Biomedical subjects

A Kusumi

Publications and source records attributed to A Kusumi.

At least 55 records · Page 3Linked to original sources

PubMed1993

Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells.

A new method of fluorescence microscopy for cell imaging has been developed that takes advantage of the spatial variations of fluorescence lifetimes in single cells as a source of image contrast, and thus it is named "fluorescence lifetime imaging microscopy (flimscopy)". Since time-resolved fluorescence measurements are sensitive to molecular dynamics and interactions, flimscopy allows the molecular information to be visualized in single cells. In flimscopy measurements, several (nanosecond) time-resolved fluorescence images of a sample are obtained at various delay times after pulsed laser excitation of the microscope's entire field of view. Lifetimes are calculated pixel-by-pixel from these time-resolved images, and the spatial variations of the lifetimes are then displayed in a pseudocolor format (flimscopy image). The total data acquisition time needed to obtain a flimscopy image with the diffraction-limited spatial resolution (approximately 250 nm) is decreased to just approximately 30 s for approximately 300 fluorescent molecules/micron2. This was achieved by developing a high-frequency (400 kHz) nanosecond-gating (9 ns full width at half height)-signal accumulation system. This technique allows the extent of resonance energy transfer to be visualized in single living cells, and is free from the errors due to variations in path length, light scattering, and the number of fluorophores that necessitate complex corrections in steady-state microfluorometry and fluorescence ratio imaging microscopy. Flimscopy was applied here to observe the extent of fusion of individual endosomes in single cells. Results revealed the occurrence of extensive fusion between primary endocytic vesicles and/or sorting endosomes, thereby raising the possibility that the biogenesis of sorting endosomes involves multiple fusions of primary endocytic vesicles.

Animals↗

PubMed1993

Age-associated increase of CD5+ B cells in the liver of autoimmune (NZB x NZW) F1 mice.

The liver has been demonstrated to be a major site for extrathymic differentiation of T cells. In this study, an identification of CD5+ B cells, which are responsible for the onset of autoimmune disease by virtue of autoantibody production, was performed in autoimmune (NZB x NZW) F1 mice. An age-associated increase of CD5+ B cells was demonstrated in the liver of these mice. Although CD5+ B cells (i.e., CD5+IgM+ and CD5+B220+) constituted a minor population of hepatic mononuclear cells (MNC) (< 5%) when mice were young (8 weeks), a large population of CD5+ B cells (10 to 30% of whole MNC) was identified in the liver of mice aged 25 to 30 weeks after the onset of disease. Such age-dependent increase of CD5+ B cells was not observed in any other strains including NZB, NZW, C3H/He and BALB/c mice. The phenotype of hepatic CD5+ B cells was the same as that of CD5+ B cells in the peritoneal cavity and spleen, showing dull-CD5, bright-IgM and dull-B220. High levels of CD5+ B cells were observed in the peritoneal cavity and liver, but not in the spleen nor in any other lymphoid organs in mice aged 30 weeks. Radioimmunoassay of autoantibodies in the 5-day culture supernatants demonstrated that hepatic MNC were unable to produce any amounts of IgM- and IgG-autoantibodies against double-stranded DNA and single-stranded DNA, despite the increased proportion of CD5+ B cells. On the other hand, peritoneal exudate cells produced only IgM-, but not IgG-, autoantibodies, whereas splenic cells were able to produce both IgM- and IgG-autoantibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1992

Age-dependent increase of extrathymic T cells in the liver and their appearance in the periphery of older mice.

