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Biomedical subjects

A Ikegami

Publications and source records attributed to A Ikegami.

At least 73 records · Page 4Linked to original sources
PubMed1985

Excited-state dynamics of bacteriorhodopsin.

Near infrared emission of bacteriorhodopsin at neutral pH and at room temperature was characterized by a large Stokes shift. This characteristic was lost in an acidic pH (approximately pH 2) where a remarkable enchancement (more than 10 times) in the fluorescence quantum yield accompanied the red shift in the main absorption band. It is suggested from fluorescence polarization measurements that the emission occurs from the first allowed excited state of the retinylidene chromophore, irrespective of pH. We suggest that the large Stokes shift observed at neutral pH is a result of a charge displacement (e.g., proton translocation) that occurs immediately after excitation, and is prevented by protonation (in the ground state) of an amino-acid residue in the protein.

Bacteriorhodopsins↗
PubMed1984

Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range.

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.

Actins↗
PubMed1984

The dynamics of lipid motion in sarcoplasmic reticulum membranes determined by steady-state and time-resolved fluorescence measurements on 1,6-diphenyl-1,3,5-hexatriene and related molecules.

Steady-state and time-resolved fluorescence anisotropy measurements were made on 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1-acyl-2-(DPH)-phosphatidylcholine (DPH-PC) incorporated into sarcoplasmic reticulum membranes. The results were analysed in terms of the 'wobbling-in-cone' model. Considerable differences in the fluorescence parameters were found. In particular TMA-DPH and DPH-PC showed a smaller cone angle, relating to the range of acyl chain motion, compared to DPH, taken to be a reflection of a difference in probe locations. The influence of the protein component was also found to restrict DPH motion more than TMA-DPH and DPH-PC. Effectiveness in assessment of perturbation of the membrane by the non-esterified fatty acid, oleic acid again revealed differences. The steady-state anisotropy decreased on addition of oleic acid; a recovery to control values was observed with DPH but not with the other probes. Time-resolved parameters followed the same pattern. The results of this work demonstrated the effectiveness of these three probes in revealing differences in membrane properties, such as protein and fatty acid perturbation of membrane lipid structure and dynamics.

Animals↗
PubMed1984

Internal motion of DNA in bacteriophages.

We have investigated internal motion of DNA in bacteriophages by measuring fluorescence anisotropy decays of intercalated ethidium. The results showed large suppression of the internal motion of the inner DNA; the interhelix interaction of the DNA in the phage head is considered to enhance the effective viscosity of the DNA rod and to restrict the angle of the internal motion. Considering that the observed internal motion arises mainly from torsional motion of the DNA, we have calculated the movable angles of the torsional motion (the standard deviation of the torsional motion) of the DNA in the phage heads. The magnitude of the calculated movable angles indicates the extent of suppression of the DNA movement in the phage head; in lambda wild type phage, the DNA is packed most rigidly in the head and the motion is found to be restricted most severely. In a deletion mutant of lambda phage, whose inner DNA content is deficient by 17.6%, steric hindrance from the interhelix DNA interaction is decreased, and the DNA can move more easily. In T4 wild type phage, although the extent of condensation of the inner DNA is the same as that in lambda wild type phage, the DNA was fairly mobile. The presence of glucosylated hydroxymethylcytosine is suggested to influence the rigidity of the inner DNA or packaging mode of the DNA in the T4 head.

Animals↗
PubMed1984

Rotational dynamics of immunoglobulins with fluorescent haptens on a membrane surface.

The rotational dynamics of rabbit immunoglobulin G with fluorescent lipid haptens on a membrane surface has been studied by nanosecond fluorescence emission anisotropic spectroscopy. It has been found that the rotational angles of the antibody are very restricted on the membrane, but that the rotation rate itself is not appreciably lower than that in solution, and is independent of the membrane fluidity.

Animals↗
PubMed1984

Dynamics of Z-form DNA.

The torsional and bending rigidities of Z-form DNA have been studied by nanosecond fluorescence anisotropy measurements of intercalated ethidium. The results suggested that Z-form DNA was considerably more flexible than B-form DNA. We have investigated the temperature dependence of the rigidity of B- and Z-form DNA and found that the temperature dependence of the torsional rigidity of Z-form DNA was remarkably lower than that of B-form DNA.

Circular Dichroism↗
PubMed1984

Dynamic structure of biological and model membranes: analysis by optical anisotropy decay measurement.

