Morphological and biochemical evidence that scrapie-associated fibrils are derived from aggregated amyloid-like filaments.
The membrane fraction from scrapie infected mouse brains was dissolved in saturated urea, centrifuged on a 10 to 50% glycerol gradient at 35,000 rpm for 24 h, and fractionated from the bottom of the tube into 11 fractions. PrP was detected throughout the gradient. However, the relative PrP concentrations of fractions 4 and 8 were the highest. The relative PrP concentration versus protein concentration of fractions 1 to 4 was higher than that of the other fractions. Scrapie infectivity also was detected in all fractions. Fractions 2, 3, 4, 7, and 8 produced the shortest incubation periods. Positively stained filamentous aggregates with sizes varying from about 40 x 60 nm to more than 4 microns were observed in fractions 2 and 4 by negative staining. These resembled amyloid filaments. Congo red-stained aggregates showed birefringence under polarized light. Aggregation of the filamentous aggregates was observed by incubation with anti-mouse SAF serum. Fine fibrils 10-18 nm in width were partially dissociated from the aggregates by brief exposure to the detergent Sarkosyl. These facts suggest that SAF are not products of self-assembly from subunit structures liberated from membranes by exposure to detergent, but exist as aggregates of amyloid-like filaments from which SAF are dissociated by detergent extraction.
Isomura
H
H
Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Japan.
Shinagawa
M
M
Ikegami
Y
Y
Sasaki
K
K
Ishiguro
N
N
D000818:Animals; D001718:Birefringence; D015153:Blotting, Western; D001921:Brain / Q000382:microbiology / Q000473:pathology* / Q000648:ultrastructure; D001923:Brain Chemistry; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003224:Congo Red / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D051379:Mice; D008813:Mice, Inbred ICR; D009419:Nerve Tissue Proteins / Q000378:metabolism; D018622:PrP 27-30 Protein; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011498:Protein Precursors / Q000378:metabolism*; D012608:Scrapie / Q000378:metabolism / Q000382:microbiology / Q000473:pathology* / Q000635:transmission; D012995:Solubility; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011498:Protein Precursors; D014764:Viral Proteins; D018622:PrP 27-30 Protein; D003224:Congo Red