The liver is a major site of generation of extrathymic T cells with unique properties (e.g., expressing intermediate TCR and containing self-reactive clones). We investigated herein whether the levels of extrathymic alpha beta T cells varied in various organs as a function of age. A systematic examination of the number of mononuclear cells in various organs of BALB/c mice revealed that the number of hepatic MNC increased with age whereas the number of thymocytes decreased. These changes were more striking in mice fed under conventional conditions than under specific pathogen-free condition. The age-dependent changes in the number of mononuclear cells in the spleen and lymph nodes were minimal. Although the total proportion of alpha beta T cells in each organ remained constant, the staining patterns of TCR-alpha beta as shown by immunofluorescence profiles varied. The most prominent change was that intermediate TCR-alpha beta cells, which constituted a small population in the liver of young mice, expanded in the liver of older mice. Intermediate TCR cells appeared even in the periphery of older mice. These findings were confirmed by the appearance of extrathymic T cells with other unique properties, e.g., double-negative CD4-8- phenotype and CD44 expression. In athymic nude mice, only intermediate TCR cells were present in the liver and periphery. An age-dependent increase of intermediate TCR cells was also seen in these mice. Taken together with the result of bromodeoxyuridine-injection experiment, which showed an intensive in vivo proliferation of cells in the hepatic sinusoids, extrathymic T cells may differentiate predominantly in the liver and appeared even to the periphery in older mice.

Age Factors↗

PubMed1992

Estrogen administration activates extrathymic T cell differentiation in the liver.

In addition to T cell differentiation in the thymus, we have recently reported that extrathymic T cell differentiation occurs preferentially in the sinusoids of the liver. Although this extrathymic pathway is relatively minor in normal mice, it becomes predominant in mice with autoimmune diseases, athymic mice, and aged mice. In the present study, injection of normal male C3H/He mice, 6-8 wk of age, with 1 mg of estrogen resulted in an increase in mononuclear cells (MNC) yielded from the liver and a drastic decrease in thymocytes approximately 10 d after such injection. This unique modulation was not observed with hydrocortisone injection (5 mg/mouse, i.p.) nor with irradiation (5 Gy/mouse). Rather, these immunosuppressive treatments induced a simultaneous decrease in cell number in both the liver and thymus. A time-kinetics study on the cell number and spontaneous cell proliferation revealed that an increase in spontaneous cell proliferation in the liver preceded the increase in the number of liver MNC, and a decrease in spontaneous cell proliferation in the thymus preceded the decrease in the number of thymocytes. At this time, an enrichment of alpha/beta T cells with intermediate T cell receptors (TCRs), including forbidden T cell oligoclones and V beta 8+ cells, which are characterized as extrathymic alpha/beta T cells with unique properties, took place in the liver. On the other hand, the thymic atrophy induced by estrogen resulted in a prominent decrease in immature double-positive (CD(4+)8+) alpha/beta T cells with dull TCRs. These results indicate that estrogen administration activates an extrathymic pathway of T cell differentiation in the liver and reciprocally inactivates the intrathymic pathway. As extrathymic T cells have unique characteristics such as autoreactivity, the present findings might be intimately related to a female predominance of autoimmune diseases and suggest a possible role of estrogen in this phenomenon.

Animals↗

PubMed1992

Details of an isolation method for hepatic lymphocytes in mice.

The liver comprises a unique lymphocyte population, i.e., extrathymic alpha beta T cells with TcR of intermediate intensity. In the present study, we attempted to determine what pretreatments were appropriate to isolate hepatic mononuclear cells (MNC) containing such intermediate alpha beta TcR cells in mice. Hepatic MNC were isolated from untreated mice and mice subjected to either bleeding or liver perfusion, and the intermediate alpha beta TcR cells in each preparation were identified. For reasons of simplicity, cell purity and cell yields, hepatic lymphocytes should be obtained from mice subjected to total bleeding. Additional information on extrathymic alpha beta T cells obtained by using the recommended method is also presented.

Animals↗

PubMed1992

Activation of extrathymic T cells in the liver and reciprocal inactivation of intrathymic T cells by bacterial stimulation.

