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Our research on proton pumping ATPases over three decades: their biochemistry, molecular biology and cell biology.

Futai M - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

Bottom Line: These two physiologically different proton pumps exhibit similarities in subunit assembly, catalysis and the coupling mechanism from chemistry to proton transport through subunit rotation.We mostly discuss our own studies on the two proton pumps over the last three decades, including ones on purification, kinetic analysis, rotational catalysis and the diverse roles of acidic luminal organelles.The diversity of organellar proton pumps and their stochastic fluctuation are the important concepts derived recently from our studies.

View Article: PubMed Central - PubMed

Affiliation: Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation, and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan.

ABSTRACT
ATP is synthesized by F-type proton-translocating ATPases (F-ATPases) coupled with an electrochemical proton gradient established by an electron transfer chain. This mechanism is ubiquitously found in mitochondria, chloroplasts and bacteria. Vacuolar-type ATPases (V-ATPases) are found in endomembrane organelles, including lysosomes, endosomes, synaptic vesicles, etc., of animal and plant cells. These two physiologically different proton pumps exhibit similarities in subunit assembly, catalysis and the coupling mechanism from chemistry to proton transport through subunit rotation. We mostly discuss our own studies on the two proton pumps over the last three decades, including ones on purification, kinetic analysis, rotational catalysis and the diverse roles of acidic luminal organelles. The diversity of organellar proton pumps and their stochastic fluctuation are the important concepts derived recently from our studies.

No MeSH data available.


Related in: MedlinePlus

Preparations of E. coli F-ATPase and its subunits.Polyacrylamide gel electrophoresis of the 4-subunit and 5-subunit F1 sectors (A), isolated F1 subunits (B), and FoF1 (C), are shown. The positions of subunits (α, β, γ, δ, ε, a, b and c) are indicated. The 5-subunit F1 (A, lane 2) could bind to Fo to reconstitute F-ATPase. The F1 sector could be reconstituted from isolated subunits (B, lanes 1, 2, 3, 4, and 5), and FoF1 (C, lane 1) could be reconstituted in liposomes capable of ATP synthesis and ATP-dependent proton transport. F1 sectors are also shown as controls (B, lane 6, C, lane 2). Modified from previous results.21)–23), 26)
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f2-82_416: Preparations of E. coli F-ATPase and its subunits.Polyacrylamide gel electrophoresis of the 4-subunit and 5-subunit F1 sectors (A), isolated F1 subunits (B), and FoF1 (C), are shown. The positions of subunits (α, β, γ, δ, ε, a, b and c) are indicated. The 5-subunit F1 (A, lane 2) could bind to Fo to reconstitute F-ATPase. The F1 sector could be reconstituted from isolated subunits (B, lanes 1, 2, 3, 4, and 5), and FoF1 (C, lane 1) could be reconstituted in liposomes capable of ATP synthesis and ATP-dependent proton transport. F1 sectors are also shown as controls (B, lane 6, C, lane 2). Modified from previous results.21)–23), 26)

Mentions: To define subunit organization, the F1 sectors with different subunit assemblies, 5-subunit (α, β, γ, δ, ε) and 4-subunit (α, β, γ, ε) F1, were purified from the soluble fraction using conventional procedures (Fig. 2A).21) Both F1 sectors had ATPase activity, but only the 5-subunit F1, the first purified active E. coli F1, could bind to washed membranes, and form F-ATPase capable of ATP synthesis and ATP-hydrolysis dependent proton transport. These results indicate that the δ subunit is essential for F1 binding to Fo. The purified F1 could be dissociated at 4 °C in the presence of chaotropic agent such as KNO3 or NaNO3, and all five subunits were purified to apparent homogeneity (Fig. 2B).22), 23) The α3β3γ complex having ATPase activity could be reconstituted from the three purified subunits in the presence of Mg2+ and ATP.22) Combination of the α3β3γ complex with the δ and ε subunits allowed reconstitution of functional F1 that could bind to Fo.23) The purified α and β subunits had nucleotide binding sites,23), 24) consistent with the results of studies on the binding of ATP analogues. Localization of the F1 sector on the cytoplasmic surface of the E. coli plasma membrane was shown using antibodies against 5-subunit F1.18)


Our research on proton pumping ATPases over three decades: their biochemistry, molecular biology and cell biology.

