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Biomedical subjects

A Ikegami

Publications and source records attributed to A Ikegami.

At least 55 records · Page 3Linked to original sources
PubMed1989

Correlation between internal motion and emission kinetics of tryptophan residues in proteins.

Time-resolved fluorescence anisotropy measurements of tryptophan residues were carried out for 44 proteins. Internal rotational motion with a sub-nanosecond correlation time (0.9 +/- 0.6 ns at 10 degrees C) was seen in a large number of proteins, though its amplitude varied from protein to protein. It was found that tryptophan residues which were almost fixed within a protein had either a long (greater than 4 ns) or short (less than 2 ns) fluorescence lifetime, whereas a residue undergoing a large internal motion had an intermediate lifetime (1.5-3 ns). It is suggested that the emission kinetics of a tryptophan residue is coupled with its internal motion. In particular, an immobile tryptophan residue emitting at long wavelength was characterized by a long lifetime (greater than 4 ns). It appears that a tryptophan residue fixed in a polar region has little chance of being quenched by neighboring groups.

Fluorescence Polarization↗
PubMed1989

Orientation of retinal in purple membrane determined by polarized Raman spectroscopy.

The orientation of the retinal molecule in the purple membrane was determined by polarized Raman spectroscopy for stacked purple membranes. The depolarization ratios of C = C stretching vibration mode were measured for three scattering geometries of purple membrane films. From the depolarization ratios we estimated the tilt angle of the transition dipole moment of retinal to the membrane normal and the rotational angle of the molecular plane along the transition dipole moment of retinal. The molecular plane of M intermediate was found to be almost perpendicular to the membrane plane. We confirmed that the tilt angle was 65 +/- 2 degrees for both bR and M intermediates.

Bacteriorhodopsins↗
PubMed1989

[Effect of trazodone (KB-831) and its metabolites on brain monoamines in rat].

The effects of trazodone (KB-831) and its metabolites on the uptake, turnover and contents of monoamines in rats were studied in comparison with those of imipramine and mianserin. Trazodone exhibited a more potent inhibitory effect on the uptake of [3H] 5-hydroxytryptamine (5-HT) into brain synaptosomes than on the uptake of [3H]norepinephrine (NE). Trazodone at 10-30 mg/kg, p.o., also inhibited the p-chloramphetamine-induced depletion of 5-HT in rat brain, but not the 6-hydroxydopamine-induced NE depletion in rat heart. Trazodone was the most selective 5-HT uptake inhibitor among the drugs tested in vitro. Its metabolite, m-chlorophenyl-piperazine (m-CPP), inhibited 5-HT and NE uptake in vitro, but not in vivo. Trazodone (100 mg/kg) and imipramine (30-100 mg/kg) inhibited the depletion of NE induced by alpha-methyl-p-tyrosine, whereas mianserin (100 mg/kg) facilitated it. At 1 hr after a single administration of each drug, an increase in 5-HT content and a decrease in 5-hydroxyindole-3-acetic acid (5-HIAA) content were observed when 30 mg/kg trazodone was used. At 100 mg/kg, trazodone increased the levels of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and decreased the NE content. m-CPP (10-30 mg/kg) produced similar effects on monoamine contents to those of trazodone. Imipramine and mianserin had no effect on monoamine contents even at a dose of 100 mg/kg. After 3 weeks of successive administration, an increase in 5-HT and a decrease in 5-HIAA were induced by trazodone and m-CPP at 1 hr, but not at 17 hr, after the final administration. Imipramine decreased the contents of NE and 5-HIAA, and its effects lasted for 17 hr. These results suggest that trazodone is a selective 5-HT uptake inhibitor and that its neurochemical profile is different from those of imipramine and mianserin.

Animals↗
PubMed1988

Bacteriorhodopsin photoreaction: identification of a long-lived intermediate N (P,R350) at high pH and its M-like photoproduct.