We recently demonstrated that the liver might be a major site of extrathymic T cell differentiation, including both alpha beta and gamma delta T cells. This extrathymic pathway in the liver, which has a relatively minor role in normal young mice, is activated in mice under bacterial stimulation. In the present study, we investigated how the extrathymic and intrathymic T cell differentiations were mutually related in mice injected intravenously with 10(8) heat-killed Escherichia coli. Three days after stimulation, extrathymic T cells in the liver were observed to be prominently activated in terms of increases in the total number of cells yielded, spontaneous cell proliferation in in vitro culture, and intermediate alpha beta TCR cells. Intermediate alpha beta TCR cells were extrathymic T cells uniquely seen in the liver. However, at the same time intrathymic T cells were profoundly inactivated, showing decreases in the number of thymocytes (more than 90% atrophy), spontaneous cell proliferation, and dull TCR cells with double positive CD4+8+ phenotype. With time, these responses were reversed and normal states were regained. These results suggested that extrathymic and intrathymic T cells are always activated or inactivated in the opposite direction, and that the liver and the thymus are dynamic immune organs. It raises the possibility that the extrathymic T cell differentiation in the liver and the intrathymic T cell differentiation may be reciprocally regulated by certain factors.

Animals↗

PubMed1992

Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method.

Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.

Animals↗

PubMed1992

Lymphotoxin activates hepatic T cells and simultaneously induces profound thymic atrophy.

We have recently demonstrated that the liver may be a major site of extrathymic T-cell differentiation. This hepatic pathway was shown to be activated in mice injected with heat-killed bacteria. It is conceivable that the resulting activation of macrophages or lymphocytes, and the production of cytokines may be responsible for a subsequent activation of hepatic T cells. In this context, we investigated the possibility of whether certain cytokines may activate hepatic T cells. It was demonstrated that the administration of lymphotoxin [tumour necrosis factor-beta (TNF-beta)] more than doubled the number of hepatic mononuclear cells (MNC) yielded 3-5 days after the treatment. More strikingly, such treatment induced profound thymic atrophy and resulted in a decrease of more than 95% in the number of thymocytes. Spontaneous proliferation in an in vitro culture of hepatic MNC from treated mice increased, and inversely such activity of thymocytes decreased. The increased number of hepatic MNC was mainly due to an increase in intermediate alpha beta T-cell receptor (TcR) cells, which are extrathymic T cells uniquely seen in the liver. On the other hand, the thymic atrophy was caused by the prompt apoptotic death of dull alpha beta TcR cells with double-positive (DP) CD4+ CD8+ phenotype. These results indicate that lymphotoxin may be one of the factors that activates extrathymic T cells in the liver and at the same time inhibits intrathymic T-cell differentiation.

Animals↗

PubMed1991

Effect of alkyl chain unsaturation and cholesterol intercalation on oxygen transport in membranes: a pulse ESR spin labeling study.

Transport and diffusion of molecular oxygen in phosphatidylcholine (PC)-cholesterol membranes and their molecular mechanism were investigated. A special attention was paid to the molecular interaction involving unsaturated alkyl chains and cholesterol. Oxygen transport was evaluated by monitoring the bimolecular collision rate of molecular oxygen and the lipid-type spin labels, tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid. The collision rate was determined by measuring the spin-lattice relaxation times (T1's) in the presence and absence of molecular oxygen with long-pulse saturation-recovery ESR techniques. In the absence of cholesterol, incorporation of either a cis or trans double bond at the C9-C10 position of the alkyl chain decreases oxygen transport at all locations in the membrane. The activation energy for the translational diffusion of molecular oxygen in the absence of cholesterol is 3.7-6.5 kcal/mol, which is comparable to the activation energy theoretically estimated for kink migration or C-C bond rotation of alkyl chains [Träuble, H. (1971) J. Membr. Biol. 4, 193-208; Pace, R. J., & Chan, S. I. (1982) J. Chem. Phys. 76, 4241-4247]. Intercalation of cholesterol in saturated PC membranes reduces oxygen transport in the headgroup region and the hydrophobic region near the membrane surface but little affects the transport in the central part of the bilayer. In unsaturated PC membranes, intercalation of cholesterol also reduces oxygen transport in and near the headgroup regions. In contrast, it increases oxygen transport in the middle of the bilayer. On the basis of these observations, a model for the mechanism of oxygen transport in the membrane is proposed in which oxygen molecules reside in vacant pockets created by gauche-trans isomerization of alkyl chains and the structural nonconformability of neighboring lipids, unsaturated PC and cholesterol in particular, and oxygen molecules jump from one pocket to the adjacent one or move along with the movement of the pocket itself. The presence of cholesterol decreases oxygen permeability across the membrane in all membranes used in this work in spite of the increase in oxygen transport in the central part of unsaturated PC-cholesterol membranes because cholesterol decreases oxygen transport in and near the headgroup regions, where the major barriers for oxygen permeability are located. Oxygen gradients across the membranes of the cells and the mitochondria are evaluated. Arguments are advanced that oxygen permeation across the protein-rich mitochondrial membranes can be a rate-limiting step for oxygen consumption under hypoxic conditions in vivo.

