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T Kouyama

Publications and source records attributed to T Kouyama.

At least 19 recordsLinked to original sources
PubMed1999

Specific lipid-protein interactions in a novel honeycomb lattice structure of bacteriorhodopsin.

In the purple membrane of Halobacterium salinarium, bacteriorhodopsin trimers are arranged in a hexagonal lattice. When purple membrane sheets are incubated at high temperature with neutral detergent, membrane vesicularization takes place, yielding inside-out vesicles with a diameter of 50 nm. The vesicular structure becomes unstable at low temperature, where successive fusion of the vesicles yields a crystal which is composed of stacked planar membranes. X-ray crystallographic analysis reveals that the bacteriorhodopsin trimers are arranged in a honeycomb lattice in each membrane layer and that neighbouring membranes orient in opposite directions. The native structure of the trimeric unit is preserved in the honeycomb lattice, irrespective of alterations in the in-plane orientation of the trimer. One phospholipid tightly bound to a crevice between monomers in the trimeric unit is suggested to act as a glue in the formation of the trimer.

Bacteriorhodopsins↗
PubMed1998

A novel three-dimensional crystal of bacteriorhodopsin obtained by successive fusion of the vesicular assemblies.

When the two-dimensional crystal of bacteriorhodopsin (bR), purple membrane, is incubated at high temperature (32 degreesC) with a small amount of the neutral detergent octylthioglucoside in the presence of the precipitant ammonium sulfate, a large fraction of the membrane fragments is converted into spherical vesicles with a diameter of 50 nm, which are able to assemble into optically isotropic hexagonal crystals when the precipitant concentration is increased. The vesicularization of purple membrane takes place under such a condition that the miscibility of the detergent to the aqueous phase becomes very low, and we suggest that it is initiated by insertion of the detergent molecules into the membrane. At low temperature, the transformation into the vesicular structure is inhibited and no large crystal is produced directly from membrane/detergent/precipitant mixtures. When a suspension of the spherical vesicles produced at the high temperature is cooled and concentrated below 15 degreesC, however, a birefringent hexagonal crystal is produced that diffracts X-rays beyond 2.5 A resolution. This new crystal belongs to the space group P622 with unit cell dimensions of a=b=104.7 A and c=114.1 A, and it is shown to be made up of stacked planar membranes, in each of which the bR trimers are arranged on a honeycomb lattice and the space among the proteins is filled with the detergent molecules and native lipids. These stacked membranes are suggested to be produced by successive fusion of the spherical vesicles. This implies that the crystallization is achieved without any step for complete solubilization of the protein. The present result offers a unique crystallization method that may be applicable to such membrane proteins that are liable to denature in the presence of an excess amount of detergent.

Bacteriorhodopsins↗
PubMed1998

Electron cryomicroscopy of bacteriorhodopsin vesicles: mechanism of vesicle formation.

We obtained vesicles from purple membrane of Halobacterium halobium at different suspension compositions (pH, electrolytes, buffers), following the procedure of Kouyama et al. (1994) (J. Mol. Biol. 236:990-994). The vesicles contained bacteriorhodopsin (bR) and halolipid, and spontaneously formed during incubation of purple membrane suspension in the presence of detergent octylthioglucoside (OTG) if the protein:OTG ratio was 2:1 by weight. The size distribution of the vesicles was precisely determined by electron cryomicroscopy and was found to be almost independent on the incubation conditions (mean radius 17.9-19 nm). The size distribution in a given sample was close to the normal one, with a standard deviation of approximately +/- 1 nm. During dialysis for removal of the detergent, the vesicles diminished their radius by 2-2.5 nm. The results allow us to conclude that the driving force for the formation of bR vesicles is the preferential incorporation of OTG molecules in the cytoplasmic side of the membrane (with possible preferential delipidation of the extracellular side), which creates spontaneous curvature of the purple membrane. From the size distribution of the vesicles, we calculated the elasticity bending constant, K(B) approximately 9 x 10(-20) J, of the vesicle wall. The results provide some insight into the possible formation mechanisms of spherical assembles in living organisms. The conditions for vesicle formation and the mechanical properties of the vesicles could also be of interest with respect to the potential technological application of the bR vesicles as light energy converters.

