Alpha-Amylase is an abundant salivary molecule that is found on the tooth surface in association with the acquired enamel pellicle. Several species of oral streptococci, including Streptococcus gordonii, have been shown to bind salivary alpha-amylase. It is hypothesized that this interaction may foster the colonization of the tooth surface by alpha-amylase binding oral bacteria and play an integral role in the formation of the dental plaques that are essential for the development of caries and periodontal disease. The long term objectives are to characterize the alpha-amylase-binding component structurally, functionally and genetically in order to help define the biological role of the alpha-amylase/strepococcal interaction in the oral cavity.
The specific aims are to (1) purify the alpha-amylase-binding component, (2) biochemically characterize the putative receptor, (3) perform structural studies on the purified component and (4) utilize molecular biological techniques to isolate and clone the gene encoding for the alpha-amylase receptor. Purification of the putative receptor will be accomplished by gel filtration and anion-exchange chromatography of trypsin and mutanolysin extracts from S. gordonii G9B. SDS-PAGE and Western blotting utilizing a polyclonal monospecific antibody to the alpha-amylase-binding component will be employed to confirm the presence of the receptor throughout the purification process. Amino acid analysis and N-terminus sequencing will be performed. Biological activity will be monitored with a dot blot assay using [125I]alpha-amylase. PCR will be employed to aid in the localization of the receptor gene and in the cloning thereof. Mutants defective in salivary alpha-amylase binding are being constructed utilizing the conjugative transposon Tn916. Radiolabelled [125I]alpha-amylase and autoradiography will be used to screen for mutants deficient in alpha-amylase binding activity. The DNA fragment harboring the transposon will be isolated into a cosmid vector and transformed into E. coli. Restriction maps and Southern blotting will be used to determine the location of the gene encoding for the putative receptor. The gene will be isolated, sequenced, and cloned. Biological activity of protein expresses by the cloned gene will be assayed as above. Structural characterization will include circular dichroism and NMR spectroscopy. Key Words: alpha-amylase, receptor, transposon, chromatography
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