An alkaline suspension of light-adapted purple membrane exposed to continuous light showed a large absorption depletion at 580 nm and a small increase around 350 nm. We attribute this absorption change to an efficient photoconversion of bR570 into a photoproduct N (P,R350), which has a major absorption maximum between 550 and 560 nm but has lower absorbance than bR570. N was barely detectable at low pH, low ionic strength, and physiological temperature. However, when the thermal relaxation of N to bR570 was inhibited by increasing pH, increasing ionic strength, and decreasing temperature, its relaxation time could be as long as 10 s at room temperature. N is also photoactive; when it is present in significant concentrations, e.g., accumulated by background light, the flash-induced absorption changes of purple membrane suspensions were affected. Double-excitation experiments showed an M-like photoproduct of N,NM, with an absorption maximum near 410 nm and a much longer lifetime than M412. It may be in equilibrium with an L-like precursor NL. We suggest that N occurs after M412 in the photoreaction cycle and that its photoproduct NM decays into bR570. Thus, at high pH and high light intensity, the overall photoreaction of bR may be approximated by the two-photon cycle bR570----M412----N----(NL----NM)----bR570, whereas at neutral pH and low light intensity it can be described by the one-photon cycle bR570----M412----N----O640----bR570.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed1988

Structure and function of bacteriorhodopsin.

Bacteriorhodopsin (bR) is a powerful light-driven proton pump. We have developed a procedure to prepare bR-containing membrane vesicles in which a pH gradient as large as 4 pH units can be generated and maintained in the light. Using such a system, we have demonstrated that bR exhibits a high proton pump activity in a wide pH region; it works well at least between pH 4 and 9.5. For the large light-induced pH change in the external medium, the presence of a high concentration (approximately 0.1 M) of magnesium or transition metal ion is required. It is suggested that the influx of magnesium ion, electrically coupled with the proton release, takes place in the light. The three-dimensional structure of bR was studied by fluorescence energy transfer techniques. It was shown that the retinal chromophore is located 10 A below a surface of purple membrane. The in-plane location and orientation of retinal was also determined; it exists in a pocket surrounded by the helices 3, 4, 5, and 6. The position of a fluorescent probe labeled to Lys 41 was determined to be near the helix 7. Based on the results obtained, we propose a model of the bR structure. The dynamic structure of bR was investigated by fluorescence depolarization techniques. It was shown that the retinal chromophore is tightly buried in a pocket within the protein. In the presence of detergents like octylglucoside, its tertiary structure can be stable near the electric isosbestic point. The rate of dissociation/association process of bR molecules is sensitive to the pH of the medium. Dimeric and/or trimeric bR can exist stably if the concentration of detergent and other solvent conditions are adequately controlled. The photoreaction of bR in purple membrane, including the dark/light adaptation, the trans photocycle and the primary photoreaction, was reported. With respect to the trans photocycle, we found that, at alkaline pH, an M-like photoproduct (NM) is generated by excitation of a long-lived photoproduct N560 which has a major absorption maximum near 560 nm. We suggest that, at alkaline pH, the overall photoreaction of bR under steady illumination is approximated by the two-photon cycle: bR570 approximately greater than M412----N560 approximately greater than NM----bR570. With respect to the primary photoreaction, we found that the fluorescence quantum yield of the near-infrared emission of bR was greatly enhanced at acidic pH (approximately pH 2).(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriorhodopsins↗
PubMed1988

Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope.

Controlled permeability can be conferred to cell membranes by exposing cells to a microsecond electric pulse of sufficient intensity (electroporation). By constructing a fluorescence microimaging system with a submicrosecond time resolution we have been able to resolve temporally and spatially the events in a single cell under a microsecond electric pulse. An enormous membrane conductance, corresponding to a loss of 0.01-0.1% of the membrane area, was observed in those membrane regions where the transmembrane potential induced by the electric pulse exceeded a critical value. The conductance decreased to a low level in a submillisecond after the pulse, leaving a moderately electroporated cell.

Animals↗
PubMed1988

Optical anisotropy decay studies of the dynamic structure of myosin filaments.

