Open AccessAminopeptidase N from Escherichia coli
Author(s) -
MURGIER Maryse,
PELISSIER Corinne,
LAZDUNSKI Andrée,
BERNADAC Alain,
LAZDUNSKI Claude
Publication year - 1977
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1977.tb11408.x
Subject(s) - spheroplast , aminopeptidase , enzyme , escherichia coli , biochemistry , cytoplasm , alkaline phosphatase , microbiology and biotechnology , biology , enzyme assay , chemistry , leucine , amino acid , gene
The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30–40%) by osmotic shock or by converting Escherichia coli K 10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75–85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N ‐ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125 I‐labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and β‐galactosidase to fluorescamine in intact cells. Only 16% of the total β‐galactosidase was labeled with this fluorescent reagent whereas 44–45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.