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T Higuti

Publications and source records attributed to T Higuti.

At least 37 records · Page 2Linked to original sources
PubMed1992

A simple, rapid method for purification of epsilon-subunit, coupling factor 6, subunit d, and subunit e from rat liver H(+)-ATP synthase and determination of the complete amino acid sequence of epsilon-subunit.

The rat liver mitochondrial epsilon-subunit, coupling factor 6, subunit d, and subunit e of H(+)-ATP synthase, which are all extra subunits with no counterparts in Escherichia coli, were purified by reverse-phase high performance liquid chromatography. The complete amino acid sequence of the rat epsilon-subunit was determined by automated Edman degradation of the whole protein and derived peptides. The protein contains 50 amino acids and has a molecular mass of 5635 kDa. It is a basic hydrophilic protein with an isoelectric point of 10.5. The sequence of the rat epsilon-subunit is highly homologous with that of the epsilon-subunit of bovine heart and slightly similar to those of the epsilon-subunit of the yeast and sweet potato mitochondria. However, it has no homology with any subunit of bacterial or chloroplast H(+)-ATP synthase.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of cDNA for the import precursor of human coupling factor 6 of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat factor 6 as a probe. The sequence was composed of 466 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions on the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame were found to consist of 108 and 76 amino acid residues with molecular weights of 12,596 and 8,969, respectively. The presequence of 32 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.

Adenosine Triphosphatases↗
PubMed1991

ATP-sensitive K+ channel in the mitochondrial inner membrane.

Mitochondria take up and extrude various inorganic and organic ions, as well as larger substances such as proteins. The technique of patch clamping should provide real-time information on such transport and on energy transduction in oxidative phosphorylation. It has been applied to detect microscopic currents from mitochondrial membranes and conductances of ion channels in the 5-1,000 pS range in the outer and inner membranes. These pores are not, however, selective for particular ions. Here we use fused giant mitoplasts prepared from rat liver mitochondria to identify a small conductance channel highly selective for K+ in the inner mitochondrial membrane. This channel can be reversibly inactivated by ATP applied to the matrix side under inside-out patch configuration; it is also inhibited by 4-aminopyridine and by glybenclamide. The slope conductance of the unitary currents measured at negative membrane potentials was 9.7 +/- 1.0 pS (mean +/- s.d., n = 6) when the pipette solution contained 100 mM K+ and the bathing solution 33.3 mM K+. Our results indicate that mitochondria depolarize by generating a K+ conductance when ATP in the matrix is deficient.

Adenosine Triphosphate↗
PubMed1991

H(+)-ATP synthase from rat liver mitochondria. A simple, rapid purification method of the functional complex and its characterization.

A novel, simple, and rapid preparative method for purification of rat liver H(+)-ATP synthase by anion-exchange HPLC was developed. The H(+)-ATP synthase purified had higher ATPase activity in the absence of added phospholipids than any preparation reported previously, and this activity was completely inhibited by oligomycin. When reconstituted into proteoliposomes, the H(+)-ATP synthase showed an ATP-dependent 8-anilinonaphthalene-1-sulfonate response and ATP-Pi exchange activity, both of which were also completely inhibited by oligomycin and an uncoupler, indicating the intactness of the H(+)-ATP synthase. An immunochemical study and a labeling experiment with N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD) demonstrated the presence of chargerin II ( a product of mitochondrial A6L DNA) and DCCD-binding protein (subunit c) in the complex. The subunits of the complex were separated into 11 main fractions by reverse-phase HPLC, and 3 of them and the delta subunit in F1 were partially sequenced. A search for sequence homologies indicated that these components were subunit b, coupling factor 6, subunit delta, and subunit epsilon. This is the first report of the existence of subunit b, factor 6, and chargerin II in H(+)-ATP synthase purified from rat liver mitochondria.

Affinity Labels↗
PubMed1990

cDNA cloning and sequencing for the import precursor of coupling factor 6 in H(+)-ATP synthase from rat liver mitochondria.

The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 458 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions of both the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame consisted of 108 and 76 amino acid residues with a molecular weight of 12,494 and 8,927, respectively. The presequence of 32 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.

Adenosine Triphosphatases↗
PubMed1990

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria.

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.

Amino Acid Sequence↗
PubMed1990

Stoichiometry of chargerin II (A6L) in the H(+)-ATP synthase of rat liver mitochondria.

Previous studies suggested that the hydrophobic protein chargerin II, which is encoded in the A6L of mitochondrial DNA, may have a key role in the energy transduction by mitochondrial H(+)-ATP synthase because an antibody against chargerin II inhibited ATP synthesis and ATP-Pi exchange, in an energy-dependent fashion. In the present work, the contents of chargerin II in the H(+)-ATP synthase purified from rat liver mitochondria and in submitochondrial particles were determined by radioimmunoassay. Results showed that the H(+)-ATP synthase contained chargerin II in a molar ratio of one to one. This is the first report on the stoichiometry of the A6L-product in mitochondrial H(+)-ATP synthase.

Animals↗
PubMed1989

Orientation of chargerin II (A6L) in the ATP synthase of rat liver mitochondria determined with antibodies against peptides of the protein.