Animals↗

PubMed1991

Development of a streak-camera-based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells.

A time-resolved microscope fluorimeter based on a synchroscan streak camera and a fast pulsed laser system has been developed to measure the fluorescence lifetime decay under the fluorescence microscope. This system allows one to measure the nanosecond fluorescence lifetimes of fluorophores in a small spot (0.8-6.3 microns diameter) in single cultured cells under a fluorescence microscope, while the cells are being viewed under a high-power objective lens. A signal acquisition time between a second and a minute was usually sufficient to obtain fluorescence decay curves with good quality for 10(3)-10(5) fluorophores localized in 1 microns 2 domain. A signal-to-noise ratio better than 30 was obtained for approximately 30,000 fluorescein-labeled band 3 molecules in a 2 microns 2 region in a single human erythrocyte ghost after signal accumulation for 30 s. The measured lifetimes for a variety of fluorescent probes attached to proteins in solution and lipids in liposomes showed a good agreement with those measured in a cuvette under standard conditions by time-correlated single photon counting. With the development of this instrument, microscope fluorimetry has become a practical, straightforward, quantitative technique for investigation of molecular processes in single cells in culture. Time-resolved microscope fluorimetry has been applied to observe fusion of liposomes in vitro and that of endosomes in single cells by monitoring resonance energy transfer. Inspection of individual liposomes and endosomes revealed the extent of fusion for each vesicle. Since the use of time-resolved microscope fluorimetry eliminates the need for subcellular fractionation or the complex correction procedures in steady-state microfluorimetry, it greatly simplifies the assay for endosome fusion in vivo. The results showed that extensive fusion of sequentially formed endosomes takes place all over the cell matrix in cultured cells. This suggests that extensive fusion with incoming endosomes takes place in many endosomal compartments, possibly sorting organelles, or that the early endosomes fuse with the preexisting network of tubular cisternae of the endosomal compartment at many points in the network. It is concluded that time-resolved microscope fluorimetry is a powerful noninvasive technique for studies of in situ biochemistry and biophysics using cells and tissues.

Animals↗

PubMed1991

Influence of phospholipid unsaturation on the cholesterol distribution in membranes.

Over the last half decade, we have studied saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes, with special attention paid to fluid-phase immiscibility in cis-unsaturated PC-cholesterol membranes. The investigations were carried out with fatty acid and sterol analogue spin labels for which reorientational diffusion of the nitroxide was measured using conventional ESR technique. We also used saturation recovery ESR technique where dual probes were utilized. Bimolecular collision rates between a membrane-soluble square-planar copper complex,3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato)copper(II) (CuKTMS2) and one of several nitroxide radical lipid-type spin labels were determined by measuring the nitroxide spin-lattice relaxation time (T1). The results obtained in all these studies can be explained if the following model is assumed: 1) at physiological temperatures, fluid-phase micro-immiscibility takes place in cis-unsaturated PC-cholesterol membranes, which induces cholesterol-rich domains in the membrane due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the cis double bond of the alkyl chains of unsaturated PC. 2) The cholesterol-rich domains are small and/or of short lifetime (10(-9) s to less than 10(-7) s). Our results also suggest that the extra space that is available for conformational disorder and accommodation of small molecules is created in the central part of the bilayer by intercalation of cholesterol in cis-unsaturated PC membrane due to the mismatch in the hydrophobic length and nonconformability between cis-unsaturated PC alkyl chains and the bulky tetracyclic ring of cholesterol.

