TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions.

A triticale cDNA encoding a prolyl aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses (∼35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only in bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.

Biochemical characterisation of prolyl aminopeptidase from shoots of triticale seedlings and its activity changes in response to suboptimal growth conditions.

Prolyl aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54kDa. Its pH and temperature optimum are pH 7.5 and 37°C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing ß-naphthylamides (ß-NA) of Ala, Phe, and Leu. The K(m) value of PAP against Pro-ß-NA was the lowest among the substrates tested and it was 1.47×10(-5)M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the aminopeptidase activity against Pro-ß-NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.

Biochemical characterization of an alkaline metallopeptidase secreted by a Pseudomonas fluorescens isolated from soil.

The extracellular endopeptidase synthesized by soil bacterium Pseudomonas fluorescens was purified to homogeneity in a four-step procedure. The enzyme was purified 45-fold, with a 20% recovery. The endopeptidase appeared to be a monomer with a molecular mass of approx. 50 kDa. The enzyme was active in the pH range of 7 to 10. The optimal activity was detected at pH 9.0 and at 42 degrees C. Enzyme activity was inhibited by EDTA, EGTA and 1,10 phenanthroline, typical metalloprotease inhibitors. Ca(2+) activated the enzyme while Zn(2+), Co(2+), Cd(2+)(in high concentration) strongly inhibited it. The presence of calcium ions strongly stabilized the enzyme with regard to thermal resistance. The amino acid sequence of fragments of the enzymatic protein determined by MS analysis revealed a high similarity to the sequences of other alkaline metalloendopeptidases of bacteria belonging to the genus Pseudomonas.