Bacteriorhodopsins↗
PubMed1998

Highly selective separation of rhodopsin from bovine rod outer segment membranes using combination of divalent cation and alkyl(thio)glucoside.

The micellization process of bovine rod outer segment (ROS) membranes is investigated utilizing a series of neutral detergents. It is found that when alkyl(thio)glucosides with an appropriate hydrophillic-lipophilic balance (e.g. octylthioglucoside) are used in combination with a divalent cation, rhodopsin is selectively extracted from ROS membranes at a specific detergent-to-membrane ratio. This allows remarkable purification of rhodopsin by a single-step solubilization, because the residual membranes are heavily aggregated in the presence of divalent cation and are therefore easily sedimented by low-speed centrifugation. The absorption spectrum of the supernatant reproducibly exhibits an A280/A500 value of 1.6, an excellent value that could rarely be obtained by chromatographic purification. The degree of purification also depends on the type of divalent cation included in the solubilization solution; specific binding of IIB-series cations (Zn2+ and Cd2+) to ROS membranes is suggested to play an important role in the solubilization process. The present result represents a unique example of selective solubilization of a specific membrane protein from highly aggregated membranes.

Animals↗
PubMed1997

Time resolved study of effect of chlorpromazine on mobility of cytochrome P-450 and phospholipids in the inner membrane of adrenocortical mitochondria.

The effects of chlorpromazine on the mobility of cytochrome P-450 and the fluidity of lipid membranes have been investigated in bovine adrenocortical submitochondrial particles (SMP). Rotational diffusion of the cytochrome was measured by observing the decay of absorption anisotropy, ra(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of ra(t) was based on a 'rotation-about-membrane-normal' model. The anisotropy decayed within 2 ms to a time independent value r3. The presence of chlorpromazine decreased the mobile population of cytochrome P-450 from 28 to 23%. The rotational relaxation time phi a of the mobile population (approximately 1100 microseconds) was, however, not significantly changed by chlorpromazine. The lipid fluidity was examined by observing time-resolved fluorescence anisotropy, rf(t), of 1,6-diphenyl 1,3,5-hexatriene (DPH). The anisotropy rf(t) decayed within 70 ns to a time independent value r infinity. The motion of DPH was analyzed based on a 'wobbling-in-cone' model. The presence of chlorpromazine decreased the cone angle from 42 degrees to 39 degrees, while the rotational relaxation time phi f (approximately 2 ns) was not significantly changed by the presence of chlorpromazine. These results demonstrate that chlorpromazine decreased the mobility of not only lipids but also membrane proteins.

Adrenal Cortex↗
PubMed1996

Fluorescence polarization study on the dynamics and location of peroxidized fluorescent phospholipids in liposomes.

Motional properties of fluorescent substances produced by lipid peroxidation by a time-resolved fluorescence polarization technique were studied. When liposomes containing phosphatidylethanolamine (PE) and linoleic hydrocarbon chain were incubated at 37 degrees C, fluorophores absorbing maximally at 360 nm and emitting near 430 nm were produced. Their fluorescence anisotropy decay measured at 23 degrees C was fitted well with a sum of a fast relaxation and a time-independent residual term. With the increase of oxidation degree, the time constant of the relaxation term increased. This may be explained by alteration in the membrane structure or by modification of the fluorescent products themselves. Information on the location of the fluorescent products was obtained when their motional property was compared with those of various extrinsic probes that were incorporated at different positions of the lipid bilayer. It was found that the motional property of the fluorescent oxidation products is similar to that of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, a rod-shaped hydrophobic probe with a charged terminal. Other probes sensing the polar region or the hydrophobic region of the membrane were characterized by a lower order parameter. It is suggested that the fluorescent oxidation products have a polar moiety located at the membrane surface and attached to the amino group of PE while the tail part being buried in the hydrophobic region of the membrane. This picture is supported by fluorescence quenching experiments with the aqueous quencher Co2+. On the other hand, fluorophores produced by the reaction of malondialdehyde and PE suggested to have a chemical structure in which the angle between the absorption and emission dipole moments is very large. On the basis of these observations, the production pathway of fluorophores in oxidized membranes is discussed.

