Jun-ichi Kishikawa 교수

General anesthetics are indispensable for effective clinical care. Although, the mechanism of action of general anesthetics remains controversial, lipid bilayers and proteins have been discussed as their targets. In this study, we focused on the relationship between cellular ATP levels and general anesthetics. The ATP levels of nematodes and cultured mammalian cells were decreased by exposure to three general anesthetics: isoflurane, pentobarbital, and 1-phenoxy-2-propanol. Furthermore, these ge

The V1- and F1- rotary ATPases contain a rotor that rotates against a catalytic A3B3 or α3β3 stator. The rotor F(1-γ) or V1-DF is composed of both anti-parallel coiled coil and globular-loop parts. The bacterial flagellar type III export apparatus contains a V1/F1-like ATPase ring structure composed of FliI6 homo-hexamer and FliJ which adopts an anti-parallel coiled coil structure without the globular-loop part. Here we report that FliJ of Salmonella enterica serovar Typhimurium shows a rotor li

The Na⁺-pumping NADH-ubiquinone oxidoreductase (Na⁺-NQR) couples electron transfer from NADH to ubiquinone with Na⁺-pumping, generating an electrochemical Na⁺ gradient that is essential for energy-consuming reactions in bacteria. Since Na⁺-NQR is exclusively found in prokaryotes, it is a promising target for highly selective antibiotics. However, the molecular mechanism of inhibition is not well-understood for lack of the atomic structural i

Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal d

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with F_oF₁ ATP synthase. When powered by ATP hydrolysis, the V₁ domain rotates the central rotor against the A₃B₃ hexamer, composed of three catalytic AB dimers adopting different conformations (AB_open, AB_semi, and AB_closed). Here, we report the atomic models of 18 catalytic intermediates of the V₁ domain of V/A-ATPase und

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V₁ domain, with proton flow through the V_o membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V₁ domain, the V_o domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the V_o domain is prevented, how

Adenosine 5'-triphosphate (ATP) is the major energy currency and is involved in many biological processes. The ATP-monitoring system for cells in animals can be helpful to study the relationship between energy metabolism and biological processes. The fluorescent ATP biosensor ATeam (ATP indicator based on Epsilon subunit for Analytical Measurements), which has been reported to monitor ATP levels in cultured cells on the basis of fluorescence resonance energy transfer (FRET), was introduced into

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconsti

ATP synthases play a crucial role in energy production by utilizing the proton motive force (pmf) across the membrane to rotate their membrane-embedded rotor c-ring, and thus driving ATP synthesis in the hydrophilic catalytic hexamer. However, the mechanism of how pmf converts into c-ring rotation remains unclear. This study presents a 2.8 Å cryo-EM structure of the V_o domain of V/A-ATPase from Thermus thermophilus, revealing precise orientations of glutamate (Glu) residues in the c<s

The Na⁺-pumping NADH-quinone oxidoreductase (Na⁺-NQR) is a key respiratory enzyme in many marine and pathogenic bacteria that couples electron transfer to Na⁺-pumping across the membrane. Earlier X-ray and cryo-EM structures of Na⁺-NQR from <i>Vibrio cholerae</i> suggested that the subunits harboring redox cofactors undergo conformational changes during catalytic turnover. However, these proposed rearrangements have not yet been confirmed. Here, we hav