Previous studies suggested that the hydrophobic protein chargerin II, which is encoded in the unidentified reading frame A6L of mitochondrial DNA (URFA6L), may have a key role in the energy transduction by mitochondrial ATP synthase because an antibody against chargerin II inhibited ATP synthesis and ATP-Pi exchange, in an energy-dependent fashion. In the present work, the orientation of chargerin II in Fo of the ATP synthase of rat liver mitochondria was examined using antibodies against peptides of chargerin II. Results showed that its N-terminal region (about 8 amino acid residues) was exposed on the surface of the C-side of Fo, but its C-terminal and charge-cluster regions were buried in Fo.

Animals↗
PubMed1988

A hydrophobic protein, chargerin II, purified from rat liver mitochondria is encoded in the unidentified reading frame A6L of mitochondrial DNA.

Previous studies showed that a hydrophobic protein called chargerin II may have a key role in energy transduction of oxidative phosphorylation, since antibody against chargerin II labeled with monoazide ethidium inhibited ATP synthesis, ATP-Pi exchange, and reversed electron flow from succinate to NAD coupled with succinate oxidation by O2. In the present work, unlabeled chargerin II was purified from intact rat liver mitochondria by high performance liquid chromatography. The purified preparation of chargerin II, which was a single protein as judged by polyacrylamide gel electrophoresis and Western blotting, was digested with lysylendopeptidase. The digest was separated on a reverse-phase column into five peptides, which all cross-reacted with the antibody against chargerin II, indicating that they were fragments of chargerin II. The sequences of two of these peptides (a total of 12 amino acids) were determined and found to be highly homologous with the sequence of the carboxyl-terminal peptide of the putative polypeptide encoded by the unidentified reading frame A6L (URFA6L) of mammalian mitochondrial DNA. The amino acid compositions of the purified preparation of chargerin II were in good accord with those of the putative product of the URFA6L. Thus, we concluded that chargerin II is encoded by the URFA6L. This is the first demonstration that the URFA6L product was identified in rat liver mitochondria and purified from the membranes.

Amino Acid Sequence↗
PubMed1987

Immunochemical study of role of chargerin II, a product of URFA6L of mitochondrial DNA in energy transduction of rat liver mitochondria.

Antibody against chargerin II [product of the unidentified reading frame A6L (URFA6L) of mitochondrial DNA] inhibited the ATP-Pi exchange reaction and reversed electron flow from succinate to NAD in mitoplasts (inner membrane plus matrix). The antibody against chargerin II caused greater inhibition on incubation with mitoplasts in the energized than the nonenergized state, suggesting that redox reactions are coupled with a conformational change of chargerin II. The present findings showed that chargerin II, the URFA6L product, may have a key role in energy transduction of mitochondrial oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed1985

Purified hydrophobic proteins, chargerins, are essential for energy transduction in oxidative phosphorylation.

Studies on anisotropic inhibitors, a unique type of inhibitor of energy transduction in oxidative phosphorylation, suggested that redox reactions generate two kinds of negative charges on the outer surface of mitochondrial inner membranes, on redox complexes and on F0, and that the inhibitors inhibit energy transduction by binding to these negative charges. Recent experiments on photoaffinity labeling of mitochondria with monoazide ethidium, which is an anisotropic inhibitor, showed that the inhibitor specifically binds to a hydrophobic protein of the membranes. In the present work the mitochondrial components labeled with monoazide ethidium were further purified and two kinds of hydrophobic proteins (apparent molecular masses, 8 and 13 kDa) were found to be specifically labeled with the inhibitor. These proteins were named chargerin I and II, respectively. Redox reactions greatly increased the molar ratio of ethidium bound to chargerin I and II in mitochondria, reflecting a conformational change of the chargerins coupled with the redox reactions. It was also shown that antibody against chargerin II specifically inhibited ATP synthesis in mitoplasts (inner membranes plus matrix) prepared from rat liver mitochondria. Thus, the present findings show that chargerins have an essential role in energy transduction in oxidative phosphorylation in rat liver mitochondria, in good accord with the conformational coupling model of the H+ pumps and ATP synthesis.

Affinity Labels↗
PubMed1984

Conformational coupling in H+-pumps and ATP synthesis--its analysis with anisotropic inhibitors of energy transduction in oxidative phosphorylation.

The analysis of anisotropic inhibitor-induced phenomena in mitochondria revealed that two kinds of negative charges are generated near surface of the C-side of mitochondrial inner membranes in the energized state, on the redox complexes (I, III & IV) and F0, respectively, and that positively charged anisotropic inhibitors (AI+) inhibit energy transduction in oxidative phosphorylation by binding to these negative charges. Thus, AI+ have two different inhibition sites in oxidative phosphorylation, the redox complexes and F0. The membrane components generating the negative charges in energized mitochondria were examined by the technique of photoaffinity labeling with monoazide ethidium, which is an AI+. Results showed that monoazide ethidium specifically binds to two kinds of hydrophobic protein (of 8 K and 13 K daltons) of mitochondria energized with succinate, and these proteins were named chargerin I and II, respectively. Chargerin I and II, which may be components of the redox complexes and Fo, seem to generate the negative charges described above, and these may be essential for H+-pumps in the redox complexes and F1 X F0. AI+ seem to inhibit ATP synthesis by binding to negatively charged sites of chargerin I and II. Based on these findings and the salient results on energy-transducing membranes obtained recently in other laboratories, a conformational model of H+-pumps and ATP synthesis in mitochondria is proposed, which is also applicable to ATP synthesis in other energy-transducing membranes and ATP-linked active transport of ions.

Adenosine Triphosphate↗