Identification and determination of aminopeptidase activities secreted by lemon balm

Abstract

UDC 615.322Using synthetic substrates, an uncomplicated and sensitive procedure for the identification and determination of extracellular aminopeptidase was developed. 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. The results show a 81. 8-88.9% intracellular and 11.1-18.2% extracellular distribution of the studied enzyme activity.Plant proteolytic enzymes play many roles in metabolism such as peptide and protein degradation, posttranslation protein modification, and other processes as well [1,2]. Proteolysis is ubiquitous in biological systems, providing a means for cells to change their protein content during development and adaptation to altered environmental conditions. Proteinases participate in the mobilization of storage proteins by transforming stored polypeptides through oligopeptides to free amino acids, which are indispensable for the development and primary and secondary metabolism of cells [3,4]. Germination and ripening of seeds and pollen is associated with the expression of various hydrolytic enzymes [5,6].In the last years, several methods for the identification and determination of the activity of aminopeptidases have been developed [5,7]. Naturally occurring or synthetic substrates may be used for these purposes [8,9].Aminopeptidases (aminoacylpeptide hydrolase EC3.4.11) catalyze the release of N-terminal amino acids from peptides or synthetic substrates. The determination of aminopeptidase activities plays an important role in many fields of basic and applied research [1, 10].The availability of a simple and rapid screening method for the detection of aminopeptidase activity is of great importance for both scientific and production purposes. One advantage is the use of synthetic substrates such as 4-(phenylazo) phenylamides (PAP-amides) and β-naphthyl amides (βNA) of the amino acids [5,11]. The aim of this work was to show that the synthetic substrates L-Arg-βNA, L-Phe-βNA, and L-Leu-βNA could be employed for the identification of the activity of extracellular plant aminopeptidases in an uncomplicated and rapid procedure, whereas L-Arg-PAP amide, L-Phe-PAP-amide, and L-Leu-PAP-amide could be employed for its determination.The synthetic substrates L-Arg-PAP amide, L-Phe-PAP amide, and L-Leu-PAP amide were used in this study to determine the intracellular and extracellular activities of aminopeptidase. The results presented are mean values ± SD of five experiments. Culture media (agar plates) with and without the substrates L-Arg-βNA, L-Phe-βNA, L-Leu-βNA, and GBC salt [12,13] were inoculated with cells from growing callus cultures and then incubated for 30-90 min. The corresponding azo dye was formed by simultaneous azocoupling of the β-naphthylamine released by the enzyme activity with Fast Garnet GBC salt (GBC salt).The activities of extracellular aminopeptidase were detected by the presence ...