| Co-Investigator(Kenkyū-buntansha) |
YAMADA Kazuhiro Kansai University, Faculty of Engineering, Lecturer, 工学部, 専任講師 (40158233)
KAWAHARA Hidehisa Kansai University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (10234105)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
The psychrotrophic ice-nucleating bactenum, Pseudomonas fluorescens KUIN-1 respond to a decrease in temperature with the induction of proteins that are classified as cold shock proteins (CSPs). In strain KUIN-1, a cold shock from 18 to 4ﾟC induced the synthesis of the 26-kDa protein. By analysis with SDS-PAGE, it was then demonstrated that the 26-kDa protein was produced by the cells after treatment at 4ﾟC.The 26-kDa protein was purified to apparent homogeneity by (NH_4)_2SO_4 precipitation and some chromatographies. The purified 26-kDa protein is composed of 6 subunits of 26,500 with a molecular mass of approximately 159,000 according to gel filtration and SDS-PAGE.The N-terminal sequence of the 26-kDa protein was Gln-Ala-Ala-Tyr-Tyr-Pro-Ala-His-His-His-GIn-Gln-Val-Gln-Glu-His-Trp-Gly-His-His-. Specifically, 26-kDa protein of the CSPs of strain KUIN-1 was very effective in protecting the cold-labile enzyme, lactate dehydrogenase against denaturation by freezing. The characteristics of
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26-kDa protein are analogous to the cold-regulated protein of the plants.
Also, the secretion of extracellular ice-nucleating material (EIM) with ice nucleation proteins (INP) from an ice-nucleating bacterium, Erwinia urehvora KUIN-3, increased in proportion to the yeast extract concentration in the ice-nucleating medium. The level of this secretion could be exhibited in terms of the ice-nucleating temperature, T_<50>(ﾟC), using the ELIZA method with the anti-Ina A antisenim. The secretions and the production on the cell surface of all ElM containing the class A, B and C stmctures were activated by the addition of yeast extract. Furthermore, based on the examination involving the deletion of various mecium components, it was found that Lys was closely associated with the secretions of EIM in the class A and B stnictures. The addition of Lys could affect the polyamine content in the ELM, thereby changing the surface charge of EIM from 5.2 to 4.9. Also, the addition of cadaverine could enhance the EIM secretion. Based on these results, the surface charge of the INP aggregate was shown to be essential for the translocation of INP and the secretion of IN.We found that this secretion, especially the secretion of a class B stnicture, was inhibited by N,N'-dicydohexylcarbodlimide, which was an H^+-ATPase inhibitor regardless of nomial growth. We also found that this secretion required ATP and the positive charge on the ElkI surface. Less
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