We applied flash-induced absorption and phosphorescence anisotropy decay methods to the study of rotational motions of myosin heads in solution), myofibrils and muscle fibers); myosin heads were selectively labeled with a triplet probe EMI (5-eosinylmaleimide). EMI-labeled subfragment 1 (S1) showed a single exponential decay of anisotropy over two decades; the analysis indicated that if S1 is modeled as a prolate ellipsoid of revolution, the major axis was 16-17 nm and the minor axis 4.7-4.5 nm. The decay curve of myosin filaments could be simulated by double-exponentials-plus-constant approximation. The data could be analyzed by a double-cone model), in which we assumed that a head part (S1), wobbles in the first cone and a rod portion next to the head also wobbles in the second cone. The semiangle of each cone was estimated to be 35 and 48 degrees, respectively. We found that myosin heads in myofibrils under relaxing conditions extensively rotated as in myosin filaments in solution. When the spacing between thick and thin filaments was artificially reduced by the increase of osmotic pressure with the addition of polyvinylpyrrolidone (PVP), restriction of the angular range of the rotational motion was observed. Under rigor conditions no motion was observed in a 10 microsecond time scale, indicating that the heads were immobilized by binding to thin filaments. Preliminary results on the rotational motions of myosin heads in muscle fibers are also reported.

Animals↗
PubMed1987

Rotational motions of myosin heads in myofibril studied by phosphorescence anisotropy decay measurements.

We studied the rotational Brownian motions of myosin heads, of which the sulfhydryl group was selectively labeled with the triplet probe 5-eosinylmaleimide, in myofibril by using flash-induced phosphorescence anisotropy decay measurements. The anisotropy decay curve under relaxing conditions consisted of a fast (submicrosecond) and a slow (a few microseconds) component and a small constant part as in the synthetic myosin filaments in solution. The decay curves could be analyzed by assuming that a head part, i.e. subfragment 1 (S1), wobbles in the first cone and a part connecting S1 and the tail of a myosin molecule of which the length is shorter than subfragment 2 (S2) wobbles in the second cone (a double-cone model); the semiangles of the former and the latter cones were about 30 degrees and 50 degrees, respectively. The rotational freedom of myosin heads was only slightly restricted by the limited space of the filament lattice in myofibrils. Under rigor conditions, no motion of myosin heads was observed in the 10-microseconds time scale.

Animals↗
PubMed1987

Changes of the DNA packaging mode during boar sperm maturation.

Changes in the mode of DNA packaging in nuclei during spermatogenesis were studied by measuring of the fluorescence anisotropy decay of an ethidium dye intercalated in the DNA in whole nuclei. The nuclei were isolated from boar spermatid or sperm cells at three distinct stages of spermatogenesis: just before the completion of a maturation process in the testis (late spermatid), immediately after a subsequent transformation into spermatozoa (caput spermatozoon), and after full maturation (cauda spermatozoon). Although these three kinds of nucleus were morphologically indistinguishable from each other, the anisotropy decay detected a clear difference. In the late spermatid nuclei, in which the replacement of histones by protamine was still in progress, the anisotropy decayed extensively. The decay suggested that the DNA in the spermatid nuclei contained very flexible regions, in which the interaction of the DNA and proteins may be weak. The rapid and extensive anisotropy decay was absent in the caput and cauda nuclei. The flexible portions must have turned into very rigid structure during transformation from the late spermatid into the caput spermatozoon.

Animals↗
PubMed1985

Increased stability of the higher order structure of chicken erythrocyte chromatin: nanosecond anisotropy studies of intercalated ethidium.

