The localization of a tri-methionine peptidase within the cell wall envelope of P̲s̲e̲u̲ḏo̲m̲o̲ṉa̲s̲ a̲e̲ṟu̲g̲i̲ṉo̲s̲a̲

The location of peptidase activity within the cell envelope structure of Pseudomonas aeruginosa has been studied. Inner and outer membrane fractions were separated on the basis of buoyant density using two consecutive sucrose gradients and identified on the basis of known markers. The majority of peptidase activity associated with the particulate fraction was found within the inner membrane fraction. The Km of tri-methionine peptidase activity in this fraction was determined to be 9.4 x 10^-4 as compared to the Km of 8.0 × 10^-4 M in crude lysates. The optimum pH for inner membrane peptidase activity is between 7.0 and 8.0; the optimum pH for crude lysate activity is 7.5. Enhancement of enzymatic activity occurs with the addition of 17 mM MgCl₂, or the addition of 33 mM MnCl₂. Solubilization attempts with certain detergents and glucosides are discussed as are the possibilities involving future isolation of the trimethionine peptidase. Some phospholipid analysis on the membranes of the cell wall structure is presented. Ramifications, for peptide transport, of the enzyme as an inner membrane component are considered. The possibility is presented that the tri-methionine peptidase functions as the peptidase activity of the signal hypothesis for protein export.