The major thrust of this proposal is the development of methodology for the definition of microheterogeneities that have become apparent from biochemical studies of amyloid-related tissue and seru proteins. These microheterogeneities are significant variables in the interpretation of immunoassays measuring amyloid proteins, and remain unknowns in our understanding of the origin of amyloid fibril subunit proteins from apparently normal serum proteins. Evidence for both polymorphisms of primary amino acid sequence and post translational modification and proteolysis have accumulated; whether the former is due to true allelic inheritance is not yet clear. Genetic factors might be operative either at the level of the biosynthesis of the presumed serum precursors-(monoclonal immunoglobulin light chains in primary, or AL amyloidosis; serum amyloid A (SAA) protein in secondary amyloidosis, and thyroxine binding prealbumin in Familial Amyloidotic Polyneuropathy (FAP) and diffuse ventricular senile cardiac amyloidosis (SCA) - at the level of the proteolytic digestion of these proteins to yield fibril subunit proteins, or at other loci yet to be defined. Elucidation of these pathogenic factors will further our understanding of the hereditary amyloidoses, and why only certain individuals develop tissue amyloid. We will utilize the techniques of two dimensional (2D) gel electrophoresis and the production of monoclonalantibodies to amyloid proteins to further define serum charge polymorphs. We will probe 2D gels of tissue proteins electrophoretically transferred onto diazotized nitrocellulose with iodinated antiserum and correlate these findings with serial sections of fresh tissue studied by immunoperoxidase staining. These techniques, combined with sensitive enzyme-linked absorbent (ELISA) assays, will be used to probe the origin of SAA polymorphism in hepatocyte cultures, and its proteolysis or degradation in Kupffer cell cultures. We will attempt to develop methods for the direct isolation and characterization of specific polymorphs and utilize these powerful techniques for the typing of amyloid in small biopsy specimens. Ultimately, these studies will provide further insights as to the pathogenesis of the amyloid diseases, and suggest alternative modalities of therapy.
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