Studies Of Purification, Properties And Physiological Role As Well As Distribution Of Several Ribosome-inactivating Proteins

1. The physiological role played by cinnamomin in the seed cell was systematically studied. Cinnamomin accumulated simultaneously with other proteins at the poststages of seed development. It did not exhibit the inhibitory effect on the protein synthesis in vivo and did not depurinate the 26 S ribosomal RNA of C. camphora. Northern and western blotting revealed that cinnamomin was specifically expressed in cotyledons. Cinnamomin degraded rapidly during the early periods of seed germination. Endopeptidase and (-mannosidase were proved to play an important role in this degradation process. Western blotting demonstrated that cinnamomin only existed in protein body, the specific cellular organelle for storage proteins. The similar properties between cinnamomin and other seed storage proteins of dicotyledons were also compared. It was concluded that cinnamomin played as a special storage protein in the seed of camphor tree. This study clarified for the first time the real physiological function played by cinnamomin and enriched the physiological function of RIP.Lamjapin, a novel type ( RIP has been isolated from kelp (Laminaria japonica A), a lower marine alga. It has a larger molecular- weight of approximate 36 kDa, pI of 8.4. Lamjapin can inhibit protein synthesis in rabbit reticulocyte lysate with the IC50 of 0.69 nM. It can depurinate at multiple sites of rat ribosomal RNA and produce the diagnostic R-fragment together with additional three larger fragments after the aniline reaction. It is a promoter factor independent RIP. It could depurinate at the A20 of SRD RNA corresponding to the A4324 of rat liver2. ribosomes. Lamjapin is the first RIP isolated from lower algae. This study broaden the distribution of RIP to the lower algal plant and it is beneficial to the further works to elucidate the function, evolutionary process and distribution of this class of proteins in the plant kindom.3. A chitinase with antifungal activity, designed as PAFC was purified from laver (Porphyra. suborbiclata). The purification procedure included the chromatographies on the phenyl-Sepharose CL-4B, chitin affinity and FPLC Mono S column. The molecular weight of PAFC is 26,868 kDa as revealed by MS. PAFC showed characteristic composition of amino acids, the secondary structure and molecular-weight of class I chitinase. It exhibited strong antifungal activity against pathogens: Fusarium graminarum and Fusarium oxysporum while displayed no activity against Pyrucalaria oryzae, Fusarium monilforme, and Coletotrichum gossypii. It promises the potential application in agriculture.4. Chitin affinity chromatography was adopted to purify (-sarcin and an antifungal protein (AFP) from the mold Aspergillus giganteus MDH 18894. Compared with the previous procedure, this method was very fast and specific. By this procedure, 4.5 mg of pure (-sarcin and 6.9 mg of pure AFP were obtained from 2 liters of culture medium. Glycochitin in the culture medium could increase the expression and the nuclease activity of (-sarcin, while had no effect on the expression and activity of AFP. It was also found that both (-sarcin and AFP exhibited the binding activity to generated chitin and no tryptophan was involved in the binding process. This property will be helpful to make clear the unknown function of (-sarcin in vivo and the uncertain mechanism of the antifungal activity of AFP.