Leucine aminopeptidase in intracytoplasmic membranes of Acinetobacter calcoaceticus.

Abstract

Cells of Acinetobacter calcoaceticus strain 69-V contain an aminopeptidase that cleaves L-leucine amide, leucylglycine or leucine hydrazide with high efficiency. Leucine 4-nitroanilide and hydrazide are hydrolyzed to less than 0.1% and 1%, resp. of leucine amide. Grown on acetate-NH4+ medium the activity of the enzyme in the cytoplasm is increased 5-fold compared with cells grown on a casamino acid medium or on yeast extract. In these cases the specific activity of the unpurified enzyme is about 5 nkat/mg for the cytoplasmic and membrane-bound enzyme species as well. Up to 30% of the aminopeptidase activity were found mainly in intracytoplasmic membranes, less in cytoplasmic membranes and only traces in outer membranes, presumably as contaminations. It is solubilized by detergents but not by high salt concentrations. An addition of antipain or Z-Ala2-Phe-CH3 before cell rupture did not change the distribution of the enzyme. A mixture of EDTA and 1.10-phenanthroline diminished the membrane-bound enzyme from 11.4% to 4.3% and leupeptin or E-64 increased it to 20%. The enzyme is regarded as leucine aminopeptidase (LAP) bound to intracytoplasmic membranes.