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Biomedical subjects

A Ikegami

Publications and source records attributed to A Ikegami.

At least 91 records · Page 5Linked to original sources
PubMed1982

On the wobbling-in-cone analysis of fluorescence anisotropy decay.

Interpretation of fluorescence anisotropy decay for the case of restricted rotational diffusion often requires a model. To investigate the extent of model dependence, two models are compared: a strict cone model, in which a fluorescent probe wobbles uniformly within a cone, and a Gaussian model, where the stationary distribution of the probe orientation is of a Gaussian type. For the same experimental anisotropy decay, analysis by the Gaussian model predicts a smaller value for the rate of wobbling motion than the strict cone analysis, but the difference is 35% at most; the cone angle obtained by the strict cone analysis agrees closely with the effective width of the Gaussian distribution. The results suggest that, when only two parameters (the rate and the angular range) are extracted from an experiment, the choice of a model is not crucial as long as the model contains the essential feature, e.g., the more-or-less conical restriction, of the motion under study. Model-independent analyses are also discussed.

Fluorescence Polarization↗
PubMed1982

Nanosecond fluorometric investigation of hydrodynamic properties of adenosine triphosphatase from thermophilic bacterium PS3.

The soluble portion (TF1) of proton-translocating ATPase from thermophilic bacterium PS3 was labeled with a fluorescent dye N-(1-pyrene)maleimide. The decay of fluorescence anisotropy of the adduct showed that TF1 in aqueous solution was characterized by a volume of equivalent sphere of 1,120 nm3. This value is 2.4 times the volume calculated from the molecular weight and partial specific volume, indicating a non-spherical shape and/or extensive hydration. A prolate ellipsoid with an axial ratio of 2 to 3 is suggested as a first approximation of the shape of hydrated TF1. The presence or absence of ATP, ADP, or Mg2+ did not alter the volume of the equivalent sphere appreciably; the probable conformational change of TF1 induced by these ligands does not lead to a gross alteration of its hydrodynamic properties.

Adenosine Triphosphatases↗
PubMed1981

The effect of cytochrome oxidase on lipid chain dynamics. A nanosecond fluorescence depolarization study.

Molecular motions in membranes composed of purified cytochrome oxidase (EC 1.9.3.1) and synthetic lipid (L-alpha-dimyristoylphosphatidylcholine or L-alpha-dioleoylphosphatidylcholine) at various ratios were investigated with a lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Nanosecond fluorescence depolarization kinetics of the probe showed that the rod-shaped probe molecules perform a fast wobbling motion (restricted rotation) in all membranes studied, presumably reflecting the motion of lipid acyl chains. At temperatures where the pure lipid was in the liquid-crystalline phase, presence of cytochrome oxidase reduced the angular range of the wobbling motion, whereas its rate, the wobbling diffusion constant, was unaffected. On the other hand, incorporation of the protein into lipid in the gel phase resulted in the increase in the wobbling diffusion constant while the range of the wobbling motion remained the same. A time-dependent view of lipid dynamics that accounts for the above findings, as well as the results of recent electron spin resonance and nuclear spin resonance studies of protein-lipid interactions, is proposed.

Animals↗
PubMed1981

Effect of double bonds on the dynamic properties of the hydrocarbon region of lecithin bilayers.