Futai M - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

Preparations of E. coli F-ATPase and its subunits.Polyacrylamide gel electrophoresis of the 4-subunit and 5-subunit F1 sectors (A), isolated F1 subunits (B), and FoF1 (C), are shown. The positions of subunits (α, β, γ, δ, ε, a, b and c) are indicated. The 5-subunit F1 (A, lane 2) could bind to Fo to reconstitute F-ATPase. The F1 sector could be reconstituted from isolated subunits (B, lanes 1, 2, 3, 4, and 5), and FoF1 (C, lane 1) could be reconstituted in liposomes capable of ATP synthesis and ATP-dependent proton transport. F1 sectors are also shown as controls (B, lane 6, C, lane 2). Modified from previous results.21)–23), 26)
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4338836&req=5

f2-82_416: Preparations of E. coli F-ATPase and its subunits.Polyacrylamide gel electrophoresis of the 4-subunit and 5-subunit F1 sectors (A), isolated F1 subunits (B), and FoF1 (C), are shown. The positions of subunits (α, β, γ, δ, ε, a, b and c) are indicated. The 5-subunit F1 (A, lane 2) could bind to Fo to reconstitute F-ATPase. The F1 sector could be reconstituted from isolated subunits (B, lanes 1, 2, 3, 4, and 5), and FoF1 (C, lane 1) could be reconstituted in liposomes capable of ATP synthesis and ATP-dependent proton transport. F1 sectors are also shown as controls (B, lane 6, C, lane 2). Modified from previous results.21)–23), 26)
Mentions: To define subunit organization, the F1 sectors with different subunit assemblies, 5-subunit (α, β, γ, δ, ε) and 4-subunit (α, β, γ, ε) F1, were purified from the soluble fraction using conventional procedures (Fig. 2A).21) Both F1 sectors had ATPase activity, but only the 5-subunit F1, the first purified active E. coli F1, could bind to washed membranes, and form F-ATPase capable of ATP synthesis and ATP-hydrolysis dependent proton transport. These results indicate that the δ subunit is essential for F1 binding to Fo. The purified F1 could be dissociated at 4 °C in the presence of chaotropic agent such as KNO3 or NaNO3, and all five subunits were purified to apparent homogeneity (Fig. 2B).22), 23) The α3β3γ complex having ATPase activity could be reconstituted from the three purified subunits in the presence of Mg2+ and ATP.22) Combination of the α3β3γ complex with the δ and ε subunits allowed reconstitution of functional F1 that could bind to Fo.23) The purified α and β subunits had nucleotide binding sites,23), 24) consistent with the results of studies on the binding of ATP analogues. Localization of the F1 sector on the cytoplasmic surface of the E. coli plasma membrane was shown using antibodies against 5-subunit F1.18)

Bottom Line: These two physiologically different proton pumps exhibit similarities in subunit assembly, catalysis and the coupling mechanism from chemistry to proton transport through subunit rotation.We mostly discuss our own studies on the two proton pumps over the last three decades, including ones on purification, kinetic analysis, rotational catalysis and the diverse roles of acidic luminal organelles.The diversity of organellar proton pumps and their stochastic fluctuation are the important concepts derived recently from our studies.

View Article: PubMed Central - PubMed

Affiliation: Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation, and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan.

ABSTRACT
ATP is synthesized by F-type proton-translocating ATPases (F-ATPases) coupled with an electrochemical proton gradient established by an electron transfer chain. This mechanism is ubiquitously found in mitochondria, chloroplasts and bacteria. Vacuolar-type ATPases (V-ATPases) are found in endomembrane organelles, including lysosomes, endosomes, synaptic vesicles, etc., of animal and plant cells. These two physiologically different proton pumps exhibit similarities in subunit assembly, catalysis and the coupling mechanism from chemistry to proton transport through subunit rotation. We mostly discuss our own studies on the two proton pumps over the last three decades, including ones on purification, kinetic analysis, rotational catalysis and the diverse roles of acidic luminal organelles. The diversity of organellar proton pumps and their stochastic fluctuation are the important concepts derived recently from our studies.

No MeSH data available.


Related in: MedlinePlus