The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.)

A proline iminopeptidase (EC. 3.4.11.5) was isolated from shoots of 3 day old seedlings. The purification procedure consisted of 5 steps: acid precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on Sepharose CL 613, twice repeated hydrophobic chromatography on Phenyl-Sepharose HP. The enzyme was purified 404.8-fold, with the specific activity of 8.5 units center dot mg(-1) of protein with recovery yield of 3 %. The purified enzyme had a molecular mass of 225 kDa estimated by gel filtration and 55.4 kDa by SDS PAGE. This indicates that native enzyme is composed of four subunits. The enzyme was specific for proline beta-naphtylamide among various amino acid beta-naphtylamides. An optimal activity was observed at 37 degrees C at pH 7.75. The enzyme was thermostable up to 37 degrees C for 30 min. The enzyme was strongly inhibited by pHMB, E-64, heavy metal ions and partially by PMSF, DFP. The results suggest that cysteine and serine residues may participate in the enzyme activity.