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A Kusumi

Publications and source records attributed to A Kusumi.

67 records · Page 4Linked to original sources
PubMed1986

Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase.

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.

Cholesterol↗
PubMed1986

Effects of very small amounts of cholesterol on gel-phase phosphatidylcholine membranes.

The mobility of 5-doxyl stearic acid spin label (5-SASL) in the gel phase of dipalmitoylphosphatidylcholine membranes between the main transition and subtransition temperatures was studied as a function of cholesterol content. Very small amounts of cholesterol (0.01-1 mol%) cause a dramatic increase in the mobility of 5-SASL. Temperature-drop experiments from 38 degrees C to 28 degrees C were made across the pretransition temperature and the rate of approach to equilibrium was measured. Cholesterol at low concentrations also affects this rate. The membrane reached equilibrium after 10 h in the absence of cholesterol, 3 h at 0.01 mol% cholesterol, and less than 10 min at 0.03 mol% cholesterol.

Cholesterol↗
PubMed1985

Detection of oxygen consumption during very early stages of lipid peroxidation by ESR nitroxide spin probe method.

Oxygen consumption during the very early stages of the spontaneous peroxidation of egg yolk phosphatidylcholine membranes was studied by monitoring the oxygen concentration in the aqueous phase of the sample using a spin-probe closed-chamber method. The method depends on the broadening by oxygen of the proton superhyperfine lines of the electron spin resonance spectra of the nitroxide radical spin probe 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxyl. It is concluded that this method is useful in monitoring lipid peroxidation and that it monitors the onset of the peroxidation process before the commonly used thiobarbituric acid assay detects the peroxidation products.

Cyclic N-Oxides↗
PubMed1984

Human autoantibodies against a desmosomal core protein in pemphigus foliaceus.

Pemphigus foliaceus (PF) is a human autoimmune disease in which antibodies are directed against the cell surface of epidermal cells with resultant blister formation. The histopathology of these blisters indicates that cells have detached from each other, and electron microscopy of early blisters shows diminished numbers, to complete loss, of desmosomes as well as abnormalities of the tonofilament-desmosome complex. In this study we demonstrate that autoantibodies from certain PF patients bind to a desmosomal core glycoprotein called desmoglein (DG) I. Proteins in extracts of normal human epidermis were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), then transferred to nitrocellulose or 2-aminophenylthioether paper for immunoperoxidase staining. Results of these immunoblots indicated that sera from 6 of 13 PF patients specifically and intensely stained an approximately 160,000 mol wt polypeptide, "PF antigen". Such staining was not seen with normal human sera or sera from patients with pemphigus vulgaris or bullous pemphigoid, two autoimmune blistering skin diseases that are clinically, histologically, and immunochemically distinct from PF. However, rabbit antiserum directed against DGI, that was isolated from bovine muzzle desmosomes, stained a polypeptide band which co-migrated with PF antigen. Furthermore, when proteins from extracts of normal human epidermis were electrophoresed in two dimensions (isoelectric focusing, then SDS-PAGE) before transfer to nitrocellulose for immunoperoxidase staining, PF antibodies and antibodies to DGI stained identical spots. Finally, PF sera as well as PF IgG that was affinity purified with PF antigen from normal human epidermis, both selectively bound to DGI extracted from bovine muzzle desmosomes. These studies demonstrate that the human autoantibodies from certain patients with PF, a disease of epidermal cell adhesion, are directed against a desmosomal core protein.

Animals↗
PubMed1984

Human aglycosyl-IgG exhibits increased hydrophobicity. Binding/fluorescence studies with 8-anilinonaphthalene-1-sulfonic acid (ANS).

