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¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1997 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1996 : ¥1,200,000 (Direct Cost : ¥1,200,000)
The cytoplasmic pH of enterococci is regulated by proton extrusion via an F-type of H^+-ATPase. The membrane level of this enzyme increases when the cytoplasm is acidified, and the increased level of the enzyme extrudes protons, resulting in the increase in the cytoplasmic pH.Thus, the regulation of the enzyme level is important for the pH homeostasis in enterococci. Our previous results have suggested that the enzyme level is regulated at the step of enzyme assembly from its subunits. In this study, we have investigated the mechanism for the regulation of enzyme assembly by the internal pH.
An F-type of H^+-ATPase consists of two parts One is the membrane bound F_0 portion which consists of three subunits. The other is the F_1 complex consisted of five subunits and the complex is attached to the F_0 portion of the membranes. We postulated two possibilities to account the regulation of the assembly. One is that F_0 and F_1 complexes are assembled independently, and the attachment of F_1
to F_0 is regulated by pH.The second one is that the F_0 complex is assembled first in the membranes, and then F_1 subunits are assembled on the F_0 portion. In the latter case, some step of th assembly can be argued to be regulated by pH.Our present results suggested that the first possibility was unlikely.
An Enterococcus hirae mutant AS17, in which glycine at 385 of beta subunit is replaced by tryptophan, was investigated. We found that the increase in the amount of the membrane bound H^+-ATPase was small in AS17 when the cytoplasmic pH decreased. Especially, the assembly of the F_1 complex was not stimulated, resulting in no increase in the enzyme level of the membranes at low pH.Both transcription and translation were suggested to be normal in this mutant. The amounts of unassembled F_1 subunits of AS17 were higher than that of the wild type strain, especially at low pH.These results suggest that the assembly of the F_1 complex is not regulated by pH in this mutant.
Based on these results, it can be argued that the assembly takes place according to the second process described above, and that the assembly of beta subunits is regulated by pH in enterococci. Less