Document Type
Thesis - University Access Only
Award Date
2003
Degree Name
Doctor of Philosophy (PhD)
Department / School
Chemistry and Biochemistry
First Advisor
Fathi T. Halaweish
Abstract
Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that inactivate ribosomes through a site-specific deadenylation of the large ribosomal RNA. RIPs have drawn great attention due to their potent antiviral and antitumor activities, which potentially can be used in medicine and plant protection. Two Cucurbitaceae species, Cucurbita foetidissima and Cucurbita texana were for the first time investigated for the presence of ribosome-inactivating proteins, and three novel RIPs were isolated and characterized. Foetidissimin, a type II RIP, was found, isolated, and characterized from Cucurbita foetidissima. Foetidissimin was isolated from the roots of Cucurbita foetidissima by size exclusion and ion exchange chromatographic methods. A non-radioactive protein synthesis inhibition assay was used to guide the separation. The molecular weight of the dimeric protein is 63 kDa and the two chains are held together by noncovalent interactions. Using a non-radioactive protein synthesis inhibition assay, foetidissimin inhibited cell-free translation in rabbit reticulocyte lysate with an LC 50 of 25.9 nM. The N-terminal sequence analysis of the first fourteen amino acids showed that the A chain of foetidissimin shares 88% similarity with a-trichosanthin, a type I RIP, and 72% similarity with A chain of nigrin F, a type II RIP. Foetidissimin II, another RIP, was also isolated and characterized from the roots of Cucurbita foetidissima. Foetidissimin II is a type2 ribosome inactivating protein with a molecular weight of 62 kDa estimated by polyacrylamide gel electrophoresis, chain A 29 kDa, and chain B 32 kDa. Cell free protein synthesis assay and in vitro antitumor assay were conducted. The IC 50 of foetidissimin II for the inhibition of protein synthesis was 251 nM, and the IC 50 s for both adenocarcinoma cells and erythroleukemia cells were 70 nM. Foetidissimin II depurinated the 28S rRNA at a specific site and after the treatment with aniline, a 450 nucleotide RNA fragment was released. Protein N-terminal sequencing indicated that both chains of foetidissimin II, especially the chain A, showed similarity to other documented RIPs. V Texanin, a type I ribosome inactivating protein, was isolated and characterized from the fruit of Cucurbita texana. Texanin is a one chain ribosome inactivating protein with a molecular weight of 28 kDa estimated by polyacrylamide gel electrophoresis. In vitro antitumor assay showed that texanin exhibited mild antitumor activity against erythroleukemia cells (IC 50 95.3 μM), but no activity against adenocarcinoma cells. In the N-glycosidase activity test, texanin depurinated the 28S rRNA at a specific site and after the treatment with aniline, a 450-nucleotide RNA fragment was released. Protein N-terminal sequencing indicated the high similarity of texanin to other documented RIPs. Several protein fractions, and purified foetidissimin and texanin exhibited trypsin inhibition activity, indicating the presence of trypsin inhibitors in the investigated plants. Further purification and confirmation of possible trypsin inhibitors in Cucurbita foetidissima and Cucurbita texana was not achieved due to the limited amount of proteins isolated.
Library of Congress Subject Headings
Cucurbita.
Proteins -- Synthesis.
Trypsin inhibitors.
Ribosomes.
Natural products.
Publisher
South Dakota State University
Recommended Citation
Zhang, Daoning, "Isolation and Identification of Ribosome-Inactivating Proteins and Trypsin Inhibitors from Cucurbita Foetidissima and Cucurbita Texana" (2003). Electronic Theses and Dissertations. 2063.
https://openprairie.sdstate.edu/etd2/2063
Rights Statement
