|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1996 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1995 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Protein content of subcellular fractions in Escherichia coli cells during heat treatment at 55ﾟC were investigated. The amount of soluble proteins in heated cells decreased and that of urea-soluble proteins, which were sedimented by high-speed centrifugation and then solubilized with 6M urea solution, increased correspondingly. In accord with this, the beta-galactosidase activity in each fraction behaved similarly. To characterize the nature of urea-soluble proteins, the sedimentary fraction was further fractionated by the sucrose density gradient centrifugation. After the heat treatment at 55ﾟC for 1 min, the cytoplasmic membrane interacted with the outer membrane, and the sedimentary intracellular proteins appeared in two fractions ; one was sedimented at the bottom and the other was cosedimented with the membranes. It was supposed therefore that the intracellular proteins was either aggregated to be sedimented, or interacted with the membranes. 2,4-Dintrophenol, an uncoupling agent of oxidative phosphorylation, was found to suppress the possible protein-membrane interaction but to enhance the sedimentation of proteins. These observations were confirmed on beta-galactosidase by Western blot analysis probed with anti-beta-galactosidase antibody. We further observed that a small amount of the intracellular proteins relased from cells during the heating period, being peaked at 45ﾟC,and 2,4-dinitrophenol also suppressed the release. The Western blot analysis also indicated the release of beta-galactosidase from the cell, although the enzyme was partially degraded.