Internal motion of the DNA in chicken erythrocyte chromatin fibers was studied by measurement of the fluorescence anisotropy decay of ethidium intercalated in the linker region. A comparison of the decay curves of the dye in chicken erythrocyte chromatin with those of calf thymus chromatin [Ashikawa, I., Kinosita, K., Jr., Ikegami, A., Nishimura, Y., Tsuboi, M., Watanabe, K., Iso, K., & Nakano, T. (1983) Biochemistry 22, 6018-6026] revealed greater suppression of nucleosome movement in chicken erythrocyte chromatin. Furthermore, the transition of this chromatin to the compact (solenoidal) structure occurred at lower solvent concentrations of Na+ or Mg2+ than those for calf thymus chromatin. These results demonstrated increased stability of the higher order structure (the solenoid) of chicken erythrocyte chromatin, which may be related to the reduction of nuclear activity in the chicken erythrocyte cell. In addition to intact chicken erythrocyte chromatin, we studied the structural transitions of H1-depleted and H1,H5-depleted chromatins. The result indicated that histone H5 of this chromatin stabilizes the higher order structure in the presence of magnesium (or divalent) cation and did not induce the transition in the solution containing only sodium cation.

Animals↗
PubMed1985

Local dielectric properties around polar region of lipid bilayer membranes.

Local dielectric constant was evaluated from the Stokes shifts of fluorescence spectra of L-alpha-dansylphosphatidylethanolamine (DPE) incorporated into liposomes made of synthetic phosphatidylcholine (dipalmitoyl or distearoyl) or bovine brain phosphatidylserine. The evaluation was established as follows. First, the Stokes shift of DPE was assured to follow Mataga-Lippert's equation and was a function of the dielectric constant and the refractive index in some standard organic solvents. Second, the change of the refractive index did not contribute much to the change in the Stokes shift. Third, the time resolved fluorescence depolarization of DPE in liposomes showed that the cone wobbling diffusion was rapid relative to the fluorescence lifetime and therefore that the dielectric relaxation did not affect the evaluation of the constant in the polar region of membranes. We then investigated the characteristics of the local dielectric constant in the polar region of the lipid bilayer and found that the dielectric constant varies between 4 and 34 depending upon calcium binding and also gel/liquid-crystal phase transition. Such large changes of the local dielectric constant were further correlated with the dynamic structure of lipid bilayer membranes measured by conventional fluorescence depolarization techniques. The large changes of dielectric constant around the polar region suggest that electrostatic interactions at this region can be altered 10-fold by such ionic or thermotropic factors and therefore that local dielectric properties can play crucial roles in membrane functions.

Dansyl Compounds↗
PubMed1985

Interaction between Ca2+ and dipalmitoylphosphatidylcholine membranes. II. Fluorescence anisotropy study.

The effect of Ca2+ on the molecular mobility in dipalmitoylphosphatidylcholine membranes was studied by steady-state and time-resolved measurements of fluorescence anisotropy. The fluorescence anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene in the hydrocarbon region indicated that the free volume of molecular rotation became more restricted when the Ca2+ concentration was increased. The decrease of the molecular mobility was observed from 1 mM Ca2+, at which the number of bound Ca2+ is much less than that of the total lipid molecules. A distinct difference between Ca2+ and Mg2+ effects suggested that the change in various membrane properties was induced by the binding of these ions. From these results we propose a long-range attractive interaction between bound Ca2+ and the polar head groups of distant phosphatidylcholine molecules.

Calcium↗
PubMed1985

Dynamic light scattering study of suspensions of purple membrane.

Purple membrane from Halobacterium halobium in suspensions has been studied by quasielastic light scattering. The intensity correlation functions of polarized scattered light were measured at various K2 values (K being the magnitude of the scattering vector), and the first cumulant Gamma of the field correlation function G1(tau) was obtained by a cumulant expansion method. The apparent diffusion coefficient Gamma /K2 did not increase monotonically with K2 values and showed a distinct anomaly in an intermediate range of K. A theoretical formulation of G1(tau) for a disc and an extremely oblate ellipsoidal shell of revolution (S. Fujime and K. Kubota, Biophys. Chem. 23 (1985) 1) was applied to the analysis of the spectra, and characteristic features of experimental spectra were well reproduced. It was suggested that a strong interference effect between scattered rays on Gamma /K2 should be attributed to a slight noncircular shape of the purple membrane and that a contribution to Gamma /K2 from membrane flexibility should be taken into account. This study will provide experimental evidence of the feasibility of membrane studies by dynamic light scattering.

Journal Article↗