PROTEOSOME OF BACILLUS SUBTILIS IN SPORE DEVELOPMENT

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During the development of a bacterial spore, and during development in most biological systems, rapid and extensive degradation of pre-existing proteins occurs. The long term objective of the proposed work is to understand the molecular details of this process in the soil/marine bacterium Bacillus subtilis, including a subtilis cells contain a proteosome like particle of 1-2 MDa and genetic evidences indicates that a gene of B. subtilis (clpQ) codes for a protein homologous to the beta subunits of other known proteosomes. In higher cells the proteosome is responsible for the ATP-dependent degradation of most cellular proteins, is involved in inflammation responses, cell cycle control, antigen presentation and apoptosis, and it has thus become an important pharmacological target. It is proposed here to purify the B. subtilis particle to homogeneity and characterize it with respect to appearance through electromicroscopy, its subunit composition by detergent gel electrophoresis, its substrate specificity, its requirement for ATP and its sensitivity, its requirement for ATP and its sensitivity towards inhibitors such at alpha-lactacystin and peptide vinylsulfones. Specific inhibitors will be used to assess the possible role of the proteosome in the hydrolysis of bulk protein during sporulation. Known deletions in the clpQ-encoded beta subunit of the proteosome will also be used to assess what effect this lesion has on the structure and reactivity of the proteosome, and on the rate of intracellular proteolysis during spore formation. In the connection studies of rates of proteolysis in Bacillus species have indicated that degradation of intracellular protein can be completely cryptic due to a failure of amino acids to exchange freely across the membranes. A similar problem is likely to occur in many biological systems, but no way exists to test for this. An important aim if this proposal is to develop a new, simple method based on the free exchange of water across all biological membranes. It is proposed to use the shift in the infrared frequency of the Amide I band of the peptide carbonyl when 18O is present, introduced by hydrolysis in the peptide in the presence of [18O] water, instead of 16O. If the method is successfully developed, it will be used to analyze the role played by proteosome in proteolysis of bulk protein.