|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1995 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1994 : ¥1,500,000 (Direct Cost : ¥1,500,000)
To clarify characteristics of Na^+ -translocating ATP synthase of a psychrophilic marine bacterium, Vibriosp. Strain ABE-1 which can synthesize ATP by electrochemical potential of Na^+ across membranes, ATPase bound to inner membrane of this bacterium was purified and characterized. From substrate specificity, sensitivity to various inhibitors for ATPases, subunit composition and molecular weights of subunits, and cross-reactivity with antibodies against several subunits of H^+-translocating F_0F_1-ATPase from Escherichia coli and V-type H^+-ATPase from mung bean tonoplast of the purified enzyme, it was concluded that it is F_0F_1-type ATPase. In contrast to cold-sensitivity of H^+-translocating F_0F_1-ATPase ubiquitously distributed in aerobic organisms, oligomeric structure of the purified ATPase which is essential to ATP synthesis was found to be disrupted above 30ﾟC.The purified ATPase was reconstituted into liposomes prepared by the sonication of phospholipids extracted from soy bean by mixing the enzyme with liposomes in the presence of Triton X-100 and the following removal of the detergent. Since ATP-dependent H^+-influx into the reconstituted proteoliposomes was observed, it was elucidated that the purified ATPase is able to translocate H^+ concomitantly with the hydrolysis of ATP.
An attempt was also made to isolate Na^+-translocating ATP synthase-deficient mutant of Vibrio sp. strain ABE-1. Neomycin-resistant mutants (155 strains) in which mutants devoid of various functions in energy metabolisms is known to be contained was isolated by sponteneous mutation. Among them, mutants sensitive to a protonophore, CCCP,(9 strains) was further selected. In vivo ATP synthesis of several strains among them was more sensitive to CCCP than that of wild strain. Membrane-bound ATPase of the CCCP-sensitive mutants exhibited properties similar to that of wild strain, but ATPase activity of a certain mutant strain was somewhat lower at neutral pHs than that of wild strain.