Demonstration of Aminopeptidase Activities Secreted byAmsonia tabernaemontana Walt. Cells

Abstract

Using synthetic substrates, an uncomplicated and sensitive procedure for the determination of extracellular aminopeptidase was developed. The studied enzyme produced by the tested plant material (calli, cell suspension culture and roots of Amsonia tabernaemontana Walt. seedlings) hydrolyzed the substrates β-naphthylamides (βNA) and 4-(phenylazo) phenylamides (PAP-amide) of the amino acids to β-naphthylamine and 4-(phenylazo) aniline, respectively, and amino acid. The β-naphthylamides of the amino acids were applied for the identification of extracellular aminopeptidase, whereas the 4-(phenylazo) phenylamides of the amino acids were used for the determination of intra- and extracellular aminopeptidase activity. By simultaneous azocoupling of β-naphthol with Fast Garnet GBC salt on agar plates a corresponding brown-red hardly water-soluble azo-dye was produced. The evaluation of dyed zones allowed the extracellular aminopeptidase activity to be assessed. No coloration of the agar medium was observed without inoculum, with heat-inactivated cells (10 min at 100 °C) or in medium inoculated without substrate. On the agar plates with substrate and sterile Amsonia seedlings, changes in coloration were observed indicating a release of aminopeptidase from the roots during germination. The results show a 91.0 % intracellular and 9.0 % extracellular distribution of aminopeptidase activity, when a cell suspension culture of A. tabernaemontana Walt. as the plant material was used. The agar plate method described permits the rapid, uncomplicated and specific detection of plant producers of extracellular aminopeptidase, which could be particularly useful in future inhibitory and/or biotechnological studies.