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Biomedical subjects

M Futai

Publications and source records attributed to M Futai.

At least 109 records · Page 6Linked to original sources
PubMed1992

Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase beta subunit of Escherichia coli.

beta Lys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of beta Lys-155 and beta Thr-156, mutants (beta Lys-155-->Ala, Ser, or Thr; beta Thr-156-->Ala, Cys, Asp, or Ser; beta Lys-155/beta Thr-156-->beta Thr-155/beta Lys-156; and beta Thr-156/beta Val-157-->beta Ala-156/beta Thr-157) were constructed, and their properties were studied extensively. The beta Ser-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had < 0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (beta Ala-155, beta Ser-155, beta Ala-156, and beta Cys-156) showed low rates of multisite (< 0.02% of the wild type) and unisite (< 1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the beta Ala-156 and beta Cys-156 enzymes and 10(2)-fold lower with the beta Ala-155 and beta Ser-155 enzymes. The beta Thr-156-->Ala or Cys enzyme showed an altered response to Mg2+, suggesting that beta Thr-156 may be closely related to Mg2+ binding. These results suggest that beta Lys-155 and beta Thr-156 are essential for catalysis and are possibly located in the catalytic site, although beta Thr-156 could be replaced by a serine residue.

Adenosine Triphosphate↗
PubMed1992

F0F1-ATPase gamma subunit mutations perturb the coupling between catalysis and transport.

We introduced mutations to test the function of the conserved amino-terminal region of the gamma subunit from the Escherichia coli ATP synthase (F0F1-ATPase). Plasmid-borne mutant genes were expressed in an uncG strain which is deficient for the gamma subunit (gamma Gln-14-->end). Most of the changes, which were between gamma Ile-19 and gamma Lys-33, gamma Asp-83 and gamma Cys-87, or at gamma Asp-165, had little effect on growth by oxidative phosphorylation, membrane ATPase activity, or H+ pumping. Notable exceptions were gamma Met-23-->Arg or Lys mutations. Strains carrying these mutations grew only very slowly by oxidative phosphorylation. Membranes prepared from the strains had substantial levels of ATPase activity, 100% compared with wild type for gamma Arg-23 and 65% for gamma Lys-23, but formed only 32 and 17%, respectively, of the electrochemical gradient of protons. In contrast, other mutant enzymes with similar ATPase activities (including gamma Met-23-->Asp or Glu) formed H+ gradients like the wild type. Membranes from the gamma Arg-23 and gamma Lys-23 mutants were not passively leaky to protons and had functional F0 sectors. These results suggested that substitution by positively charged side chains at position 23 perturbed the energy coupling. The catalytic sites of the mutant enzymes were still regulated by the electrochemical H+ gradient but were inefficiently coupled to H+ translocation in both ATP-dependent H+ pumping and delta mu H+ driven ATP synthesis.

Amino Acid Sequence↗
PubMed1992

Involvement of a non-proton pump factor (possibly Donnan-type equilibrium) in maintenance of an acidic pH in lysosomes.

Change of the internal pH of isolated lysosomes was measured with fluorescein isothiocyanate-dextran. In buffer of pH 7.0, isolated lysosomes had an acidic pH of about 5.5, which decreased to pH 5.2 on addition of ATP. Addition of bafilomycin inhibited the acidification by H(+)-ATPase and resulted in an increase of the internal pH to 5.5 due to passive diffusion of protons across the lysosomal membrane. However, no further alkalization was observed. The acidic pH (pH 5.5) of isolated lysosomes could be maintained for at least 48 h in the absence of ATP, but increased gradually to pH 5.9-6.4 upon incubation with monovalent cations (K+ or Na+), amines, or ionophores. These results suggest that a non-proton pump factor (possibly Donnan equilibrium) is involved in maintaining the acidic pH of isolated lysosomes.

Animals↗
PubMed1992

Mapping of antigenic sites to monoclonal antibodies on the primary structure of the F1-ATPase beta subunit from Escherichia coli: concealed amino-terminal region of the subunit in the F1.

To analyze relationships between the ternary and primary structures of the beta subunit of Escherichia coli F1 ATPase, we prepared two monoclonal antibodies beta 12 and beta 31 against the beta peptide. These antibodies bind to the beta subunit but do not bind to the F1 ATPase, resulting in no inhibition of the ATPase activities. Several different portions of the beta subunit peptide were prepared by constructing expression plasmids carrying the corresponding DNA segment of the beta subunit gene amplified by the polymerase chain reaction. Western blotting analysis using these peptides revealed that the antibodies bound to a peptide of 104 amino acid residues from the amino terminal end, which is outside the previously estimated catalytic domain between residues 140 and 350. These results indicated that the amino terminal portion of the maximal 104 residues is not exposed to the surface of the F1 ATPase. The binding spectrum of the antibodies to the subunit from various species including Vibrio alginolyticus and thermophilic bacterium PS3 indicated possible epitope sequences within the 104 residues. The ternary structure of the beta subunit, in terms of cleavage sites by endopeptidases, was analyzed using the antibodies. A 43-kDa peptide without binding ability to beta 12 and beta 31 appeared upon cleavage by lysyl endopeptidase. The results suggested that lysyl residues from around 70 to 100 from the amino terminus are exposed to the surface of the beta subunit.