Fluorescence Polarization↗
PubMed1995

Dispersion state of phospholipids and fluorescence production with peroxidation in organic solvents: investigated by time-resolved fluorescence technique.

Fluorescent substances were found to be produced efficiently when phospholipids containing phosphatidylethanolamine (PE) and linoleic chains were autoxidized in non-polar solvents. By using these fluorescent substances as intrinsic probes, the dispersion state of phospholipids was investigated in various organic solvents. Fluorescence anisotropy decay measurements indicated that the aggregation size of phospholipids was much larger in hexane than in chloroform, methanol and tert-butyl alcohol. The average diameter of phospholipid aggregates in hexane was calculated to be 4-6 nm, which was dependent on the lipid composition. A consistent result was obtained when N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-gly cer o-3- phosphoethanolamine (NBD-PE) was used as an extrinsic probe. Comparison of the fluorescence data with small-angle X-ray scattering (SAXS) data suggested that a reverse micellar structure of phospholipids formed in hexane. It was shown that phospholipid aggregation enhanced the extent of peroxidation as well as the production yield of fluorescent substances of phospholipid.

Chromatography, Thin Layer↗
PubMed1994

Domain motion in actin observed by fluorescence resonance energy transfer.

Actin is composed of two well-separated globular domains which are further subdivided into two subdomains [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C. (1990) Nature 347, 37-44]. Subdomains 1 and 2 constitute the small domain, and subdomains 3 and 4 comprise the large domain. In order to test a hinge bending domain motion in actin such as observed in many kinases, fluorescence resonance energy transfer between two probes attached to each of the two domains was measured by steady-state and time-resolved fluorometers. The adenine base is bound in a hydrophobic pocket between subdomains 3 and 4, and Tyr-69 is located at subdomain 2. In the present study, the adenine moiety was labeled with a fluorescence donor, epsilon ATP, and tyrosine-69 was labeled with the energy acceptor, dansyl chloride. Assuming the random orientation factor k2 = 2/3, the distance between epsilon-adenine moiety and dansyl chloride attached to Tyr-69 in G-actin was determined to be 2.46 nm from steady-state fluorescence measurements. The addition of DNase I did not appreciably change the distance (less than 0.1 nm). The distance decreased to 2.27 nm during polymerization by the addition of phalloidin under physiological salt conditions. On the other hand, time-resolved fluorescence energy transfer measurements have been used to investigate a distribution of distances for a donor-acceptor pair. In G-actin, the mean distance between probes was 2.79 nm with a full width at half-maximum of 3.91 nm, indicating a large number of conformational substates in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Significance of amino groups of phosphatidylethanolamine in phospholipid peroxidation of mixed liposomes.

The effect of distribution of phosphatidylethanolamine (PE) in multilamellar vesicles (MLV) on their peroxidative susceptibility was studied. Liposomes containing various ratios of L-alpha-dilinoleoyl phosphatidylcholine (DiLinPC) and L-alpha-dilinoleoyl phosphatidylethanolamine (DiLinPE) were peroxidized by ferrous ion or a water-soluble radical initiator 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH). The oxygen consumption rate was compared with the formation kinetics of fluorescent products. Inclusion of PE in liposomes was found to accelerate the Fe(2+)-dependent peroxidation. Modification of the amino group of PE with 2,4,6-trinitrobenzenesulfonic acid (TNBS) inhibited Fe(2+)-induced oxygen consumption in the late stage where fluorescent substances formed. A possible role of the amino group of PE on lipid peroxidation is discussed, especially in terms of its reactivity with oxidation products and subsequent formation of fluorescent substances.

Amidines↗
PubMed1994

Molecular organization and dynamics in bacteriorhodopsin-rich reconstituted membranes: discrimination of lipid environments by the oxygen transport parameter using a pulse ESR spin-labeling technique.

