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M Futai

Publications and source records attributed to M Futai.

At least 181 records · Page 10Linked to original sources
PubMed1986

Purification and properties of an auxin-binding protein from maize shoot membranes.

An auxin-binding protein was purified from membranes of maize shoots including the coleoptiles, leaf rolls and mesocotyls. The method of Ca2+-promoted sedimentation of membrane particles was adopted for large-scale preparation. The auxin-binding protein was solubilized from the acetone-washed membranes, and purified by successive chromatographies on DEAE-Sephacel, 1-naphthylacetic acid-linked AH-Sepharose 4B, and Sephadex G-100 columns. The yield of the purified protein was about 0.2 mg from 1 kg of shoots. The binding protein exists as a dimer with a subunit molecular weight of 21,000, and possesses one auxin-binding site per dimer. The preparation also contains a minor molecular form with a subunit molecular weight of 20,000. The auxin-binding protein is not a hydrophobic protein, as judged from its amino acid composition and solubility. The circular dichroic (CD) spectrum of the binding protein resembles the spectrum anticipated from the beta-structures, and shows no alpha-helix characteristic in the secondary structure. The CD spectral changes induced on the binding of auxin and its antagonist seem to be related to the receptor function. The affinity of the binding protein for auxin is dependent on pH, with an optimum at pH 5.0, while the binding protein is unstable below pH 6. We discuss here the intracellular localization of the auxin-binding protein from the view point of the controversial pH-dependence of the binding affinity and stability.

Amino Acids↗
PubMed1985

ATP synthesis and hydrolysis by a hybrid system reconstituted from the beta-subunit of Escherichia coli F1-ATPase and beta-less chromatophores of Rhodospirillum rubrum.

Photophosphorylation and ATPase activities were restored to beta-less Rhodospirillum rubrum chromatophores by their reconstitution with purified beta-subunits of either R. rubrum F1-ATPase (Rr beta) or Escherichia coli F1-ATPase (Ec beta). In the homologous reconstituted system both activities were restored to the same extent, whereas in the hybrid system ATP synthesis was restored to about 10% when the hydrolysis was restored to 200%. This difference in rates of synthesis and hydrolysis was not due to any general uncoupling effect of Ec beta leading to an increased membrane permeability to protons, because with both hybrid and homologous systems an identical light-induced quenching of quinacrine fluorescence was observed. They differed, however, in ATP-driven quenching of quinacrine fluorescence, which was much lower in the hybrid system. These results suggest that the hybrid has a decreased capacity for proton-translocation through the membrane-bound Fo channel during ATP hydrolysis, and probably also during ATP synthesis. The very high ATPase activity of the hybrid system indicates that it might enable the released protons to leak to the outside medium rather than to move inside through the Fo channel. The activities restored by Rr beta and Ec beta exhibit a similar sensitivity to dicyclohexylcarbodiimide, but different sensitivities to oligomycin and to an anti-E. coli F1 (EcF1) antibody. Oligomycin inhibited only the homologous R. rubrum system whereas anti-EcF1 was a much more effective inhibitor of the hybrid system. It is therefore concluded that Rr beta plays a role, that the Ec beta cannot fulfill, in conferring oligomycin sensitivity to the RrFo X F1-ATP synthase-ATPase complex.

Adenosine Triphosphate↗
PubMed1985

Deletion of seven amino acid residues from the gamma subunit of Escherichia coli H+-ATPase causes total loss of F1 assembly on membranes.

