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Y Mukohata

Publications and source records attributed to Y Mukohata.

At least 19 recordsLinked to original sources
PubMed1994

The light-driven proton pump, cruxrhodopsin-2 in Haloarcula sp. arg-2 (bR+, hR-), and its coupled ATP formation.

Haloarcula sp. arg-2, a natural bacterial isolate from Andes heights, has a light-driven proton pump but not a light-driven anion pump. We have cloned and sequenced the gene encoding for the proton pump which has been named cruxrhodopsin-2. The gene consists of 768 bp encoding 255 amino acids with a molecular mass of 27,544 Da. The deduced amino acid sequence of cruxrhodopsin-2 is 77%, 50%, 48% and 48% identical to those of cruxrhodopsin-1, bacteriorhodopsin, archaerhodopsin-1 and archaerhodopsin-2, respectively. The charged amino acids important for the proton pump function were conserved among all these molecules. Cruxrhodopsin-2 accounted for 0.05 nmol/mg protein in arg-2, which was 20-30-fold less than the proportion of bacteriorhodopsin in Halobacterium salinarium R1M1. In contrast to R1M1, under anaerobic conditions, arg-2 showed light-induced proton extrusion concomitant with an increase in ATP level without transient proton uptake. Dicyclohexylcarbodiimide enhanced the rate and extent of proton extrusion and inhibited ATP formation in the light. The apparent stoichiometry of H+/ATP was estimated to be more than three in this natural bR+hR- strain.

Adenosine Triphosphate↗
PubMed1994

Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs.

Composition of retinal isomers in three proton pumps (bacteriorhodopsin, archaerhodopsin-1, and archaerhodopsin-2) was determined by high performance liquid chromatography in their light-adapted and dark-adapted states. In the light-adapted state, more than 95% of the retinal in all three proton pumps were in the all-trans configuration. In the dark-adapted state, there were only two retinal isomers, all-trans and 13-cis, in the ratio of all-trans: 13-cis = 1:2 for bacteriorhodopsin, 1:1 for archaerhodopsin-1, and 3:1 for archaerhodopsin-2. The difference in the final isomer ratios in the dark-adapted bacteriorhodopsin and archaerhodopsin-2 was ascribed to the methionine-145 in bacteriorhodopsin. This is the only amino acid in the retinal pocket that is substituted by phenylalanine in archaerhodopsin-2. The bacteriorhodopsin point-mutated at this position to phenylalanine dramatically altered the final isomer ratio from 1:2 to 3:1 in the dark-adapted state. This point mutation also caused a 10 nm blue-shift of the adsorption spectrum, which is similar to the shift of archaerhodopsin-2 relative to the spectra of bacteriorhodopsin and archaerhodopsin-1.

Adaptation, Physiological↗
PubMed1992

Photophosphorylation elements in halobacteria: an A-type ATP synthase and bacterial rhodopsins.

Photophosphorylation in halobacteria is carried out by two rather simple elements: an A-type ATP synthase and light-driven ion-pumping bacterial rhodopsins. The unique features of halobacterial ATP synthase, mostly common to archaebacteria (A-type), and of new members of the bacteriorhodopsin family are introduced along with studies performed in the authors' laboratory. This is the story of how we found that the A-type ATP synthase is close to V-type ATPase but far from F-type ATPase, although all three ATPases are believed to have the same ancestor. Archaerhodopsins, the new members of the proton-pumping retinal proteins, were found in Australian halobacteria and have been used in a comparative study of bacterial rhodopsins.

Adenosine Triphosphatases↗
PubMed1991

A vacuolar ATPase and pyrophosphatase in Acetabularia acetabulum.

Vacuole-rich fractions were isolated from Acetabularia acetabulum by Ficoll step gradient centrifugation. The tonoplast-rich vesicles showed ATP-dependent and pyrophosphate-dependent H(+)-transport activities. ATP-dependent H(+)-transport and ATPase activity were both inhibited by the addition of a specific inhibitor of vacuolar ATPase, bafilomycin B1. A 66 kDa polypeptide present in the preparation cross-reacted with the anti-IgG fractions against the alpha and beta subunits of Halobacterium halobium ATPase and with the antibody against the A subunit (68 kDa subunit) of mung bean vacuolar ATPase. A 56 kDa polypeptide present in the vacuole preparation showed cross-reactivity with the antibody against the B subunit (57 kDa) of mung bean vacuolar ATPase but not with the anti-beta subunit of H. halobium ATPase. A 73 kDa polypeptide cross-reacted with the antibody against inorganic pyrophosphatase of mung bean vacuoles. These results suggest that vacuolar membrane of A. acetabulum equipped energy transducing systems similar to those found in other plant vacuoles.