Amino Acid Sequence↗
PubMed1992

Vacuolar type H(+)-ATPase genes: presence of four genes including pseudogenes for the 16-kDa proteolipid subunit in the human genome.

Genes for the human vacuolar type H(+)-ATPase proteolipid (16-kDa) subunit were cloned and their nucleotide sequences were determined. Comparison of the deduced sequences indicated that at least four genes including pseudogenes are present in the human genome. One of them corresponded to that for the 16-kDa subunit expressed in HeLa cells. The coding sequence was separated by two introns. The second intron was located in the DNA segment giving a loop between the second and third transmembrane helices, supporting the idea that the 16-kDa subunit was evolved by gene duplication. The primary sequence determined from the second clone had a termination codon behind the third transmembrane helix. Possible translation products from the other two clones had no putative acidic residues essential for proton transport function of the 16-kDa subunit. Thus, it is interesting to know whether these genes are transcribed, since they may have unique cellular functions.

Base Sequence↗
PubMed1992

Sequence motif in control regions of the H+/K+ ATPase alpha and beta subunit genes recognized by gastric specific nuclear protein(s).

A nuclear protein(s) from rat or pig stomach recognized a conserved sequence in the 5'-upstream regions of the rat and human H+/K(+)-ATPase alpha subunit genes. A gel retardation assay suggested that part of the binding site was located in the TAATCAGCTG sequence. No nuclear proteins capable of the binding could be detected in other tissues of rat (liver, brain, kidney, spleen and lung) or pig liver. The sequence motif (GATAGC) located 5'-upstream of the beta-subunit gene also seemed to be recognized by the same protein, because the binding of nuclear protein to the sequence motifs in the alpha and beta subunits was mutually competitive. Considering the sense-strand sequence of the binding motif in the alpha-subunit gene, we conclude that (G/C)PuPu(G/C)NGAT(A/T)PuPy is a core sequence motif for the gastric specific DNA binding protein (PCSF, parietal cell specific factor).

Adenosine Triphosphatases↗
PubMed1992

Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector.

The roles of the Escherichia coli H(+)-ATPase (FoFl) delta subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23----end, Gln-29----end, Gln-74----end) and missense (Gly-150----Asp) mutants had very low ATPase activities, indicating that the delta subunit is essential for the binding of the Fl portion to Fo. The Gln-176----end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174----end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu-175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr-11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150----Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the delta subunit is necessary, but that individual residues may not have strict functional roles.

Amino Acid Sequence↗
PubMed1992

Assembly of a hybrid from the alpha subunit of Na+/K(+)-ATPase and the beta subunit of H+/K(+)-ATPase.

Messenger RNA for the alpha subunit of Torpedo californica Na+/K(+)-ATPase was injected into Xenopus oocytes together with that of the beta subunit of rabbit H+/K(+)-ATPase. The Na+/K(+)-ATPase alpha subunit was assembled in the microsomal membranes with the H+/K(+)-ATPase beta subunit, and became resistant to trypsin. These results suggest that the H+/K(+)-ATPase beta subunit facilitates the stable assembly of the Na+/K(+)-ATPase alpha subunit in microsomes.

Adenosine Triphosphatases↗
PubMed1992

Role of the amino terminal region of the epsilon subunit of Escherichia coli H(+)-ATPase (F0F1).

Escherichia coli strain KF148(SD-) defective in translation of the uncC gene for the epsilon subunit of H(+)-ATPase could not support growth by oxidative phosphorylation due to lack of F1 binding to Fo (M. Kuki, T. Noumi, M. Maeda, A. Amemura, and M. Futai, 1988, J. Biol. Chem. 263, 17, 437-17, 442). Mutant uncC genes for epsilon subunits lacking different lengths from the amino terminus were constructed and introduced into strain KF148(SD-). F1 with an epsilon subunit lacking the 15 amino-terminal residues could bind to F0 in a functionally competent manner, indicating that these amino acid residues are not absolutely necessary for formation of a functional enzyme. However, mutant F1 in which the epsilon subunit lacked 16 amino-terminal residues showed defective coupling between ATP hydrolysis (synthesis) and H(+)-translocation, although the mutant F1 showed partial binding to Fo. These findings suggest that the epsilon subunit is essential for binding of F1 to F0 and for normal H(+)-translocation. Previously, Kuki et al. (cited above) reported that 60 residues were not necessary for a functional enzyme. However, the mutant with an epsilon subunit lacking 15 residues from the amino terminus and 4 residues from the carboxyl terminus was defective in oxidative phosphorylation, suggesting that both terminal regions affect the conformation of the region essential for a functional enzyme.

Amino Acid Sequence↗
PubMed1992

The role of V-ATPase in neuronal and endocrine systems.