Rotational Brownian motion of molecules in membranes can be "visualized" by time-resolved measurement of the decay of anisotropy of various flash-induced optical signals, such as fluorescence, phosphorescence, delayed fluorescence, transient absorption, or fluorescence depletion. The basic principles of the various forms of anisotropy measurement are illustrated in a unified manner. In organized structures such as membranes, rotational motion is restricted in angular range. Methods of analysis of observed optical anisotropy decays for the case of restricted rotation are described; the emphasis is laid on the separate estimation of the two important parameters, the range and rate, that characterize the restricted rotation. Practical aspects of the analytical procedures are also discussed. As an example of application, recent work from the authors' laboratory is reviewed: dynamic structures of lipid hydrocarbon chain region of membranes have been revealed by time-resolved fluorescence depolarization studies. A lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated in model and biological membranes of known compositions. The decay of fluorescence anisotropy indicated that the rod-shaped probe molecules wobbled in the membranes with a wobbling diffusion constant around 0.1 rad2/nsec, presumably reflecting the dynamics of surrounding lipid chains. The effects of temperature, ions, lipid chain unsaturation, cholesterol, and protein on the range and rate of wobbling were examined with model membranes. The dynamic structure of biological membranes was found to be basically similar to that of the bilayer of unsaturated phospholipid; proteins and cholesterol act mainly as barriers that reduce the angular range of wobbling motion.

Animals↗
PubMed1984

Anisotropic electric properties of purple membrane and their change during the photoreaction cycle.

Purple membrane suspension shows two different orientations in electric fields of different frequencies. The orientation at low frequencies (less than or equal to approximately 10 Hz), with the membrane surface perpendicular to the electric field, is due to permanent dipole moment of the membrane and the orientation at high frequencies (greater than or equal to approximately 100 Hz), with the surface parallel to the electric field, is due to induced dipole moment. By quantitative analysis of these orientations, we determined the permanent dipole moment and the polarizability. Both values varied according to the membrane size: the permanent dipole moment ranged from 500 kD to 10 MD and was proportional to the square of the diameter of the membrane. The polarizability ranged from 1 X 10(-13) to 1 X 10(-11)cm3 and was proportional to the third to fourth power of the diameter. Because the permanent dipole moment was proportional to the area of the membrane, we could determine permanent dipole moment per bacteriorhodopsin. By determining the actual membrane size under electron microscopy, we got 98 D/bacteriorhodopsin. We also concluded that the direction of the permanent dipole moment was from the cytoplasmic to the extracellular side. These values, however, were strongly dependent on the ionic strength in the medium, suggesting a screening effect due to counter ions near the membrane surface. We evaluated the screening effect and showed about a four-charge difference between the two sides of the purple membrane. Under illumination, we found that the permanent dipole moment decreased from 98 to 63 D/bacteriorhodopsin. From the best-oriented sample, we also concluded that the angle of retinal against the axis normal to the membrane surface was greater than 68.6 degrees.

Bacteriorhodopsins↗
PubMed1983

Internal motion of deoxyribonucleic acid in chromatin. Nanosecond fluorescence studies of intercalated ethidium.

We have investigated the internal motions of DNA in a nucleosome core particle and chromatin by measuring the nanosecond fluorescence depolarization of intercalated ethidium. Assuming that the observed anisotrophy decay originates from the torsional motion of DNA, we have analyzed the dynamics of DNA in a nucleosome core particle and in chromatin in detail. The results suggest that DNA in a nucleosome core particle has a torsional rigidity similar to that of DNA in solution and that even at the point of the ionic bonds between DNA and a histone octamer the torsional motion of DNA is not completely inhibited. On the other hand, the dynamics of linker DNA in chromatin were found to reflect the overall structural state of the chromatin: the motion of linker DNA was suppressed as the structure of chromatin turned from an extended state to a condensed one. This indicates that, in solenoidal chromatin, nucleosome movements in chromatin are largely suppressed. Furthermore, the result may suggest that the torsional rigidity of linker DNA is increased as it is forced to bend in solenoidal chromatin.

Animals↗
PubMed1983

Small-angle X-ray scattering study of adenosine triphosphatase from thermophilic bacterium PS3.