Human monoclonal, aglycosyl-IgG produced in vitro in the presence of tunicamycin, was compared with its native and acid pH-altered counterparts for their respective abilities to bind the fluorescent hydrophobicity probe, 8-anilinonaphthalene sulfonate. A novel technique based on continuous-flow dynamic dialysis (Sparrow et al., 1982, Anal. Biochem. 123:255-264) allowed binding studies under non-equilibrium conditions. While the native IgG conformation exhibits two, weak ANS binding sites (ca. 10(3) l/mol), aglycosyl-IgG has one weak and one moderate affinity (least squares average Ka = 2 X 10(4) l/mol) site, and the acid conformer binds yet another two ANS molecules with moderate affinity (4 X 10(4) l/mol). Increases in affinity and in the number of sites correlate roughly with increased relative percent fluorescence by conventional fluorimetry. The fluorescence lifetime of ANS bound to altered IgGs is about 10% longer (T2 = 15 nsec by time-resolved fluorimetry) than that for native IgG. All populations also exhibit a rapid decay component (T1 = 3 nsec) analogous to that seen for ANS in 50% aqueous dioxane. Results are discussed in relation to structural role(s) for IgG-linked heterosaccharides.

Anilino Naphthalenesulfonates↗
PubMed1984

Complement C1q binding affects spin-labeled heterosaccharides of rabbit antibodies in immune but not artificial immunoglobulin G aggregates.

IgG anti-hapten antibodies were purified from the sera of rabbits homozygous for allotypic determinants d11 and d12 in the constant region of the heavy chain. Correlative with this determinant is the absence (d11) or presence (d12) of an oligosaccharide chain just below the hinge region of the IgG molecule. Both d11 and d12 molecules contain a complex heterosaccharide chain located near the carboxyl terminus of the second constant region domain. The two populations of IgG antibodies were thus selectively labeled with the spin probe Tempamine in their second constant region domains by reductive amination primarily of terminal N-acetylneuraminic acid residues. Chemical and enzymatic cleavages showed about 80% of the attached spin labels were N-acetylneuraminic acid-associated. Analysis of probe adducts by ESR spectrometry showed the presence of slower and faster moving subcomponents. Formation of immune complexes by antigen induces slight but significant restrictions of spin label mobility for both d11 and d12 IgG molecules. This restriction is qualitatively different from that seen in glutaraldehyde-, carbodiimide-, or ethanol-induced aggregates of the same IgG antibodies. The addition of purified complement C1 subcomponent C1q to immune aggregates resulted in marked immobilization of spin labels, the rotational correlation time of which was 30-40 mu s for both d11 and d12 molecules (evaluated by saturation transfer spectroscopy). A similar spin probe immobilizing effect is not seen when C1q binds to chemically aggregated IgG antibodies (which also do not activate C1). A novel model is proposed in which C1q is hypothesized to juxtapose Fc moieties in a discrete fashion required for subsequent C1 activation processes mediated by immune complexes.

Animals↗
PubMed1982

Spin-label saturation-transfer electron spin resonance detection of transient association of rhodopsin in reconstituted membranes.

Rotational diffusion of rhodopsin in reconstituted membranes of phosphatidylcholines of various alkyl chain lengths has been measured by using saturation-transfer electron spin resonance spectroscopy as a function of temperature and lipid/rhodopsin mole ratio. For dipalmitoyl-phosphatidylcholine, the rotational correlation time is 20 microseconds at physiological concentration, the same as in rod outer segment (ros) membranes. Dilution reduces the time to 10 microseconds, a value that is ascribed to well-dispersed monomeric rhodopsin. Use of phospholipids with longer or shorter chains results in sharply increased rotational correlation times. It is concluded that rhodopsin molecules are transiently associated in both reconstituted and ros membranes and that the nature of the association is determined by lipid type and composition.

Animals↗
PubMed1982

Oxygen transport parameter in membranes as deduced by saturation recovery measurements of spin-lattice relaxation times of spin labels.

Spin-lattice relaxation time (T1) measurements of nitroxide radical spin labels in membranes have been made by using the saturation-recovery technique. Stearic acid and sterol-type labels were used as probes of dimyristoylphosphatidylcholine liposomes from 0 degrees C to 36 degrees C. In the absence of oxygen, the range of variation of T1 over all samples and conditions is about a factor of 3. Heisenberg exchange between oxygen and spin labels is an effective T1 mechanism for the spin labels. The full range of variation of T1 in the presence of air is about a factor of 100. It is suggested that the oxygen transport parameter W = T1(-1) (air) - T1(-1) (N2) is a useful new monitor of membrane fluidity that reports on translational diffusion of small molecules. The values of W change at the prephase and main phase transitions and vary in complex ways. Arguments are advanced that the data are indicative of anisotropic translational diffusion of oxygen.

Diffusion↗