Acetabularia↗
PubMed1991

Archaerhodopsin-2, from Halobacterium sp. aus-2 further reveals essential amino acid residues for light-driven proton pumps.

We have isolated a retinal protein which differs from bacteriorhodopsin and archaerhodopsin and pumps out as many protons in the light as those proton pumps. We tentatively named it archaerhodopsin-2. We have cloned and sequenced the gene that encodes archaerhodopsin-2. The gene consists of 780-bp nucleotides for 259 amino acids with a molecular mass of 27,937 Da. The amino acid sequence of archaerhodopsin-2 is 56% identical to bacteriorhodopsin and 88% to archaerhodopsin, with a few gaps of a few amino acids in both cases. Although the amino acid sequence of archaerhodopsin has revealed 157 conserved residues common to bacteriorhodopsin, the sequence of archaerhodopsin-2 reduces that number to 133. Of these, 38 amino acids are also common to chloride pumps and 24 to all bacterial retinal proteins known to date.

Amino Acid Sequence↗
PubMed1991

The ATP synthase of Halobacterium salinarium (halobium) is an archaebacterial type as revealed from the amino acid sequences of its two major subunits.

The head piece of the A-type ATP synthase in an extremely halophilic archaebacterium, namely Halobacterium salinarium (halobium), is composed of two kinds of subunit, alpha and beta, and is associated with ATP-hydrolyzing activity. The genes encoding these subunits with hydrolytic activity have been cloned and sequenced. The putative amino acid sequences of the alpha and beta subunits deduced from the nucleotide sequences of the genomic DNA consist of 585 and 471 residues, respectively. The amino acid sequence of the alpha subunit of the halobacterial ATPase is 63 and 49% identical to the alpha subunits of ATPases from two other archaebacteria, Methanosarcina barkeri and Sulfolobus acidocaldarius, respectively. The sequence of the beta subunit is 66 and 55% identical to the beta subunits from these respective organisms. The homology between the alpha and beta subunits is around 30%. In contrast, the sequences of the halobacterial ATPase is less than 30% identical to F1 ATPase when any combination of subunits is considered. However, they are greater than 50% identical to a eukaryotic vacuolar ATPase when alpha and a, beta and b combinations are considered. These data fully confirm the first demonstration of this kind of relationship which was achieved by immunoblotting with an antibody raised against the halobacterial ATPase. We concluded that the archaebacterial ATP synthase is an A-type and not an F-type ATPase. This classification is also demonstrated by a "rooted" phylogenetic tree where halobacteria locate close to other archaebacteria and eukaryotes and distant from eubacteria.

Amino Acid Sequence↗
PubMed1991

Australian Halobacteria and their retinal-protein ion pumps.

Halophiles collected in Western Australia have been found to be examples of extremely halophilic rod-shaped archaebacteria, members of the genus Halobacterium. Most of them contain retinal proteins, and these proteins differ from one another and also from both bacteriorhodopsin (bR) and halorhodopsin [and sensory rhodopsins (sR)] isolated from Halobacterium salinarium (halobium), as revealed by their peptide maps and amino acid sequences. However, these retinal proteins still have the ability to pump protons or chloride ions in the light. These new ion pumps, designated archaerhodopsins (aR) [Mukohata et al. (1988) Biochem. Biophys. Res. Commun. 151, 1339-1345], are almost identical in terms of their molecular sizes and transient photochemical properties to the ion pumps identified previously. Differences are found in the: (1) apparent extinction coefficient of dark/light-adapted aR-2; (2) titration profiles at acidic pH of the absorption spectra of all aRs; and (3) circular dichroism spectra, which are influenced by the coexistent isoprenoid bacterioruberin. The amino acid sequences of two proton pumps from the Australian halobacteria, namely aR and aR-2, are approximately 90% homologous and both sequences are about 60% homologous with that of bR. Hydropathy plots suggest that these pumps also have a seven-helical structure similar to that of bR. The amino acid residues are highly conserved in the helical regions, in particular in the case of helices C and G (91 and 84%, respectively), among the three proton pumps.

Amino Acid Sequence↗
PubMed1990

Archaebacterial ATPases: relationship to other ion-translocating ATPase families examined in terms of immunological cross-reactivity.

