PAIRED HELICAL FILAMENT AND PLAQUE AMYLOID PROTEINS

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The paired helical filaments (PHFs) composing the neurofibrillary tangles have been the subject of chemical investigation for over fifteen years yet their nature still remains clouded. A method, designated the anhydrous, boiling formic acid (ABFA) technique, has been developed, which completely solubilizes and denatures the PHFs to make them amenable to chemical analysis. A solubilized concentrate of PHFs has been fractionated on Sepharose-4B and the peak PHF "core" or backbone protein fractions analyzed by lithium dodecyl sulfate-PAGE (LDS-PAGE). This gives numerous polymeric bands and an apparent monomeric molecular weight of 74 kd for the PHF backbone protein, a value in close agreement to that of the beta protein gene product, 79 kd. Electrophoresis in 8 M urea reduces the number of bands suggesting that they are non-covalent polymers resulting from hydrophobic interaction: Amino acid analyses show that the formic acid method produces modification of only the amino acids, cysteine and cystine. Initial amino acid sequence analysis revealed the N-terminus of the PHF backbone protein to be blocked. This investigation, therefore, will provide a complete amino acid sequence of the PHF backbone protein by utilizing chemical and enzymic cleavage to produce overlapping peptides for amino acid sequence analyses and mass spectroscopy to identify the amino terminal blocking moiety. Only methodology that provides acceptable quantitative yields from starting material will be employed. The present data on the composition of the amyloid fibrils composing the "senile" plaque is conflicting although the beta protein or the beta protein precursor appears by immunohistochemical studies to be a major component. An investigation similar to that of the above will therefore, also be performed on homogeneous preparations of "senile" plaques which are also solubilized by the ABFA method in order to determine definitively the molecular weight and amino acid composition and sequence of the amyloid fibril protein composing them.