Purification And Characterization Of A Serine Protease Correlated Leaf Senescence In Wheat Leaves

Dark-induced senescing leaves of Yangmai158(Triticum aestivum L., Yangmai158) for48h were used as the experimental material in this study. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography, gradient gel electrophoresis and gel cut.Then the characters of the protease and the primary-structure were further studied. The main research results were as following:1. Isolation, purification and MS identification of the proteaseSenescence induced leaves was homogenized and centrifuged, the supernatant was incubated at50℃, pH5.5Na-acetate buffer, for0.5h, a lot of proteins (especially Rubisco) which were not stable were eliminated. Then centrifuged and the supernatant was precipitated with ammonium sulfate (from50%to70%saturation). The precipitate solution was desalted by filtering through a Sephadex G-25column. Then the desalted fraction was applied onto a Q Sepharose column, eluted with a linear gradient of0-0.6mol·L-1NaCl, and fractions of the flow-through containing the protease activity were pooled and desalted by ultrafiltration. The Q-Sepharose fraction was isolated by5%-20%gelatin-gradient-PAGE and5%-20%natural gradient-PAGE separately, then cut off four protein bands(labled as1,2,3,4from the top to down) from the natural gradient-PAGE according to the location of the white bands on gelatin-gradient-PAGE. Slices were homogenized with a mortar and pestle with1ml buffer and the supernatant was used as purified enzyme. The purified enzyme was detected by natural gradient-PAGE and the activity of the bands detected by gelatin-gradient-PAGE. The results showed that there were four separated protein bands on the gel which were silver stained and the second band had activity which assayed by gelatin-gradient-PAGE. At the same time, the four separated protein bands was send to the National Center of Biomedical Analysis, Academy of Military Medical Sciences (Beijing, China) for mass spectrometry analyses. The result of MS identification showed that the second band is a subtilisin-like serine protease.2. Biochemical characters identification of the proteaseThe biochemical characters of the pure protease were further studied, the results showed that the activity almost could be inhibited completely by serine proteinase inhibitor (AEBSF and PMSF), and could be partly inhibited by EGTA(about30%activity was leaved), it indicated that this protease was a kind of subtilisin-like serine protease which depend on metal; but it could not be inhibited by acidic proteinase inhibitor (pepstatin), metal proteinase inhibitor (EDTA;1,10-phenanthroline), cysteine proteinase inhibitor (E-64), serine protease TPCK and cysteine/serine protease inhibitor(leupeptin); the optimum pH was about7, the optimum temperature of the protease was about50℃;.and it still had20%activity after incubated at70℃for1h; In order to study the character of the protease deeply, we chose9kind of chemicals to deal with the protease, and the results as following:the protease was not sensitive to1%SDS,1mol/Lurea,25%Ethanol,25%DMSO,0.2%Tween80and0.2%TritonX-10,25%Isopropanol could partly inhibit its activity(about50%), but80mM DTT and25%methanol could inhibit most of its activity (less than10%activity remained).In conclusion, the protease purifided from wheat senescing is a kind of quite stable and new subtilisin-like serine protease depending on metal.