The motional properties of the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene have been assessed in liposomes of various lecithins: dipalmitoyl-phosphatidylcholine (DPPC), dioleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1- palmitoyl-2-linoleoylphosphatidylcholine (PLPC), and 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC). The temperature dependence of the steady-state fluorescence anisotropy was determined within the range of 10-50 degrees C. Nanosecond measurements were also made and the results analyzed by a model of wobbling diffusion confined within a cone. In this treatment the cone angle (theta c) obtained relates to the degree of order of the fatty acyl chains, and the wobbling diffusion constant (Dw) relates to the rate of motion of the hydrocarbon chains. Theta c was increased (order decreased) and Dw increased (rate increased) for the first double bond (POPC) as compared to the fully saturated DPPC. In all four unsaturated lecithins the temperature dependences of theta c and Dw were similar: theta c increased by about 4-5 degrees, and Dw increased by a factor of 1.5 per 10 degrees C. DPPC in the liquid-crystalline phase also showed a similar trend. The absolute values, on the other hand, showed some differences: for theta c, DOPC (78 degrees at 37 degrees C) greater than PAPC (75 degrees) greater than POPC (70 degrees) congruent to PLPC (69 degrees) greater than DPPC (20 degrees), and for Dw, PAPC (0.28 ns-1) greater than DOPC (0.22 ns-1) congruent to PLPC (0.24 ns-1) greater than POPC (0.21 ns-1) greater than DPPC (0.05 ns-1). The differences among the unsaturated lecithins are relatively small suggesting that the first double bond introduced into a lecithin plays the most important role at physiological temperatures. In 1:1 mixtures of the disaturated lecithin, DPPC, with the unsaturated lecithins, the temperature of the phase transition of the former was lowered and broadened, by approximately the same amount, irrespective of the differences in unsaturation.

Chemical Phenomena↗
PubMed1981

Dynamic structure of biological membranes as probed by 1,6-diphenyl-1,3,5-hexatriene: a nanosecond fluorescence depolarization study.

A fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, was incorporated in four different biological membranes, the purple membrane of Halobacterium halobium, human erythrocyte membrane, rabbit sarcoplasmic reticulum membrane, and rat liver mitochondrial membrane. Time-resolved fluorescence depolarization of the probe suggested that the rotational Brownian motion of the probe in the membranes was restricted in the angular range. The motion of the rod-shaped, lipophilic probe molecule, expected to reflect closely the motion of neighboring lipid hydrocarbon chains, was analyzed in terms of the wobbling-in-cone model in which the major axis of the probe was assumed to wobble freely in a cone of semiangle theta c with a wobbling diffusion constant Dw. At 35 degrees C, Dw in the four membranes, in the above order, ranged between 0.048 and 0.15 ns-1 and theta c between 31 and 53 degrees. From the rotational rate Dw, the viscosity against the wobbling motion was calculated to be 0.9-0.3 P. When the temperature was raised from 10 to 35 degrees C, Dw in all membranes increased approximately 3-fold, corresponding to activation energies of 7-8 kcal/mol, and theta c increased by about 10 degrees, except for the purple membrane in which the angular range remained narrow. The same characteristic temperature dependence has been found in many model membrane systems that contain unsaturated lecithins, suggesting an important role of unsaturated phospholipids in the dynamic structure of the lipid hydrocarbon chain region of biological membranes at physiological temperatures. Comparison with model systems suggests that proteins and cholesterol act mainly as barriers that narrow the angular range.

Animals↗
PubMed1981

Nanosecond time-resolved fluorescence investigations of temperature-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicles and solubilized systems.

Intrinsic and lipid phase transition-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicle and solubilized systems were examined by the fluorescence lifetime of N-(1-anilinonaphthyl-4)-maleimide conjugated with the enzyme. The time-dependent fluorescence intensity of N-(1-anilinonaphthyl-4)-maleimide attached to cytochrome oxidase was described as a triple exponential decay. Both the intrinsic and lipid phase transition-induced conformational changes were detectable in plots of the average lifetime against temperature. In most cases a peak occurred at the temperature of the conformational change. The time-dependent emission anisotropy showed that N-(1-anilinonaphthyl-4)-maleimide embedded in cytochrome oxidase in phosphatidylcholine vesicles underwent a rapid restricted wobbling within a cone. The half-angle of the cone was around 30 degrees for cytochrome oxidase in dimyristoyl phosphatidylcholine vesicles.

Animals↗
PubMed1980

Fluorescent probe study of temperature-induced conformational changes in cytochrome oxidase in lecithin vesicle and solubilized systems.

