Our general interest is in the study of specific proteins from procaryotes which have to be transferred across or inserted into a membrane in order to reach their distinct final location. The aim of this proposal is to investigate first the location(s) of a specific enzyme, alkaline phosphatase (APase) in Bacillus licheniformis MC14, to determine the physical relatedness of the enzyme from the various locations and finally to investigate the process by which it is secreted. The active enzyme has been localized on the inside of the membrane but is also secreted through the membrane. This secreted enzyme has been found associated with the outer surface of the membrane, as a soluble species released upon protoplast formation and in the culture medium. The enzymes from all four locations appear to be immunologically identical. This proposal involves the studies of the in vivo synthesized APase(s), protoplast secretion studies and in vitro synthesis of the APase. The purified in vivo synthesized APase from the various locations will be characterized according to size, N-terminal, peptide mapping and N-terminal sequenced. Metabolically competent protoplasts will be studied to determine if they secrete APase (active or inactive, monomer or dimer). If they do secrete APase, protein synthesis will be stopped by chloramphenicol, the protoplasts labeled with 125I using the lactoperoxidase method, the membrane-bound polyribosomes containing nascent APase polypeptide chains isolated by immunoprecipitation and the nascent chains checked for 125I label. In vitro synthesis of the APase, analysis of the product and comparison of the in vivo synthesized product is planned.