Studies On Cytotoxicity And Themo-stability Of Cinnamomin

Part I: Refolding of Partially Thermo-unfolded Cinnamomin A-chain Mediated by B-chain. The pure cinnamomin A-chain is easy to denature compared to that in the mixture of A- and B-chain or in intact cinnamomin molecule either being stored at 4℃ or heated. When being heated at 45℃ for 20 min, the A-chain generates partially unfolded intermediate and loses its tertiary structure as monitored by circular dichroism (CD) and tryptophan fluorescence, thus resulting in the inactivity of its RNA N-glycosidase albeit it retains most of its secondary structures. This partially unfolded intermediate is sensitive to protease, exhibiting properties of a molten globule. The changes in conformation and activity are irreversible upon cooling. The partially unfolded intermediate can fully restore its RNA N-glycosidase activity in presence of cinnamomin B-chain. The phenomenon that the cinnamomin B-chain mediates the refolding of partially unfolded A-chain probably plays important role in the intracellular transport of the cytotoxic protein, i.e., keeping the structural stability of A-chain and refolding partially unfolded A-chain that occasionally appeared in the process of intracellular transport, to avoid the destiny of proteolysis that occurs to most denatured proteins in cell. Part II: The B-chain causes the lower cytotoxicity of intact cinnamomin to BA/F3b cells. The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with that of ricin. Cinnamomin A-chain exhibits the RNA N-glycosidase activity in inhibiting in vitro protein synthesis similarly compared with that of ricin, whereas the cytotoxicity of intact cinnamomin to BA/F3βcells is obviously lower thanintact ricin. In order to demonstrate that it is the B-chains of the two RIPs bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3βcells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3βcells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA. Scatchard analyses of the binding of the two B-chains indicate that the two RIPs share similar binding sites with different affinity.Part III: Cleavage of Supercoiled Circular Double-stranded DNA Induced by a Eukaryotic Cambialistic Superoxide Dismutase. Besides the known role in protecting cells from toxicity of oxidative stress, camphor cambialistic SOD induce the cleavage of supercoiled double-stranded DNA into the nicked and linear DNA. It can not cleave linear DNA (linearized pGEM-4Z DNA orλphage DNA) and E. coli 5S ribosomal RNA, demonstrating there is no contamination of DNase or RNase in the cambialistic SOD preparation. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). These results show that the dismutation activity is crucial for the supercoiled DNA-cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) demonstrates that both activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA induced by the cambialistic SOD are random.