A protein-bound label, N-(1-anilinonaphthyl-4)-maleimide (ANM), was used to investigate conformational changes in bovine heart cytochrome oxidase. The fluidity of cytochrome oxidase vesicles was monitored by a lipophilic probe, 1,6-diphenyl-1,3,5-hexatriene. The fluroescence intensity and emission anisotropy of these probes were examined between 4 and 60 degrees C in enzyme--dipalmitoyllecithin vesicles, in enzyme--dimyristoyllecithin vesicles, in enzyme--dioleoyllecithin vesicles, and in the soluble enzyme. The temperature-dependent changes in these quantities indicated that there were two types of conformational changes in oxidized cytochrome oxidase: one was attributed to an intrinsic enzyme conformation change which occurred around 20 degrees C, and the other was attributed to a conformational change induced by the lipid phase transition. Although ANM-reactive subunits of cytochrome oxidase in these four lecithin vesicle and solubilized systems were different from each other, subunit I always reacted with ANM in preference to other subunits.

Animals↗
PubMed1980

Magneto-orientation of lecithin crystals.

For the purpose of clarifying the magneto-orientation mechanism of several biological membrane systems, xylene-suspended thin-layered single crystals of lecithin, which is a major component of membranes, were subjected to magnetic fields of various strengths, and the orientation behavior of the crystals was analyzed. Lecithin crystals oriented in such a way that the directions of both the hydrocarbon chains and the phosphorylcholine group of the lecithin molecule were perpendicular to the magnetic field, suggesting that the crystals were diamagnetically biaxial. In contrast, hydrocarbon chains of phospholipids in many biological membranes are known to orient parallel to magnetic fields. The value of the volume diamagnetic susceptibility anisotropy, delta chi (i.e. the difference between susceptibilities parallel and perpendicular to the direction of the hydrocarbon chain in the lecithin crystals), estimated from the magneto-orientation behavior was about -9 X 10(-8) cgs. This is distinctly larger than that of vesicles of egg yolk lecithin measured by Boroske and Helfrich [Boroske, E. & Helfrich, W. (1978) Biophys. J. 24, 863-868] and also larger than that of single crystals of polyethylene, which was measured for comparison by the same technique as that used for lecithin in this study. The differences between these values of delta chi seem to result from the contribution of the polar head (i.e., the part of the lecithin molecule other than the two hydrocarbon chains), which is ordered, less ordered, or deleted in the respective cases above, although mainly from the contribution of the hydrocarbon chains, which are ordered in crystals and disordered in vesicles.

Crystallography↗
PubMed1979

Stabilizing effect of cholesterol on phosphatidylcholine vesicles observed by ultrasonic velocity measurement.

The temperature dependence of the ultrasonic velocity was measured in sonicated vesicles of dipalmitoylphosphatidylcholine by varying the content of cholesterol. When cholesterol is incorporated, an anomalous dip of the ultrasonic velocity gradually smeared out. At the same time, the ultrasonic velocity of the membrane increased remarkably above 30 degrees C due to the increase of the bulk modulus by about 15%. On the other hand, the ultrasonic velocity and the bulk modulus decreased below 30 degrees C. Comparing the cholesterol-incorporated membrane with vesicles of bovine brain sphingomyelin and human erythrocyte membrane, we discuss the role of cholesterol in biological membranes in terms of the stability of the membrane as a barrier.

Chemical Phenomena↗
PubMed1979

Effects of temperature on cytochrome oxidase activity in solubilized form and in lipid vesicle systems.

Isolated mammalian cytochrome oxidase gave an Arrhenius plot with a break (Tb) at about 20 degrees C when assayed in a medium containing Emasol. The activation energies above and below 20 degrees C were 9.3 (EH) and 18.9 kcal/mol (EL), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature Tt = 40 degrees C), dimyristoyl phosphatidylcholine (DMPC, Tt = 23 degrees C) and dioleoyl phosphatidylcholine (DOPC, Tt = -22 degrees C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36 degrees C with E = 22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with Tb = 23 degrees C. EH and EL were 6.6 and 15.8 kcal/mol, respectively. The plot for the oxidase-DMPC also showed a break (Tb = 26 degrees C) with EH = 6.6 and EL = 26.6 kcal/mol. These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the "hot" and "cold" conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.

