NR ABLN
AU Bolton,D.C.; Bendheim,P.E.; Marmorstein,A.D.; Potempska,A.
TI Isolation and structural studies of the intact scrapie agent protein
QU Archives of Biochemistry and Biophysics 1987 Nov 1; 258(2): 579-90
PT journal article
AB Purification of the scrapie agent by methods using digestion with proteinase K yields a protein product, PrP-27-30, with an apparent mass of 27-30 kDa (D. C. Bolton et al. (1982) Science 218, 1309-1311; S. B. Prusiner et al. (1982) Biochemistry 21, 6942-6950). In contrast, a 33-37 kDa glycoprotein, HaSp33-37, was the major protein component isolated from scrapie-affected hamster brain by a procedure that did not use protease digestion. The purified fractions containing HaSp33-37 had greater than 10(11) LD50 units of the scrapie agent per milligram of protein. Proteinase K digestion of HaSp33-37 gave a product indistinguishable from PrP-27-30 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The amino acid sequence of the first 22 residues of HaSp33-37 was determined. The sequence coincided with that predicted for the N-terminus of the precursor to PrP-27-30 (K. Basler et al. (1986) Cell 46, 417-428; N. K. Robakis et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6377-6381) after processing by signal protease. HaSp33-37 was digested with N alpha-tosyl-L-phenylalanine chloromethyl ketone-trypsin to produce a 29-32 kDa protein fragment; following digestion this fraction retained complete biological activity. The amino terminal sequence of the 29-32 kDa protein corresponded to a position intermediate between the amino termini of HaSp33-37 and PrP-27-30. We conclude that HaSp33-37 is the intact form of the scrapie agent protein and that PrP-27-30 is produced by proteinase K degradation when this enzyme is introduced during isolation of the scrapie agent.
IN Die Autoren reicherten das scrapie Agens auf eine Konzentration von mehr als 100 Millionen LD50 Einheiten pro µg Protein an. Mit einer Prozedur ohne Proteinase K isolierten sie aus dem gereinigten scrapie Agens Prionproteine mit scheinbaren Molekularmassen von 33-37 kDa. Eine nachträgliche Behandlung mit Proteinase K reduzierte durch Anspaltung des Aminoterminus die scheinbare Molekularmassen der Prionproteine auf 27-30 kDa.
MH Amino Acid Sequence; Amino Acids/analysis; Animal; Brain/*microbiology; Electrophoresis, Polyacrylamide Gel; Hamsters; Molecular Sequence Data; Molecular Weight; PrPsc Proteins; Prions/*pathogenicity; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S.; Viral Proteins/*isolation & purification
AD Department of Molecular Biology, New York State Office of Mental Retardation and Development Disabilities, Staten Island 10314.
SP englisch
PO USA