A mutant gene for the gamma subunit of H+-translocating ATPase was cloned from Escherichia coli mutant NR70 isolated by B. P. Rosen [J. Bacteriol. 116, 1124-1129 (1973)]. Determination of its nucleotide sequence revealed a deletion of 21 base pairs between nucleotide residues 64 and 84, resulting in a deletion of seven amino acid residues (LysAlaMetGluMetValAla) from the amino-terminal portion. This deletion resulted in the loss of a hydrophobic domain of the subunit estimated by an analysis of its hydropathic character. Since F1 subunits are reported not to be assembled on the normal F0 portion of NR70, it is concluded that the hydrophobic domain deleted in the mutant subunit is important for assembly of the F1 portion. Introduction of a plasmid pNR70 carrying the mutant allele of NR70 into a wild-type strain gave no recombinants resistant to neomycin. This result suggested that the neomycin-resistant phenotype is not directly related to the defect in the gamma subunit of NR70.

Amino Acid Sequence↗
PubMed1985

Change of inhibitor sensitivities of Escherichia coli F1-ATPase due to a mutational substitution of Phe for Ser at residue 174 of the beta subunit.

The F1-ATPase from the uncD11 mutant of E. coli (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., & Futai, M. (1980) J. Biochem. 88, 695-703), showed different enzymological properties from the wild-type enzyme. The mutant F1-ATPase had biphasic kinetics and essentially the same Km values as the wild-type enzyme, although its Vmax values were lower. The mutant enzyme showed altered sensitivities to dicyclohexylcarbodiimide (DCCD), azide and quercetin; it was less sensitive than the wild-type to quercetin and DCCD, and its Mg2+-dependent ATPase activity was slightly more resistant to azide than that of the wild-type, whereas its Ca2+-dependent activity was more sensitive. On the other hand, the mutant and wild-type F1 were inhibited equally by 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). The fact that the Mg2+- and Ca2+-dependent F1-ATPase activities of the wild-type and mutant responded differently to quercetin and azide suggested that their mechanisms of action were different. Previous studies (Noumi, T., Mosher, M.E., Natori, S., Futai, M., & Kanazawa, H. (1984) J. Biol. Chem. 259, 10071-10075) indicated that Ser is replaced by Phe at residue 174 of the beta subunit of the mutant. Thus the Ser residue or its neighboring area(s) may constitute the binding site of DCCD, quercetin and azide.

Amino Acid Sequence↗
PubMed1985

Escherichia coli mutants possessing an Li+-resistant melibiose carrier.

Escherichia coli K-12 strains in the absence of the lactose carrier grew on the disaccharide melibiose as the sole source of carbon. The presence of 0.1 mM Li+ in the medium strongly inhibited growth of such cells, and Li+-resistant mutants appeared after several days of incubation. These mutants showed altered cation coupling to melibiose transport via the melibiose carrier. Cotransport between H+ and melibiose was lost in the mutants, although Na+-melibiose cotransport was retained. We observed no Li+-melibiose cotransport. Therefore, these mutants represent a new type of cation-coupling mutants of the melibiose carrier.

Biological Transport↗
PubMed1985

Alteration in cation specificity of the melibiose transport carrier of Escherichia coli due to replacement of proline 122 with serine.

The structural genes (melB) for the melibiose carrier of five mutants of Escherichia coli showing altered cation specificity for melibiose transport were cloned. The mutations were mapped in a 248-base-pair DNA fragment by a recombinational assay by using the mutants transformed with hybrid plasmids carrying various portions of the wild-type melB gene. The nucleotide sequences of the corresponding DNA fragments derived from mutated melB genes were determined, and the amino acid sequences of the carrier were deduced. Proline 122 was replaced with serine in the melibiose carrier of all five mutants (which were isolated independently). We conclude that this amino acid replacement caused the alteration in cation specificity (loss of coupling to H+) of the melibiose carrier.

Amino Acid Sequence↗
PubMed1984

A phenylalanine for serine substitution in the beta subunit of Escherichia coli F1-ATPase affects dependence of its activity on divalent cations.