Synaptic vesicles have important roles in the neural transmission at nerve terminals: the storage and the controlled exocytosis of neurotransmitters. At least two different factors are responsible for the concentration process: the vacuolar-type H(+)-ATPase (V-ATPase), establishing an electrochemical gradient of protons, and specific transport systems for transmitters. We will discuss our recent progress on the energy-transducing systems in synaptic vesicles: (1) structural aspects of V-ATPase; (2) energy coupling of transport of transmitters; (3) reconstitution of transporters; (4) effects of neurotoxins and neuron blocking agents; (5) function of synaptic-vesicle-like microvesicles from endocrine tissues.

Adenosine Triphosphatases↗
PubMed1992

Escherichia coli ATP synthase (F-ATPase): catalytic site and regulation of H+ translocation.

We discuss our recent results on the Escherichia coli F-ATPase, in particular its catalytic site in the beta subunit and regulation of H+ transport by the gamma subunit. Affinity labelling experiments suggest that beta Lys-155 in the glycine-rich sequence is near the gamma-phosphate moiety of ATP bound at the catalytic site. The enzyme loses activity upon introduction of missense mutations in beta Lys-155 or beta Thr-156 and changes catalytic properties upon introduction of other mutations. By analysis of mutations and their pseudo revertants, residues beta Ser-174, beta Glu-192 and beta Val-198 were found to be located near the glycine-rich sequence. The combined approaches of chemical labelling and genetics have been fruitful in visualizing the structure of the catalytic site. Analysis of mutations in the gamma subunit suggests that this subunit has an essential role in coupling catalysis with proton translocation.

Amino Acid Sequence↗
PubMed1991

One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters.

About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes. The recombinant plasmid pBWU13 carries the unc operon for F0F1. When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential. Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis. F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification.

Adrenal Medulla↗
PubMed1991

The rat H+/K(+)-ATPase beta subunit gene and recognition of its control region by gastric DNA binding protein.

The rat gastric H+/K(+)-ATPase beta subunit gene was cloned, and its nucleotide sequence was determined. The coding region is separated by 6 introns, whereas the related human Na+/K(+)-ATPase beta subunit gene was shown to have 5 introns (Lane, L.K., Shull, M.M., Whitmer, K.R., and Lingrel, J.B. (1989) Genomics 5, 445-453). The positions of introns 1, 2, and 5 of the two genes were the same. The similarities in intron/exon organizations and primary structures (30-40% identical residues) suggest that the beta subunit genes for H+/K(+)-ATPases were derived from a common ancestor. The upstream region of the rat H+/K(+)-ATPase beta subunit gene contains direct repeat sequences and palindromes, potential binding sites for RNA polymerase II and E4TF1, and CACCC box sequences. Gel retardation assay demonstrated that the stomach, but not other tissues (liver, brain, kidney, spleen, and lung), has a nuclear protein(s) capable of binding to the regions upstream of the potential RNA polymerase II binding sites (TATA box). The nuclear protein(s) are suggested to recognize three tandem GATAGC sequences and may be important for controlled transcription of the H+/K(+)-ATPase beta subunit gene in gastric parietal cells.

Adenosine Triphosphatases↗
PubMed1991

Chromaffin granule H(+)-ATPase has F1-like structure.

Isolated H(+)-ATPase from chromaffin granules was reconstituted into liposomes and the resultant proteoliposomes were further purified by Ficoll density gradient centrifugation. Studies by electron microscopy showed that proteoliposomes had particle structures (average diameter, about 10 nm) on their outer surface. These particles could be removed from the proteoliposomes by cold treatment. Immuno-electron microscopy showed that these particles were recognized by antibodies against the hydrophilic sector of the enzyme. These results indicate that the H(+)-ATPase has a peripheral membrane structure similar to that of F1-ATPase.

Animals↗
PubMed1991

Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.

Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H(+)-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic (Bowman, E.M., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7972-7976). Effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 microM bafilomycin A1. The effect was revealed by washing the cells. Both studies using 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysomal pH of A431 cells increased from about 5.1-5.5 to about 6.3 in the presence of 1 microM bafilomycin A1. The pH increased gradually in about 50 min. In the presence of 1 microM bafilomycin A1, 125I-labeled epidermal growth factor (EGF) bound to the cell surface at 4 degrees C was internalized normally into the cells at 37 degrees C but was not degraded at all, in marked contrast to the rapid degradation of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irrespective of the addition of bafilomycin A1. These results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo.

Anti-Bacterial Agents↗
PubMed1991

Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase.

A sequence motif in the beta subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta Gly149 by Ser suppressed the effect of the beta Ser174----Phe mutation (defective H(+)-ATPase), but beta Gly150----Ser substitution did not have this effect. A single mutation (beta Gly149----Ser or beta Gly150----Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta Gly149----Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca(2+)-dependent activity, but had the wild-type level of Mg(2+)-dependent activity with active oxidative phosphorylation. Introduction of a beta Gly149----Ser or beta Gly150----Ser mutation with the beta Ser174----Phe mutation also lowered the Ca(2+)-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta Thr156----Ala or Cys mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity. The possible location of the catalytic site including divalent cation binding site(s) is discussed.

Amino Acid Sequence↗