Murine Amyloid Fibril Protein: Isolation, Purification and Characterization

Murine amyloid has been produced by four different induction methods ( Mycobacterium butyricum, casein, casein plus Freund's adjuvant and endotoxin-induced mouse amyloidosis) in several strains and obtained from mice with "spontaneous" amyloidosis. The amyloid fibrils have been concentrated from spleen and liver. Electron microscopy of all of these preparations reveals the amyloid fibril to be 100 Å in width and composed of two parallel filaments, each measuring 35-40 Å in width and having the appearance of a twisted ribbon. X-ray diffraction of all preparations reveals a "backbone" spacing at 4.75 Å and a "side chain" spacing at 11 Å indicating a "β-pleated sheet" structure. Identification of the major protein component of amyloid fibril concentrates was made by combined use of sodium dodecyl sulfate polyacrylamide disc electrophoresis, labeling of the protein with 3 H-tryptophan and Sepharose 4B gel filtration. Purification of the amyloid protein from spontaneous amyloidosis liver was accomplished by sequential gel filtration with 5 M guanidine in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns. The material is a unique glycoprotein with a molecular weight of 7200, a high content of dicarboxylic and short chain amino acids, a significant amount of tryptophan and an unreactive NH 2 -terminal amino acid, tentatively identified as pyrrolid-2-one-5-carboxylic acid. There are no methionine, half-cystine, hydroxylysine or hydroxyproline residues. Murine amyloid protein, therefore, has striking similarities to many human amyloid protein preparations. It differs from the human proteins in the similarity of molecular weights of different preparations.