Cholesterol↗

PubMed1990

Microimmiscibility and three-dimensional dynamic structures of phosphatidylcholine-cholesterol membranes: translational diffusion of a copper complex in the membrane.

Saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes have been studied, with a special attention paid to fluid-phase immiscibility in cis-unsaturated phosphatidylcholine (PC)-cholesterol membranes as previously proposed and to the three-dimensional structure of the membrane. The investigation was carried out with dual probes: a membrane-soluble, square-planar copper complex, (3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato]copper(II) (CuKTSM2), and one of several nitroxide radical lipid-type spin-labels. Bimolecular collision rates between metal ion and spin-label were determined by measuring the nitroxide spin-lattice relaxation times (T1's) in the presence and absence of CuKTSM2 by use of saturation-recovery ESR techniques, and from these measured rates, translational diffusion coefficients of CuKTSM2 were estimated. Profiles of the collision rate across the membrane bilayer were obtained with Tempocholine phosphatidic acid ester, 5-doxylstearic acid, 16-doxylstearic acid, and cholesterol-type spin-labels as a function of cholesterol mole fraction, length and unsaturation of acyl chains, and temperature. In the liquid-crystalline phase of saturated PC membranes, incorporation of cholesterol decreases the collision rate at all depths in the membrane, and the effect of cholesterol is smallest in the middle of the bilayer. In trans-unsaturated PC membranes, a cholesterol-induced decrease of the collision rate was also observed, except in the head-group regions. In cis-unsaturated PC membranes, virtually no effect of cholesterol was observed on the collision rate, either with phospholipid-type spin-labels or with cholesterol-type spin-labels. This result is in clear contrast with our previous observation, in which the effect of cholesterol in cis-unsaturated PC membranes is small on the alkyl-chain motion of phospholipid-type spin-labels but large on the wobbling rotational diffusion of cholesterol-type spin-labels [Pasenkiewicz-Gierula, M., Subczynski, W. K., & Kusumi, A. (1990) Biochemistry 29, 4059-4069]. A model is proposed to explain these results in which the fluid-phase immiscibility is prevalent in cis-unsaturated PC-cholesterol membranes, but where cholesterol-rich (cholesterol oligomeric) domains are small (several lipids) and/or of short lifetime (10(-9) s to less than 10(-7) s). It is suggested that this microimmiscibility arises from the structural nonconformability between the rigid cholesterol ring structure and the rigid bend at the cis double bonds in PC alkyl chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstanes↗

PubMed1990

Rotational diffusion of a steroid molecule in phosphatidylcholine-cholesterol membranes: fluid-phase microimmiscibility in unsaturated phosphatidylcholine-cholesterol membranes.

Rotational diffusion of androstane spin-label (ASL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of the chain length and unsaturation of the alkyl chains, cholesterol mole fraction, and temperature for a better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. Special attention was paid to the differences in the cholesterol effects on ASL motion between saturated and unsaturated PC membranes. ASL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines were obtained for various PC-cholesterol membranes in the liquid-crystalline phase. Cholesterol decreases both the cone angle and the wobbling rotational diffusion constant for ASL in all PC membranes studied in this work. The cholesterol effects are the largest in DMPC membranes. An increase of cholesterol mole fraction from 0 to 30% decreases the rotational diffusion constant by a factor of 9-15 (depending on temperature) and the cone angle by a factor of about 2. In dioleoyl-PC membranes, addition of 30 mol % cholesterol reduces both the rotational diffusion constant and the cone angle of ASL by factors of approximately 2.5 and approximately 1.3, respectively, while it was previously found to cause only modest effects on the motional freedom of phospholipid analogue spin probes [Kusumi, A., Subczynski, W. K., Pasenkiewicz-Gierula, M., Hyde, J. S., & Merkle, H. (1986) Biochim. Biophys. Acta 854, 307-317]. It is proposed that fluid-phase microimmiscibility takes place in dioleoyl-PC-cholesterol membranes at physiological temperatures, which induces cholesterol-rich domains in the membrane, partially due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the C9-C10 cis double bond of the alkyl chains of dioleoyl-PC. The mechanism by which cholesterol influences the lipid dynamics in the membrane is different between saturated and unsaturated PC membranes.

