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T Mogi

Publications and source records attributed to T Mogi.

At least 109 records · Page 6Linked to original sources
PubMed1987

Structure-function studies on bacteriorhodopsin. III. Total synthesis of a gene for bacterio-opsin and its expression in Escherichia coli.

We have chemically synthesized a DNA duplex of 757 base pairs which encodes the entire protein sequence of mature bacterio-opsin of Halobacterium halobium. The main aim of the synthesis was to facilitate site-specific mutagenesis in all parts of the gene by replacement of short restriction fragments by their counterparts containing the required nucleotide changes. Therefore, 30 unique restriction sites that are fairly evenly spaced were introduced in the synthetic DNA. A total of 28 oligonucleotides ranging in size from 21 to 69 nucleotides were synthesized corresponding to both strands. The entire gene was assembled from four synthetic fragments of 25, 268, 219, and 245 base pairs. The correctness of the nucleotide sequence was confirmed by sequencing the fragments as well as the complete gene. When expressed under the control of PL promoter in Escherichia coli, the synthetic and the native genes gave similar amounts of bacterio-opsin. Attempts to increase expression of the synthetic gene by introducing codons that are preferred in E. coli or by introduction of a synthetic transcription terminator were without significant effect.

Bacteriorhodopsins↗
PubMed1987

Bacteriorhodopsin mutants containing single tyrosine to phenylalanine substitutions are all active in proton translocation.

To study the possible role of the tyrosine residues in proton translocation by bacteriorhodopsin, we have replaced these residues individually by phenylalanine. The required codon changes were introduced in the bacterioopsin gene by replacement of appropriate restriction fragments by synthetic counterparts containing the desired nucleotide changes. The denatured opsin polypeptides obtained by expression of the mutant genes in Escherichia coli were purified and treated with a mixture of detergents, phospholipids, and retinal in a previously established renaturation procedure. All of the mutant proteins folded to regenerate bacteriorhodopsin-like chromophores. Three mutants with tyrosine to phenylalanine substitutions at positions 57, 83, and 185 regenerated the chromophore more slowly than the wild-type protein, and two of these mutants, Phe-57 and -83, showed slightly blue-shifted chromophores. When reconstituted into liposomes all of the mutant proteins with single Tyr----Phe substitutions pumped protons at rates and levels comparable to those of the wild-type bacteriorhodopsin. We conclude that single substitutions of tyrosine by phenylalanine do not affect folding, retinal binding, or light-dependent proton pumping in bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1986

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12.

Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate (pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product). Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.

Amino Acid Transport Systems, Neutral↗
PubMed1984

Mechanism of proline transport in Escherichia coli K12. I. Effect of a membrane potential on the kinetics of 2H+/proline symport in cytoplasmic membrane vesicles.

The stoichiometric coupling mechanism of the membrane potential (delta psi) in the reaction of H+/proline symport was investigated kinetically, using cytoplasmic membrane vesicles of the proline carrier-overproducing strain of Escherichia coli MinS/ pLC4 -45. When a delta psi was imposed across the cytoplasmic membrane by respiration, the Michaelis constant of transport (Kt) was lowered to about 1 microM, which was 2 orders of magnitude smaller than that of passive influx and efflux, and the maximum velocity (Vmax) was concomitantly enhanced as an exponential function of delta psi. Thermodynamically, the carrier translocated proline with a stoichiometry of 2 mol of protons versus 1 mol of substrate when driven by a delta psi at pH 8.0. Data on the delta psi dependence of Vmax of proline transport could be explained quantitatively by the Geck-Heinz hypothesis (Geck, P., and Heinz, E. (1976) Biochim, Biophys. Acta 443, 49-63). A symmetrical model of the 2H+/proline symport via formation of a carrier/H+/substrate (CH+H+S) intermediate is proposed. In this model, the effect of delta psi on the Kt was resolved as stimulation of formation of a transport intermediate, whereas the effect of delta psi on the Vmax was explained by enhancement of translocation of loaded carriers between the two sides of the membrane.

Biological Transport↗
PubMed1984

Mechanism of proline transport in Escherichia coli K12. II. Effect of alkaline cations on binding of proline to a H+/proline symport carrier in cytoplasmic membrane vesicles.

The substrate binding reaction of the proline carrier was investigated in nonenergized conditions using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The binding activity specifically required both alkaline cations (X+), Na+ and Li+, and protons. The Na+-dependent binding activity was dependent on the proline carrier, which is the product of the putP gene, and was not affected by ionophores and energy transduction inhibitors. The parameters of proline binding were determined by double reciprocal plots in reaction media with various combinations of Na+ and H+ concentrations. The apparent dissociation constant was greatly affected by the Na+ and H+ concentrations of the medium and could be expressed as a combination of the reciprocals of the Na+ and H+ concentrations, while the maximum number of binding sites remained constant. The characteristics of proline binding to the carrier can be explained by a mechanism in which the unloaded carrier forms a carrier/H+/X+ (CH+X+) complex by a random equilibrium and only the CH+X+ complex binds substrate in nonenergized conditions, as proposed for the Na+/H+/glutamate symport carrier of E. coli B ( Fujimura , T., Yamato , I., and Anraku , Y. (1983) Biochemistry 22, 1954-1959).

Amino Acid Transport Systems, Neutral↗
PubMed1984

Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier.

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.

Amino Acid Transport Systems, Neutral↗
PubMed1984

[Three surgically treated cases of spontaneous rupture of the esophagus with reference to a new surgical approach--eversion stripping and esophagogastrostomy through the posterior mediastinum].

Three surgical cases of spontaneous rupture of the esophagus were reported. Case 1 was a 56-year-old man who was admitted 3 days after the onset was treated with open drainage. He had no complications whatsoever 8 years after the treatment. In case 2 (a 55-year-old man), eversion stripping of the esophagus and gastrostomy were performed 2 months after diagnosis, but esophageal reconstruction was not successful. He died of acute congestive heart failure 5 years after surgery. The Third case was a 60-year-old women whose rupture was confirmed 11 days after the onset. Cervical esophagostomy, gastrostomy and jejunostomy were performed 16 days after the rupture. Thereafter, esophageal eversion stripping and esophagogastrostomy through the posterior mediastinum were successfully carried out 2 months after the first surgery. She had no postoperative complications. Eversion stripping of the esophagus with esophagogastrostomy through the posterior mediastinum is an effective and safe method for some advanced cases of spontaneous esophageal rupture. We have not found any reports of surgical cases with spontaneous esophageal rupture treated by this approach in the literature.

Esophageal Diseases↗
PubMed1976

Total rupture of the left main bronchus successfully repaired nine years after injury.

A patient who sustained a traumatic rupture of the left main bronchus that was successfully repaired nine years later is presented, and reference is made to reports of similar instances in which operation has been performed more than five years after injury. Pulmonary function following operation improved progressively and proved the delayed repair to have been a reasonable decision.

Adult↗