S.; Support, U.S. Gov't, P.H.S.; Viral Proteins/*isolation & purification/metabolism
AD Stanley B. Prusiner, Department of Neurology and Biochemistry and Biophysics, University of California, San Francisco 94143, California
SP englisch
PO USA
OR Prion-Krankheiten 6
ZF kritische Zusammenfassung von Roland Heynkes