This research is part of an effort to better understand the molecular mechanisms underlying human nervous system development and function, as well as the pathogenesis of certain neurogenetic disorders. Our studies have focused on structural and active site properties of the human neuron specific proteins, lysosomal hydrolases, other proteins (particularly those peptides and proteins that interact with excitable membranes), receptors and venom toxins. Proteins are purified from both human and animal tissues using affinity chromatography, electrophoretic separation, and high performance liquid chromatography. We continue to refine and develop state-of-the-art microsequencing techniques employing both gas-phase and solid-phase protein sequencers. We are presently evaluating the use of capillary zone electrophoresis for the isolation of low amounts of proteins/peptides and DNA from biological materials. Amino acid sequence analysis is carried out on neuronal and non-neuronal proteins and venom toxins. Peptide maps of both normal and mutant proteins are generated using chemical (cyanogen bromide) and enzymatic (trypsin, thermolysin, V8 protease) cleavage. The identification of carbohydrate attachment sites, sulfhydryl residues, and intra-chain disulfide residues is used to predict protein structure. Alkylating agents and enzyme inhibitors are used to define active sites. From the primary protein sequence, hydrophobic and hydrophilic domains of the protein are identified. Information obtained from these protein structure studies permits the design of oligonucleotides and peptides that are synthesized for collaborative research involving antibody production, cDNA cloning, DNA sequence analysis and in vitro mutagenesis, and that are directed toward developing gene therapy.
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