Adenosine triphosphatase from the thermophilic bacterium PS3(TF1) has been studied by solution X-ray scattering. A structural change in TF1 caused by the binding of ADP was observed by examining the difference between the radii of gyration of the unligated and ligated forms. The radius of gyration of the unligated TF1 was found to be 49.5 +/- 0.3 A, and it decreased by approximately 3% after ligation with ADP. The positions and the amplitudes of a subsidiary maximum and a shoulder in the scattering profile showed subtle change on nucleotide binding. The lower limit of the maximum length of TF1 was determined to be 165 A for the unligated form and 150 A for the ligated form. The shape analysis of TF1 was performed by model calculations for simple triaxial bodies or their complexes. Among the various models tested, the one that gave the best fit with the experimental data consisted of seven ellipsoids of revolution; six identical ellipsoids with semi-axes: a = b = 18.5 A and c = 74 A. arranged hexagonally, and the other with a = b = 28 A and c = 45 A, located below the other six on the 6-fold axis. On the basis of this model it was suggested that there is a structural change on ligation with nucleotides, consisting of a shrinkage of the six long ellipsoids by 6% along their major axes.

Adenosine Diphosphate↗
PubMed1983

Possible association of NADPH-cytochrome P-450 reductase and cytochrome P-450 in reconstituted phospholipid vesicles.

A fluorescent probe, N-(1-anilinonaphth-4-yl)-maleimide (ANM), was specifically labeled to SH group(s) in the hydrophilic moiety of NADPH-cytochrome P-450 reductase at a ratio of 1 +/- 0.1 ANM/mol of protein. The ANM-labeled reductase and P-450 were reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles in which all of the enzymes were functionally active. The reconstitution of the mixed-function oxidase system was found to be strongly dependent on both the lipid to protein molar ratio and phospholipid composition. The interactions of ANM-labeled reductase with P-450 in proteoliposomes were investigated by perturbation of the fluorescence of ANM. Upon incorporation of P-450 into the phospholipids vesicles (ANM-reductase/P-450/lipids identical to 1:1.4:800), a significant decrease of total fluorescence intensity and slight increase of emission anisotropy of ANM were observed. In the average fluorescence lifetime of ANM bound with reductase, an appreciable change was shown between the absence and presence of P-450 in the vesicles. These data provide clear evidence that significant molecular interactions occur between the two proteins in a membranous reconstituted system.

Animals↗
PubMed1983

Fluorescence energy transfer studies of transmembrane location of retinal in purple membrane.

A diffusion-enhanced energy transfer technique was employed for the determination of transmembrane location of the retinal chromophore in the purple membrane. Theoretical considerations showed that the rate of energy transfer from an energy donor embedded within a membrane to acceptors dissolved in solvent could be described by an analytical function of the distance a of closest approach between the donor and acceptor, if the "rapid-diffusion limit" was attained. The criterion for this limit was given by the relation: (RO)6 much less than 20D tau Da4, where RO is the characteristic distance of energy transfer, D is the diffusion coefficient of the acceptor and tau D is the fluorescence lifetime of the donor in the absence of acceptor. By photo-reduction of the purple membrane with sodium borohydride, the retinal chromophore was converted to a highly fluorescent derivative, which showed a broad emission band in the visible region. From analysis of the fluorescence decay curves of the photo-reduced purple membrane in the presence of various concentrations of cobalt-ethylenediamine tetraacetate (Co-EDTA: energy acceptor), the depth of the chromophore from the membrane surface was estimated to be 8 (+/-3) A. This result was supported by investigations of energy transfer processes in a system where the native purple membranes and the photo-reduced membranes were stacked in parallel: the energy acceptor in this system was the native retinal chromophore.

Bacteriorhodopsins↗
PubMed1983

Dynamics of DNA in chromatin and DNA binding mode to core protein.

We have studied the dynamics of DNA in nucleosome core particles and in the linker region of chromatin using nanosecond fluorescence anisotropy decay measurements of intercalated ethidium. DNA in the core undergoes torsional motions to the same extent as the linker DNA in extended chromatin. We therefore concluded that the binding of DNA to the histone octamer is relatively weak or limited to a few points; stretches of at least several tens of base pairs exist which can move as freely as DNA in solution.

Animals↗
PubMed1983

Internal motion of F-actin in 10(-6)-10(-3) s time range studied by transient absorption anisotropy: detection of torsional motion.

By means of laser flash photolysis, the transient absorption anisotropy (TAA) of the triplet probe, 5-iodoacetamide-Eosin, labeling rabbit skeletal F-actin was measured in the 10(-6)-10(-3) s time range. The TAA curve at 20 degrees C showed a relatively slow decay phase covering several hundred microseconds and a large residual anisotropy (approximately 0.1 at 2 ms). After analysis with Barkley & Zimm's formula, it was concluded that the TAA of Eosin-F-actin can be approximated by the anisotropy decay due to torsional motion of F-actin.

Actins↗