A mutant (KF11) of Escherichia coli H+-translocating ATPase (F1-F0) has a single point mutation in the beta subunit of F1 that has lost 90% of its Mg2+-dependent ATPase activity (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M. (1980) J. Biochem. (Tokyo) 88, 695-703). The mutation was mapped at about the 500th nucleotide residue from the 5' end of the beta subunit gene by a genetic recombination test on the physical map of the cistron coding for the beta subunit. The mutant allele of KF11 (uncD11) was cloned on a hybrid plasmid (pKF11) via DNA isolated from a lambda uncD11 transducing phage. Restriction fragments of pKF11 containing the estimated mutation site were subjected to polyacrylamide gel electrophoresis under conditions where strands were separated into single strands. The two strands of a DNA segment, which was shown to carry an altered base, showed anomalous migration compared with those from the wild-type fragment. The results confirmed the result of mapping of the altered site by genetic tests. On the basis of these results, the nucleotide sequence of the mutated gene was determined, and a single base change of the 524th cytosine to thymine resulting in a phenylalanine for serine substitution at residue 174 of the beta subunit was found. This result, together with results on the altered properties of F1 from KF11 reported previously, indicates that residue 174 is essential for the Mg2+-dependent ATPase activity of F1 but not for the Ca2+-dependent ATPase activity.

Amino Acid Sequence↗
PubMed1984

Replacement of serine 373 by phenylalanine in the alpha subunit of Escherichia coli F1-ATPase results in loss of steady-state catalysis by the enzyme.

The mutant allele (uncA401) of the gene for the alpha subunit of Escherichia coli F1-ATPase was cloned from the total DNA of the mutant AN120 on a hybrid plasmid pAN120. Determination of the DNA sequence of the alpha subunit gene from pAN120 revealed a single base change of cytosine at nucleotide residue 1118 to thymine and indicated that serine 373 was replaced by phenylalanine. It has been reported that the mutant F1 is defective in a step of steady-state catalysis, whereas its single turnover process is normal (Kanazawa, H., Noumi, T., Matsuoka, I., Hirata T., and Futai, M. (1984) Arch. Biochem. Biophys. 228, 258-269). Thus, we concluded that serine 373 in the alpha subunit is essential for steady-state catalysis by F1-ATPase.

Alleles↗
PubMed1984

Nucleotide sequence of the promoter region of the melibiose operon of Escherichia coli.

The nucleotide sequence of the promoter region of the melibiose operon of E. coli was determined. Consensus sequences for the -35 region, the Pribnow box and the binding site for cyclic AMP receptor protein were found in this region. The possible secondary structure of this DNA region was very similar to that of the promoter region of the lactose operon. A possible initiation ATG preceded by a Shine-Dalgarno sequence with proper spacing was present just downstream of the promoter region. The possible sequence of 52 amino acid residues in the NH2 terminus of the alpha-galactosidase were determined.

Amino Acid Sequence↗
PubMed1984

Detection of ATP-dependent conformational change in the F1 portion and beta subunit of Escherichia coli H+-ATPase using 8-anilinonaphthalene-1-sulfonate.

The bindings of ATP to Escherichia coli coupling factor ATPase (F1) and its isolated beta subunit were studied with 8-anilinonaphthalene-1-sulfonate (ANS). The fluorescence of ANS increased upon addition of F1 or the beta subunit, and this fluorescence was quenched on addition of ATP. Thus, ANS bound to the hydrophobic region of F1 or the beta subunit, and the binding of ATP was detected as a quenching of ANS fluorescence. The quenching of the fluorescence suggested that F1 or the beta subunit underwent a conformational change on binding to ATP. With and without ATP, 5.0 and 3.6 mol of ANS, respectively, bound to 1 mol of F1. On the other hand, with or without ATP about 1 mol of ANS bound to beta. The fluorescence quenching was specific for ATP and was not observed with GTP or CTP. It was dependent on pH, being higher at acidic pH, but it was not enhanced by MgCl2. The dissociation constants of F1 and the beta subunit for ATP were estimated to be 10(-4)-10(-5) M. The significance of these binding sites is discussed in relation to the mechanism of the ATPase.

