Development of the RFHR-PAGE and the Comprehensive Analysis of Nucleic Acid Binding Proteins

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1. We developed the radical-free and highly reducing method of two dimensional polyacrylamide gel electrophoresis (RFHR-PAGE) for the analysis of nucleic acid binding proteins and basic proteins, in particular, ribosomal proteins. In the period of this grant, several points including the chilling system and the migration conditions were improved, resulting the highly sensitive and quantitative ability. The possibility of the practical use is being examined in cooperation with some manufactures.1. In the last two years, ribosomal proteins were prepared from cells of forty-three prokaryotes and nine eukaryotes and analyzed by the RFHR-PAGE.The obtained two dimensional spot pattern of ribosomal proteins was extremely characteristic of each species. These results showed that the two dimensional spot patterns are effective for not only the identification but also the evolutional classification of species.3. Several hundreds fragments of nucleic acid binding proteins which exist in the insoluble cell fraction ("cell debris") in E.coli were separated by the RFHR-PAGE, and it was proved that these fragments were produced by protease VII.Furthermore a new ribosomal protein C which we previously found in E.coli ribosomes was also cut by protease VII and transferred to ribosomal protein L31 artificially. Therefore L31, an artifact, does not exist in living cells. Protein C is an intrinsic ribosomal protein and should be called L31 instead of old L31, an artificial fragment of protein C.