Immunological cross-reactivity among three types of H(+)-ATPases, that is, three archaebacterial ATPases, the F1-ATPase from thermophilic bacterium PS3 (TF1) and the vacuolar membrane ATPase from Saccharomyces cerevisiae, was examined by means of immunoblot analyses. The three archaebacterial ATPases were very similar in immunological cross-reactivity, suggesting that they belong to the same family of ATPases. Cross-reaction was also observed between the ATPase from Sulfolobus acidocaldarius, one of the three archaebacteria, and TF1. S. cerevisiae vacuolar ATPase reacted with the antibodies prepared against each of the three archaebacterial ATPases, but did not react with the antibody against TF1. Electron microscopic examination revealed that the oligomeric structure of Sulfolobus ATPase was very similar to that of F1-ATPase. These results, taken together, suggest that the archaebacterial ATPases share close structural similarities with the vacuolar ATPases, and, to a lesser degree, with the F0F1-ATPases.

Adenosine Triphosphatases↗
PubMed1989

Isolation of a gene that encodes a new retinal protein, archaerhodopsin, from Halobacterium sp. aus-1.

We have cloned and sequenced the gene that encodes archaerhodopsin, a light-driven H+ pump in Halobacterium sp. aus-1 (Mukohata, Y., Sugiyama, Y., Ihara, K., and Yoshida, M. (1988) Biochem. Biophys. Res. Commun. 151, 1339-1345). The nucleotide sequence of this gene contained an open reading frame which corresponded to a protein of 260 amino acids with a molecular mass of 27,851 daltons, including a precursor sequence of 6 amino acids at the amino terminus and 2 amino acids at the carboxyl terminus. The deduced amino acid sequence of archaerhodopsin exhibited 59 and 32% homology to the sequences of bacteriorhodopsin and halorhodopsin, respectively, from Halobacterium halobium. Three charged residues (Asp-121, Asp-218, and Lys-222) are conserved in the transmembrane segments among the three retinal proteins. Residues Asp-91 and Asp-102 which, it has been suggested, may be essential for the pumping of protons (Mogi, T., Stern, L. J., Marti, T., Chao, B. H., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,4148-4152) are conserved between archaerhodopsin and bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1988

Proteoliposomes with right-side-out oriented purple membrane/bacteriorhodopsin require cations inside for proton pumping.

Proteoliposomes were prepared by sonication of phospholipids and blue membranes (cation-free purple membranes carrying little activity of light-driven proton pumping) in an acidic medium of very low ionic strength. The majority of the bacteriorhodopsin population in these proteoliposomes was in the right-side-out (as in living cells) orientation as judged from the resultant polypeptides after papain digestion. By raising the pH of sonication, the population of right-side-out oriented bacteriorhodopsin decreased, and consequently that of the inversely oriented one increased. In KCl and NaCl up to certain concentrations or in choline chloride even at high concentrations, in the light, the proteoliposomes with right-side-out bacteriorhodopsin did not pump protons, whereas those with inversely oriented bacteriorhodopsin did. The former began to pump only after cations were likely incorporated/permeated into the proteoliposome and reached the carboxyl terminal (cytosol) side of bacteriorhodopsin/purple membrane.

Bacteriorhodopsins↗
PubMed1988

The gamma-subunit of ATP synthase from spinach chloroplasts. Primary structure deduced from the cloned cDNA sequence.

cDNA clones encoding the gamma-subunit of chloroplast ATP synthase were isolated from a spinach library using synthetic oligonucleotide probes. The predicted amino acid sequence indicated that the mature chloroplast gamma-subunit consists of 323 amino acid residues and is highly homologous (55% identical residues) with the sequence of the cyanobacterial subunit. The positions of the four cysteine residues were identified. The carboxyl-terminal region of the chloroplast gamma-subunit is highly homologous with those of the gamma-subunits from six other sources (bacteria and mitochondria) sequenced thus far.

Amino Acid Sequence↗
PubMed1987

The halobacterial H+-translocating ATP synthase relates to the eukaryotic anion-sensitive H+-ATPase.

The H+-translocating ATP synthase of Halobacterium halobium (Y. Mukohata and M. Yoshida (1987) J. Biochem. 102, 797-802) includes a catalytic moiety of 320 kDa which is isolated as an azide-insensitive ATPase (T. Nanba and Y. Mukohata (1987) J. Biochem. 102, 591-598). The polyclonal antibody against this archaebacterial ATPase cross-reacts with the anion-sensitive H+-ATPase of red beet, Beta vulgaris, tonoplast as well as with another archaebacterial ATPase from Sulfolobus acidocaldarius. The affinity is much higher than to F1-ATPase from spinach chloroplasts or to Ca2+-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle.

Archaea↗
PubMed1987

Activation and inhibition of ATP synthesis in cell envelope vesicles of Halobacterium halobium.