Animals↗
PubMed1978

Effect of cholesterol on the molecular motion in the hydrocarbon region of lecithin bilayers studied by nanosecond fluorescence techniques.

Effects of cholesterol on the dynamic structure of the hydrocarbon region of dipalmitoyllecithin vesicles were examined. Decays of the emission anisotropy and the fluorescence intensity of 1,6-diphenyl-1,3,5-hexatriene embedded in lecithin-cholesterol vesicles were measured over a temperature range of 10--60 degrees C. The emission anisotropy decreased rapidly with time and then leveled off. The rotational motion of the probe was analyzed by a model of wobbling diffusion confined in a cone. Cholesterol (10--50 mol%) decreased the cone angle in the liquid-crystalline phase and increased it in the gel phase. In the presence of 33 mol% cholesterol, the wobbling diffusion constant increased in the gel phase and changed little in the liquid-crystalline phase. The viscosity in the cone decreased in the gel phase and remained almost unchanged in the liquid-crystalline phase in the presence of 33 mol% cholesterol. The total fluorescence intensity followed a singel exponential decay independently of the cholesterol content 0--50 mol%.

Chemical Phenomena↗
PubMed1978

Ultrasonic studies of lipid bilayer. Phase transition in synthetic phosphatidylcholine liposomes.

The ultrasonic velocity at 3 MHz and the density in the nonsonicated and sonicated liposomes of dipalmitoylphosphatidylcholine have been measured in the temperature range from 0 degrees C to 55 degrees C. The results indicate that nonsonicated multilamellar vesicles undergo a weak first order transition which is analogous to the nematic-isotropic transition of liquid crystals. A sharp change in the ultrasonic velocity associated with the first order transition disappears when the multilamellar vesicles are sonicated. The bulk modulus of the lipid bilayer calculated from the ultrasonic velocity and the density of sonicated liposomes has a value of 3.0 X 10(10) dyne/cm2 at 20 degrees C, reaches a minimum value of 2.1 X 10(10) dyne/cm2 at its transition temperature and increases slightly to 2.2 X 10(10) dyne/cm2 at 50 degrees C.

Chemical Phenomena↗
PubMed1977

Dynamic structure of lipid bilayers studied by nanosecond fluorescence techniques.

Molecular motions in liposomes of dipalmitoyl-phosphatidylcholine (DPPC) were studied by nanosecond fluorescence techniques. As a fluorescent probe for the hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used. Time courses of fluorescence intensity IT(t) and emission anisotropy r(t) of DPH embedded in DPPC liposomes were measured at various temperatures. The value of the fluorescence lifetime tau obtained froma single exponential decay of IT(t) was somewhat higher than that in liquid paraffin below the transition temperature Tt and decreased above Tt. Higher values of tau below Tt indicate the almost complete hydrophobic environment. The decay curves of r(t) were separated into two phases: an initial fast decreasing phase of the order of one nanosecond and a second almost constant phase. This indicates that the orientational motion of DPH in the hydrocarbon region is described by a wobbling diffusion restricted by a certain anisotropic potential. The results were analyzed on the model that the wobbling diffusion is confined in a cone with a uniform diffusion constant. Though temperature dependence of the cone angle was sigmoidal, that of the wobbling diffusion constant was like the exponential function. The change in the cone angle at Tt was sharper than that in the wobbling diffusion constant at Tt. Estimated values of the viscosity in the cone were an order of magnitude smaller than the values of "microviscosity" which were estimated from the steady-state emission anisotropy without considering the restrictions on the rotational motion.

Benzene Derivatives↗