Biochemical Characteristics, Synthesis Localization And Separation Of A Protease Related To The Degradation Of Large Subunit Of Rubisco In Wheat

In our previous research, we found a kind of protease closely binding to Rubisco (Ribulose1,5-bisphosphate carboxylase/oxygenase) could degrade its large subunit (LSU) to51KDa. On the basis of former studies, using the leaf of wheat (Triticum aestivum L.cv.3E158) and Yangmai158(Triticum aestivum L., Yangmai158) as material, Rubisco holoenzyme was first obtained by gel cut after non-denaturing gradient polyacrylamind gel electrophoresis (GPAGE), and then the degradation of LSU and characters of the protease were identified by conventional and improved SDS-PAGE. The biochemical characteristics, synthesis localization and separation of the protease were mainly studied, And a preliminary attempt about its purification was conducted. The main results are summarized as following:1. The biochemical characteristics of the proteaseAfter non-denaturing gradient (5%-10%) gel electrophoresis of crude enzyme solution from wheat leaves, the high purity Rubisco holoenzyme was got by gel cut acording the location of Rubisco on the gel. When the solutions of Rubisco holoenzyme were incubated at30～60℃for2h, we found that there was a optimal activity of the protease which could degradate LSU at40～50℃. The effects of different concentrations of ATP on the degradation of LSU showed that the LSU could be quickly degraded into many small fragments with appending1mmol·L-1ATP.Selected the firstborn leaf as material, the Rubisco holoenzyme got by gel cut was incubated at40℃for6h, Detected, the degradation fragments of LSU could be clearly observed, While this degradation phenomenon of LSU could be eliminated by the treatment of Rubisco holoenzyme boiled for5minutes. The results above showed that the protease binding closely to Rubisco, which could degrade LSU, had existed in the young leaf of wheat.2. The synthesis location of of the proteaseTwo kinds of protein synthesis inhibitor-cycloheximide (inhibitor of nuclear gene coding proteins) and lincomycin (inhibitor of chloroplast gene coding proteins) respectively, treated the wheat leaves under dark condition in vitro. The degradation of LSU in crude enzyme solution from the leaf treated by different concentrations (0.01,0.1and1mg/ml) of the inhibitors was detected by SDS-PAGE. The results showed that lincomycin possessed more pronounced inhibition of LSU degradation. In addition, the inhibition displayed better in the treatments with lmg/ml of inhibitors.The leaves treated with lmg/ml of the two inhibitors and the mixed solution under dark for1day, Rubisco holoenzyme obtained by gel cut after GPAGE was soaked in the pH7.5Tris-HCl buffer and pH5.5citrate buffer system, and the LSU degration in soaked solutions was detected by SDS-PAGE. The results showed that, under the two pH conditions, lincomycin could more restrain the degradation of LSU. In addition, the longer the treatment time carried through, the stronger the degree of LSU degradation inhibition of lincomycin at lmg/ml was. So, we could conclud that, the protease synthesized during the dark-induced, was encoded mainly by the chloroplast genes.3. Separation of the protease closely binding to the Rubisco.The Rubisco in the Rubisco extraction solution from gel-cut after GPAGE was separated by the mono-Q strong anion-exchange chromatography, desalting, concentration and SDS-PAGE. The results showed that, Rubisco degradation would continue to carry out after strong anion exchange chromatography. That is to say, the protease, which could degrade LSU, was still closely binding to the Rubisco.The Rubisco extraction from gel-cut was Electrophoresis by nonfull-denatured SDS-PAGE, in which the sample was not boiled, SDS was took off with TritionX-100. After that, the LSU, SSU, and other components in the Rubisco holoenzyme were first obtained by gel cut. All the other ingredients were added to the LSU, and detected respectively, we can found that the protease that degraded LSU could be separated from the Rubisco extraction after SDS-PAGE. In addition, When the Rubisco extraction from gel-cut was detected by Gel-SDS-PAGE, results showed that there was more than one protease to be able to degrade LSU in Rubisco extraction, and a protease, that molecular weight is higher than the LSU, had more important role in LSU degradation.