Escherichia coli↗
PubMed1984

Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coli.

The nucleotide sequence of the melB gene coding for the melibiose carrier in Escherichia coli has been determined. The melibiose carrier is predicted to consist of 469 amino acid residues, resulting in a protein with a molecular weight of 52,029. The predicted carrier protein is highly hydrophobic (70% nonpolar amino acid residues). The hydropathic profile suggests that there are 10 long hydrophobic segments in the primary structure of the carrier protein. Most of them seem to traverse the membrane. Although the hydropathic profile of the melibiose carrier is similar to that of the lactose carrier as a whole, homology in the primary structure between the two carriers is very low. Furthermore, no homology in the nucleotide sequence is found in the structural genes for the two carriers. However, the nucleotide sequences of the intergenic regions are very similar between the melibiose operon and the lactose operon. There is a typical intercistronic regulatory sequence in the 3'-flanking region of the melB as well as in that of the lacY, which suggests the presence of another gene downstream of the melB.

Amino Acid Sequence↗
PubMed1984

Comparison of F1's of oxidative phosphorylation from Escherichia coli and Salmonella typhimurium and demonstration of interchangeability of their subunits.

The peripheral membrane portion (SF1) of proton-translocating ATPase of Salmonella typhimurium and its alpha, beta, and gamma subunits were purified and compared with the same portion (EF1) from Escherichia coli. The alpha, beta, and gamma subunits of these F1's were found to be mutually interchangeable, and all possible combinations of the three subunits from EF1 and SF1 showed ATPase activity. Both F1's could bind functionally to the integral membrane part (F0) of either bacterium, suggesting that F0 and F1 are interchangeable in these two bacteria and thus that the two F1's are closely similar at the level of subunit structure. However, SF1 differed from EF1 in some enzymological properties such as its specific activity and susceptibilities to sodium dodecyl sulfate and methanol. The specific ATPase activity of EF1 was more than twice that of SF1, and hybrid enzymes containing the beta subunit of EF1 had higher activity than other hybrids. Amino acid analysis suggested that the primary structures of the alpha subunits of the two F1's are less homologous than those of the beta subunits. Thus, the primary structure of the alpha subunit may be more species specific than that of the beta subunit.

Amino Acids↗
PubMed1984

Physical and genetic characterization of the melibiose operon and identification of the gene products in Escherichia coli.

We have identified hybrid plasmids carrying the melibiose operon of Escherichia coli in a colony bank of Clarke and Carbon (Tsuchiya, T., Ottina, K., Moriyama, Y., Newman, M., and Wilson, T. H. (1982) J. Biol. Chem. 257, 5125-5128). Using one of the plasmids as a starting material, the DNA fragments containing the melibiose operon were recloned in a vector pBR322. Restriction maps were prepared, and several DNA segments were subcloned into pBR322. Genetic complementation tests and recombination analyses using those plasmids and melA- and melB- mutants as well as biochemical analyses of mel mutants transformed with those plasmids enabled us to determine the physical location of promoter, melA, and melB on the DNA segment. The size of the melAB region was about 3,000 base pairs. Gene products were identified using maxicells harboring plasmids carrying the melibiose operon. The apparent molecular weight of the alpha-galactosidase (coded by melA) was about 50,000 and that of the melibiose carrier (coded by melB) was about 31,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The melibiose carrier was also identified as a 30,000-dalton protein in reconstituted proteoliposomes which possessed melibiose transport activity.

Disaccharides↗
PubMed1984

Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1.

A plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H+-ATPase) of Escherichia coli causes growth inhibition of wild-type cells. Insertion of a transposable element in this plasmid released this inhibitory effect. In analyzing this inhibitory effect, we determined the insertion points at the nucleotide-sequence level of transposable elements on 30 independent derivatives of pKY159 . Insertions of IS1, IS5, and gamma delta were found between the promoter and the gene for a possible component of 14,000 daltons of the H+-ATPase. Of 31 insertions, 26 were of IS1 and were located at the same site, indicating that this site is a hotspot for IS1 insertion and that IS1 insertion is much more frequent than that of IS5 or gamma delta in this region. Four different sites for IS1 insertion were found; in two of these an 8 base pair (bp) duplicate of the target sequence ( AAAAACGT and AAACGTTG ) was generated, while in the other two a 9 bp duplicate was found. In all cases in this study the nucleotide sequence of IS1 was the same as that of IS1-K. In the two cases with an 8 bp duplicate in different sites, a common 6 bp sequence ( AAACGT ) was found. These results suggested that generation of the 8 bp duplicate is related to the common sequence rather than a mutation in IS1 suggested by Iida et al. (1981) and also suggested that the essential length of the duplicate is 8 bp or less than 8 bp. A 6 bp sequence ( GTGATG ) homologous to the end portion of IS1 was found at the hotspot , but not at other sites, suggesting that this homology contributed to the high frequency of IS1 insertion.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

F1ATPase of Escherichia coli: a mutation (uncA401) located in the middle of the alpha subunit affects the conformation essential for F1 activity.

F1ATPase from the Escherichia coli mutant of H+-ATPase, AN120 (uncA401), has less than 1% of the wild type activity and has been shown to be defective in the alpha subunit by in vitro reconstitution experiments. In the present study, the mutation site was located within a domain of the subunit by recombinant DNA technology. For this, a series of recombinant plasmids carrying various portions of the alpha subunit gene were constructed and used for genetic recombination with AN120. Analysis of the recombinants indicated that the mutation site could be located between amino acid residues 370 and 387. The biochemical properties of the mutant F1 were analyzed further using the fluorescent ATP analog DNS-ATP (2'-(5-dimethylaminonaphthalene-1-sulfonyl)-amino-2'-deoxy ATP). The single turnover process of E. coli F1ATPase proposed by Matsuoka et al. [(1982) J. Biochem. 92, 1383-1398.] was compared in the mutant and wild type F1's. Mutant F1 bound DNS-ATP and hydrolyzed it as efficiently as wild type F1. Results showed that binding of ATP to a low affinity site, possibly in the beta subunit, caused decrease of fluorescence of DNS-ATP in the wild type F1 and that this effect of ATP binding was inhibited by DCCD (dicyclohexyl carbodiimide). However, this effect was not inhibited by DCCD in the mutant F1, suggesting that in the proposed process some step(s) after ATP binding to the low affinity site differed in the mutant and wild F1's. When Pi was added to F1 bound to DNS-ATP or to aurovertin, a fluorescent probe capable of binding to the beta subunit, the opposite changes of fluorescence of these probes in the mutant and wild type F1's were observed, suggesting that the conformational change induced by phosphate binding was altered in the mutant F1. On the basis of the estimated mutation site and the biochemical properties of the mutant F1, the correlation of the domain of this site in the alpha subunit with the function of F1 ATPase is discussed.

Adenosine Triphosphate↗
PubMed1984

Solubilization and reconstitution of membrane proteins of Escherichia coli using alkanoyl-N-methylglucamides.

Alkanoyl-N-methylglucamides, nonionic detergents, were utilized to solubilize membrane proteins of Escherichia coli and were used to reconstitute them into liposomes. First, critical micelle concentrations (CMC) of nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide were determined to be 25 mM and 7 mM, respectively, by photometric assay. Then solubilization and reconstitution of the melibiose transport carrier were performed using these detergents at concentrations above the CMC. Melibiose counterflow activity was observed with the proteoliposomes reconstituted from the extracted proteins and phospholipids. The proton-translocating ATPase complex (F1-F0) was also solubilized with these detergents. These results indicate that nonanoyl- and decanoyl-N-methylglucamide are useful detergents for solubilization and reconstitution of membrane proteins.

Biological Transport↗