Cholesterol↗

PubMed1989

Oxygen permeability of phosphatidylcholine--cholesterol membranes.

Oxygen transport in phosphatidylcholine-cholesterol membranes has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed at various distances from the membrane surface using long-pulse saturation recovery ESR techniques. The collision rate was estimated for tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameter across the membrane were obtained as a function of cholesterol mol fraction and temperature in L-alpha-dimyristoylphosphatidylcholine ([ Myr2]PtdCho) and L-alpha-dioleoylphosphatidylcholine ([ Ole2]PtdCho) membranes. Membrane oxygen permeability coefficients were estimated from oxygen transport parameter profiles. At approximately 30 degrees C, the oxygen permeability coefficients in the presence and absence of 50 mol % cholesterol are 22.7 and 125.2 cm/s, respectively, for [Myr2]PtdCho membranes, and 54.7 and 114.2 cm/s, respectively, for [Ole2]PtdCho membranes (compared with 60-80 cm/s for water layers with the same thicknesses as the membranes). The major results in the liquid-crystalline phase are as follows: (i) In the absence of cholesterol, membranes are not barriers to oxygen transport. (ii) Addition of 50 mol % cholesterol decreases oxygen permeability by a factor of approximately 5 and approximately 2.5 in [Myr2]PtdCho and [Ole2]PtdCho membranes, respectively. The resistance to oxygen transport is located in and near the polar headgroup regions in the membrane. (iii) Cholesterol increases oxygen transport in the central regions of [Ole2]PtdCho membranes.

Cholesterol↗

PubMed1988

Regulation of band 3 mobilities in erythrocyte ghost membranes by protein association and cytoskeletal meshwork.

Rotational diffusion of erythrocyte anion channel protein band 3 was measured in ghost membranes by observing time-resolved phosphorescence anisotropy decays of eosinyl-5-maleimide covalently attached to the protein. Experiments were carried out under conditions similar to those employed by Tsuji and Ohnishi (1986) for translational diffusion measurement of band 3 [(1986) Biochemistry 25, 6133-6139] to allow direct comparison of rotational and translational diffusion of band 3. Detailed analysis of diffusive properties of band 3 in ghost membranes was made on the basis of these rotational and translational diffusion data. Rotational diffusion measurements indicated that there are at least three populations of band 3 molecules with high, low, and no rotational mobilities in the time scale of 10(-4)-10(-2) s. These populations are in equilibrium, and the fractional ratios are strongly temperature dependent. At 26 degrees C, 44% of band 3 molecules are mobile (16% have an average rotational correlation time of 0.19 ms, and 28% have an average correlation time of 2.4 ms), and 56% are immobile. These results correlate well with translational diffusion data which indicated 40% mobile and 60% immobile fractions of band 3. The rotational diffusion data together with the translational diffusion data by Tsuji and Ohnishi (1986) and Golan and Veatch [(1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2537-2541] suggest that immobilization of band 3 is largely caused by binding of band 3 oligomers to ankyrin, which abolishes both rotational and translational diffusion of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed1988

Rotational diffusion of a steroid molecule in phosphatidylcholine membranes: effects of alkyl chain length, unsaturation, and cholesterol as studied by a spin-label method.