The characteristics of ATP synthesis in cell envelope vesicles of Halobacterium halobium were further studied. The results confirmed the previous conclusion (Mukohata et al. (1986) J. Biochem. 99, 1-8) that the ATP synthase in this extremely halophilic archaebacterium can not be an ordinary type of F0F1-ATPase, which has been thought to be ubiquitous among all the aerobic organisms on our biosphere. The ATP synthesis was activated most in 1 M NaCl and/or KCl, and at 40 degrees C, and at 80 mM MgCl2 where F0F1-ATPase loses its activity completely. The synthesis was negligible at 10 degrees C, and at 5 mM MgCl2. The Km for ADP was about 0.3 mM in the presence of 20 mM Pi, 1 M NaCl, 80 mM MgCl2, and 10 mM PIPES at pH 6.8 and 20 degrees C. The ATP synthesis was not inhibited by NaN3 and quercetin (specific inhibitors for F0F1-ATPase) or vanadate (for E1E2-ATPase) or ouabain (for Na+,K+-ATPase) or P1,P5-di(adenosine-5')pentaphosphate (AP5A, for adenylate kinase). The ATP synthesis was not inhibited by modification (pretreatment) with NaN3 or 5'-p-fluorosulfonylbenzoyladenosine (FSBA). On the contrary, the ATP synthesis was rather non-specifically inhibited by N-ethylmaleimide (NEM), trinitrobenzenesulfonate (TNBS), phenylglyoxal, and pyridoxal phosphate. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) as well as N,N'-dicyclohexylcarbodiimide (DCCD) was found to be a specific inhibitor at least partly, because the NBD-Cl inhibition was partly prevented by ADP added to the modification mixture.

Adenosine Diphosphate↗
PubMed1987

A mechanism of respiratory control: studies with proteoliposomes containing cytochrome oxidase and bacteriorhodopsin.

Both beef heart cytochrome oxidase and bacteriorhodopsin of Halobacterium halobium were reconstituted into liposomes by the sonication-cholate dialysis method. The proteoliposomes showed the respiratory control ratio of 4.2, and steady-state illumination of the vesicles lead to the 2.7-fold stimulation of the oxidase activity in the absence of uncouplers. The light-stimulated state 4 respiration increased with light intensity, but light had no effect on the oxidase activity that had been relieved by addition of uncouplers. Proteoliposomes with the photosensitive oxidase activity were also obtained when cytochrome oxidase vesicles were fused with bacteriorhodopsin vesicles in the presence of calcium chloride, and the extent of photoactivation was maximally 1.4-fold. The light-induced respiratory release was observed even in the presence of valinomycin or nigericin, indicating that the oxidase activity was sensitive to both the membrane potential and the pH gradient. We propose as a mechanism of the respiratory control that the process of proton transport to the reaction center for water formation is the rate limiting step for the cytochrome oxidase activity.

Bacteriorhodopsins↗
PubMed1987

A membrane-bound ATPase from Halobacterium halobium: purification and characterization.

An ATPase was newly identified on the inner face of the plasma membrane of the extremely halophilic archaebacterium Halobacterium halobium. The enzyme was released into an alkaline EDTA solution and purified by several chromatographic steps in the presence of sulfate at 1 M or over. The molecular weight of the native enzyme was around 320,000; it is most likely composed of two pairs (alpha 2 beta 2) of 86,000 (alpha) and 64,000 (beta) subunits. The enzyme hydrolyzed ATP and other nucleoside triphosphates but neither ADP nor AMP. The enzyme required divalent cations, among which Mn2+ was most effective (Mg2+ activated 35% of Mn2+). The ATPase activity was optimum at pH between 5.5 and 6, particularly in a nearly saturated Na2SO4 (or Na2SO3) solution, while it was very low in a chloride salt solution even at 4 M at any pH. The Km value for ATP was 1.4 mM and the K1 value for ADP (competitive to ATP) was 0.08 mM. Neither azide (a specific inhibitor for F0F1-and F1-ATPase) nor vanadate (for E1E2-ATPase) inhibited the enzyme. The ATPase was stable at high concentrations of sulfate. At low concentrations of salts, or at low temperatures even in high NaCl concentrations, the enzyme was inactivated. Although the ATPase isolated here from halobacterial membrane has such unusual characteristics, it is the most probable candidate for the (catalytic part of) halobacterial ATP synthase, which differs from F0F1-ATPase/synthase (Mukohata et al. (1986) J. Biochem. 99, 1-8; Mukohata and Yoshida (1987) J. Biochem. 101, 311-318).

Adenosine Triphosphatases↗