Comparison of the catalytic activities of members of the preproricin gene family of Ricinus communis
Leshin, Jonathan Andrew.
Thesis (Ph.D.)--Georgetown University, 2008.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. Ribosome-inactivating proteins (RIPs) enzymatically depurinate an adenine in the ricin-sarcin loop of the 28s ribosomal RNA. Previous studies using Southern blots have indicated the presence of multiple copies of the preproricin gene in the Ricinus communis genome and a survey of a 4X draft coverage of the genome shows the presence of seven full length genes, one of which is ricin and six of which are ricin-like proteins (RLP). However, other than ricin, none of the genes have been examined for expression in the plant. The purpose of this research project was to increase our knowledge of RLPs by measuring and comparing the catalytic activities of ricin a-chain (RTA) and the a-chain of all six of the RLPs, using a rabbit reticulocyte lysate assay. The immunocrossreactivity to anti-ricin a-chain antibodies was examined using western blot. The mechanism of action was confirmed by RNA gel electrophoresis. The relative Km was determined by measuring the intensity of aniline cleavage fragments.; A rabbit reticulocyte lysate assay was used to compare the IC50 value of these toxins by measuring the decrease in production of luciferase after the addition of the toxin. While most of the toxins had an IC50 in the low nanomolar range close to 5 nM, one toxin, RLP5 was 15-fold less active with an IC50 of 75 nM. To examine the immunocrossreactivity of the RLPs, a western blot was performed using a monoclonal anti-ricin a-chain antibody (NR-843). Only RTA, RLP3 and RLP4 could be visualized on a western blot. This indicates that not all RLPs present in the plant will react with NR-843. Using RNA gel electrophoresis combined with aniline reactivity against exposed phosphoribose backbones, it was determined that RTA and all six RLPs can deadenylate RNA in the ricin-sarcin loop. Lastly, measuring the concentration of the fragment released by the RLPs, it was determined that RTA and all six RLPs share a similar relative Km.
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