LOCALIZATION OF ALANYL AMINOPEPTIDASE AND LEUCYL AMINOPEPTIDASE IN CELLS OF PSEUDOMONAS-AERUGINOSA BY APPLICATION OF DIFFERENT METHODS FOR PERIPLASM RELEASE

Various methods for the isolation of periplasm were examined and compa red with regard to the complete release of known periplasmic marker en zymes and the contamination of the periplasm by cytosol for Pseudomona s aeruginosa PAO1 as a significant Gram-negative test strain. The aim of the investigations was to clarify the exact localization of alanyl aminopeptidase (AAP) and leucyl aminopeptidase (LAP) of this microorga nism and to evaluate these methods. The osmotic shock of NOSSAL and HE PPEL (1996) was the most effective method with the lowest contaminatio n by the cytosolic marker enzyme malic enzyme, but some proteins, whic h are located near the inner side of the cytoplasmic membrane, can be released additionally into the periplasm. All other procedures like ch loroform or polymyxin treatment, the magnesium chloride washing of int act bacteria and spheroblasting by lysozyme in the presence of EDTA or magnesium chloride resulted only in a partial, sometimes only very lo w release of periplasm. The periplasmic enzymes are bound either more by hydrophobic or more by ionic interactions to the cell envelope and show a different behaviour with the different releasing agents. These methods are useful for a further differentiation between really peripl asmic proteins and those proteins, which were false positive found in periplasm as a result of the osmotic shock. Our results show that AAP from Pseudomonas aeruginosa is a periplasmic enzyme with hydrophobic i nteractions to the cytoplasmic membrane, corresponding to the early re sults of LAZDUNSKI and MURGIER for Escherichia coli (LAZDUNSKI et al. 1975 a and b, MURGIER et al. 1977), and LAP is cytosolic, but located near the cytoplasmic membrane. The AAP is not a real amphipatic membra ne protein, as could be demonstrated by phase separation experiments w ith Triton X-114.