A goal of these studies is to better understand the molecular mechanisms underlying human nervous system development and function, as well as the pathogenesis of certain inherited brain disorders. Our studies have focused on the structural and active-site properties of proteins found in the nervous system including neurotropic peptides/proteins, lysosomal hydrolases, and other proteins/peptides which interact with excitable membranes, receptors and venom toxins. Proteins from human and animal tissues are purified by liquid chromatography (ion-exchange, gel permeation and affinity techniques), high performance liquid chromatography (HPLC) and electrophoretic separation. State-of-the-art microsequencing analysis (gas-phase, liquid-phase and solid-phase), amino acid analysis and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) are performed to characterize proteins. Both isoelectric-focusing and molecular weight determination of picomole amounts of proteins are carried out by capillary-zone electrophoresis. Peptide maps of both normal and mutant proteins are generated using chemical (mild acid, cyanogen bromide) and enzymatic (trypsin, AspN, V8 proteases) cleavage and analyzed by HPLC. Several novel scorpion venom neurotoxins, which interact with synaptosomal structures such as human calcium and potassium channels in excitable membranes, are being studied. We are also investigating the association of amyloid peptides with both high and low density lipoproteins from cerebrospinal fluid in Alzheimer's disease. Our research emphasis is on studies that define post-translational processes not identifiable by DNA sequence analysis, e.g. carbohydrate attachment, protease cleavage, and phosphorylation that could be responsible for abnormal brain function. By a combination of methods described above, we have identified a major allergen from avocado. The avocado allergen was sequenced, cloned and recombinantly expressed to allow biochemical characterization. With our previous identification of a major allergen from latex, which was homologous to a kiwi fruit protein, we have established a strong connection between latex allergy and food allergies. This may provide a viable approach to desensitization therapy for both latex and food allergic patients. Recently, we have identified the salivary neurotropic factor Gustin as a carbonic anhydrous isozyme (CAVI) and shown it to function as a stimulator of taste-bud formation. Several new long chain potassium channel blocking peptides have been characterized from scorpion venom both directly from the protein and more recently by analysis of their cDNA sequence. Additionally, a novel defensin-like peptide has been identified.
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