Molecular organization and dynamics in protein-rich membranes have been studied by investigating transport (diffusion-concentration product) of molecular oxygen at various locations in reconstituted membranes of bacteriorhodopsin (BR) and L-alpha-dimyristoylphosphatidylcholine. Oxygen transport was evaluated by monitoring the bimolecular collision of molecular oxygen with four types of nitroxide lipid spin labels placed at various locations in the membrane. The collision rate was estimated from the spin-lattice relaxation times (T1's) measured at various oxygen partial pressures by analyzing the short-pulse saturation recovery ESR signals. CD spectra and decay of polarized flash-induced photodichroism of bacteriorhodopsin indicated that BR molecules are monomers in reconstituted membranes with a lipid/BR molar ratio of 80 (80-rec) and are 25% monomers and 75% trimers plus oligomers of trimers when the lipid/BR ratio is 40 (40-rec). In the 80-rec, the lipid environment is homogeneous on a microsecond scale (T1), probably because the exchange rate of lipids between the bulk and the boundary regions is greater than the T1 relaxation rate (approximately 10(6) s-1). The oxygen collision rate in the hydrophobic region of the 80-rec membrane is smaller by a factor of 1.6 than in that of the lipid membrane without BR, and the effect of BR in decreasing the collision rate is independent of the "depth" in the hydrophobic region. In the 40-rec, two collision rates were observed, one of which is close to those for purple membrane (or the gel-phase membrane), while the other is about the same as was measured in the 80-rec.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed1994

Polyhedral assembly of a membrane protein in its three-dimensional crystal.

A novel ordered assemblage of bacteriorhodopsin, a transmembrane protein functioning as a light-driven proton pump, is found in its three-dimensional crystal. Atomic force microscope images of the crystal surface reveal that spherical protein clusters with a diameter of approximately 50 nm are hexagonally close-packed. Electron micrographs of mechanically disintegrated crystals show that the inside of the protein cluster is filled with the mother liquor. The crystal is made up of hollow protein clusters. When disintegrated crystals are illuminated in the presence of a lipophilic anion, a significant alkalization of the external medium occurs. This result indicates that the protein cluster contains native lipids and that the cytoplasmic side of the protein faces the external medium. X-ray diffraction patterns and the observed diameter of the spherical shell suggest that approximately 200 bacteriorhodopsin trimers are aligned on a polyhedral surface lattice. Another remarkable feature of the spherical assemblies of bacteriorhodopsin is that they fuse with each other at low ionic strength and occasionally form a tubular or doughnut-like structure. The concept of membrane protein polymorphism is introduced on the basis of these observations, and it is used to describe the dynamic structure of some other biological membranes.

Bacteriorhodopsins↗
PubMed1994

Value of thallium-201 early reinjection for assessment of myocardial viability.

To assess the efficacy of early reinjection for predicting post intervention improvement in thallium-201 (T1) uptake and regional wall motion, we reinjected a small dose of T1 following post-stress imaging and obtained reinjection early images (10 min after early reinjection) and reinjection delayed images (3 hr afterwards) in 40 patients who were referred to us for revascularization (group I). Twenty-nine patients in group I also underwent conventional stress-redistribution T1 scintigraphy (group II). Conventional stress-redistribution T1 scintigraphy was repeated after intervention. Contrast left ventriculography was performed before and after intervention and changes in regional wall motion were assessed in 22 of 40 patients. In group I, the predictive value for improvement and no improvement (the accuracy) of reinjection early images in perfusion was 83%, while that of reinjection delayed images was 91%. Furthermore, the accuracy of reinjection early images in regional wall motion was 80%, while it was 91% for reinjection delayed images. In group II, the accuracy in perfusion was 78% and the value in regional wall motion was 70%. Both accuracy in perfusion and in regional wall motion obtained from reinjection delayed images were significantly higher than the values in group II (p < 0.05). These data suggest that early reinjection is useful for predicting postintervention thallium uptake and regional wall motion.

