The Extraction And Purification Of Plant Ribosomal Inactivating Protein, And Its Effects On The Apoptosis Of Leukemia Cells

Ribosomal inactivating protein (RIPs) are a group of enzymes of plant origin, which de-adenylate the major ribosomal RNA ,making it unable to bind the elongation factor 2 (EF2) and thus arresting protein synthesis. RIPs can be divided in two main classes depending on their subunit composition. Typeâ…  RIPs are single chain polypeptides with a molecular mass of about 30KD and highly basic isoelectric point (pH(9.5). Typeâ…¡ RIPs are heterodimeric proteins consisting of an A-chain, functionally equivalent to the Typeâ… enzymes, and a lectin-like B-chain which recognises cell surface sugar residues allowing the uptake of the protein and resulting in a cytotoxic effect. Interest in RIPs include their use as abortifacients and toxic components of immunotoxins targeted at malignant cell, antiviral agents. Recently, more and more concentrations have been focused on the anti-tumor effect of typeâ… RIPs. Because there are many kinds of plant containing typeâ… RIPs, people used to isolate and purify it through the classic way that ion-exchange chromatography had been widely used. But, this method is so complicated and costly that its application has been highly limited. In the study presented in this paper we managed to obtain typeâ… RIP with high purity from the seeds of bitter guord. Cibacron blue F3GA is a kind of dye which could bind the protein containing a nucleotide-binding domain specificly, thus A economical and effective method has been carried into execution in which the psuedo-ligand affinitive chromatography and gel chromatography were applied to isolate and purify the typeâ… RIP. Then the typeâ… RIP extracted from the bitter gourd has been used to treat the human leukemia HL-60 cells toexplore its cytotoxicity .The results and conclusions are summarized as follows: 1. Using ammonium sulfate precipitation, Blue Sepharose CL-6B psuedo-ligand affinitive chromatography and Sephadex G-75 gel chromatography, we obtained the typeâ… RIP with high purity, the feasibility of this method has been approved by SDS-PAGE results.2. After the treatment of HL-60 cells with 40(g/ml typeâ… RIP the cell viability decreased dramatically. At the same time, Flow cytometry and TUNEL test showed 40(g/ml typeâ… RIP induced apoptosis profoundly. 3. Using RT-PCR and Western-blot tests,in typeâ… RIP-treated HL-60 cells as compared with control HL-60 cells, the expression of caspase-3 and Bax were found to be up-regulated notably while the protein expression of Bcl-2 was down-regulated prominently . All the results above mentioned indicate that typeâ… RIP is a sort of effective apoptosis-inducing agent and the caspase-3 is a vital molecule in the process of apoptosis induced by typeâ… RIP, at the same time, Bcl-2 and Bax probably function as a regulator in the activation of caspase-3.