Rotational diffusion of cholestane spin-label (CSL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of chain length and unsaturation of alkyl chains, cholesterol mole fraction, and temperature for better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. CSL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines are obtained for various membranes in the liquid-crystalline phase. The wobbling diffusion constant decreases in the order dilauroyl-PC greater than dimyristoyl-PC greater than dioleoyl-PC approximately dipalmitoyl-PC greater than distearoyl-PC greater than dioleoyl-PC/cholesterol = 3/1 greater than dioleoyl-PC/cholesterol = 1/1 membranes. Activation energy for the wobbling diffusion of the long axis of CSL is strongly dependent on alkyl chain length, unsaturation, and cholesterol mole fraction. It decreases with decrease in alkyl chain length and by introduction of unsaturation in the alkyl chains. In dioleoylphosphatidylcholine membranes, activation energy decreases by a factor of approximately 3 in the presence of 50 mol % cholesterol. Activation energy for wobbling diffusion of CSL in phosphatidylcholine membranes is smaller than the activation energy for translational diffusion of a phospholipid. The former is more dependent on alkyl chain length and unsaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkanes↗

PubMed1987

Dynamic fluorescence quenching studies on lipid mobilities in phosphatidylcholine-cholesterol membranes.

Bimolecular collision rate of 8-anilinonaphthalene-1-sulfonic acid (ANS) and the nitroxide doxyl group attached to various carbons on stearic acid spin labels (n-SASL) in phosphatidylcholine-cholesterol membranes in the fluid phase was studied by observing dynamic quenching of ANS fluorescence by n-SASL's. The excited-state lifetime of ANS and its reduction by the n-SASL doxyl group were directly measured by the time-correlated single photon counting technique to observe only dynamic quenching separately from static quenching and were analyzed by using Stern-Volmer relations. The collision rate of ANS with the n-SASL doxyl group ranges between 1 X 10(7) and 6 X 10(7), and the extent of dynamic quenching by n-SASL is in the order of 5-much much greater than 6- greater than 7- less than 9- less than 10- less than 12- less than 16-SASL (less than 5-SASL) in dimyristoylphosphatidylcholine (DMPC) membranes. Collision rate of 16-SASL is only 10% less than that of 5-SASL. Since the naphthalene ring of ANS is located in the near-surface region of the membrane, these results indicate that the methyl terminal of SASL appears in the near surface area frequently, probably due to extensive gauche-trans isomerism of the methylene chain. The presence of 30 mol% cholesterol decreases the collision rate of ANS with 12- and 16-SASL doxyl groups but not with the 5-SASL doxyl group in DMPC membranes. On the other hand, in egg-yolk phosphatidylcholine membranes, inclusion of 30 mol% cholesterol does not affect the collision of ANS with either 5-SASL or 16-SASL doxyl groups, in agreement with our previous observation that alkyl chain unsaturation moderates cholesterol effects on lipid motion in the membrane (Kusumi et al., Biochim. Biophys. Acta 854, 307-317). It is suggested that dynamic quenching of ANS fluorescence by lipid-type spin labels is a useful new monitor of membrane fluidity that reports on various lipid mobilities in the membrane; a class of motion can be preferentially observed over others by selecting a proper spin label, i.e., rotational diffusion of lipid about its long axis and translational diffusion by using 5-SASL, wobbling motion of the lipid long axis by using 7-SASL or androstane spin label, and gauche-trans isomerism by using 16-SASL.

Anilino Naphthalenesulfonates↗

PubMed1986

Fc:Fc interactions revealed by spin-labeled IgG heterosaccharides in model immune complexes.

Dynamic properties of spin-labelled heterosaccharides in the Fc-region of murine monoclonal antihapten immunoglobulin G were studied in model immune complexes (IC) as a function of the IC size. Model IC dimers, trimers and oligomers were formed using bivalent photoaffinity antigens. The ESR spectrum exhibits two components. The rotational correlation time of the less-immobilized species is shorter than 10(-10) sec, and that of the more-immobilized component is in the order to 10(-9) approximately 10(-8) sec depending on the IC size. Fraction of the more-immobilized spin labels increases, and the mobility of this component decreases with increase in IC size (i.e., mobility: monomers approximately equal to dimers greater than trimers much greater than immune-complex precipitates). These data strongly suggest the existence of Fc:Fc interactions in IC, and provide the basis for a model in which such interactions underlie the initial mechanism by which the information of antigen binding to Fab region is transferred into organized Fc:Fc association structure for IgG effector activities.

Animals↗