Aged↗
PubMed1993

Molecular structural studies of effector functions of a mouse immunoglobulin G that lacks the entire CH1 domain: small-angle X-ray scattering, nanosecond fluorescence depolarization and stable isotope-aided NMR analyses.

Molecular structural studies are reported of a short-chain mouse IgG2a antibody that lacks the entire CH1 domain. We have recently shown that (1) this short-chain antibody comprises two components in which the inter light-chain disulfide bridge does and does not exist, and (2) these two components are different in the constitutive complement-activating activity [Mizutani et al. (1993) J. Immunol. 150, 131-138]. Structures were compared for these two components on the basis of small-angle X-ray scattering, nanosecond fluorescence depolarization and isotope-aided NMR data. It has been discussed how the presence and absence of the inter light-chain disulfide bridges affect the complement-activating activity of the two components of the short-chain antibody.

Animals↗
PubMed1992

Photoreaction of bacteriorhodopsin at high pH: origins of the slow decay component of M.

The absorption spectrum of light-adapted purple membrane in 3 M KCl is dependent on temperature even in the room temperature region. Temperature-induced difference spectra at various pH values suggested that the trans isomer of bacteriorhodopsin, bR570, is in thermal and/or photodynamic equilibrium with several different conformers. The major second conformer occurring at neutral pH had the same spectroscopic properties as the 13-cis isomer, and its content at 35 degrees C was estimated to be more than 20%. Heterogeneity in the protein conformation became more significant above pH8, where temperature-induced difference spectra exhibited a negative peak at 580 nm and a positive peak at 296 nm. This absorption change is very similar to that observed upon the formation of the N intermediate, suggesting that an N-like conformer occurs at high pH and temperature. A significant temperature dependence was also seen in the M decay kinetics at high pH, which were described by two decay components; i.e., the fast decaying M (Mf) was predominant at low temperature, but the amplitude of the slow component (M(s)) increased with increasing temperature. It is suggested that M(s) is generated upon excitation of the N-like conformer, in which the residue (Asp-96) usually acting as a proton donor to the Schiff base is deprotonated. The N-like conformer could be N itself, because M(s) was enhanced when N was accumulated by background light. A strong correlation between the amplitude of M(s) and the concentration of N was also revealed by the accumulation kinetics of Mf, M(s), and N after the onset of continuous actinic light.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed1990

Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin.

Bacteriorhodopsin-containing vesicles that were able to alkalize the extravesicular medium by greater than 1.5 pH units under illumination, i.e., inside-out vesicles, were reconstituted by reverse-phase evaporation with Halobacterium halobium polar lipids or exogenous phospholipids. Acid titration of a dark-adapted sample was accompanied by a color change from purple to blue (pKa = 2.5-4.5 in 0.15 M K2SO4), and alkali titration resulted in the formation of a red species absorbing maximally at 480 nm (pKa = 7 to greater than 9), the pKa values and the extents of these color changes being dependent on the nature of lipid. When a vesicle suspension at neutral or weakly acidic pH was irradiated by continuous light so that a large pH gradient was generated across the membrane, either a purple-to-blue or a purple-to-red transition took place. The light-induced purple-to-red transition was significant in an unbuffered vesicle suspension and correlated with the pH change in the extravesicular medium. The result suggests that the purple-to-red transition is driven from the extravesicular side, i.e., from the C-terminal membrane surface. In the presence of buffer molecules outside, the dominant color change induced in the light was the purple-to-blue transition, which seemed to be due to a large decrease in the intravesicular pH. But an apparently inconsistent result was obtained when the extravesicular medium was acidified by a HCl pulse, which was accompanied by a rapid color change to blue. We arrived at the following explanation: The two bR isomers, one containing all-trans-retinal and the other 13-cis-retinal, respond differently to pH changes in the extravesicular and the intravesicular medium. In this relation, full light adaptation was not achieved when the light-induced purple-to-blue transition was significant; i.e., only the 13-cis isomer is likely to respond to a pH change at the N-terminal membrane surface.

Bacteriorhodopsins↗