Biomedical subjects

M Setaka

Publications and source records attributed to M Setaka.

At least 37 records · Page 2Linked to original sources

PubMed1990

Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa.

A (H+ + K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation. The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na+ + K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H+ + K+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (p-NPPase). The (H+ + K+)-ATPase had a pH optimum of 7.4 and was stimulated by Tl+, K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM, respectively, at this pH. On the other hand, monovalent cations such as Na+, Li+ and (CH3)4N+ as well as divalent cations such as Cu2+, Ca2+, Ba2+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently. Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H+ + K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed a major band (greater than or equal to 90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [gamma-32P]-ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H+ + K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.

Adenosine Triphosphatases↗

PubMed1987

Antibody to platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF).

Specific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As the hapten we used the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Antibody activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycerophosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.

Animals↗

PubMed1986

Effects of chlorpromazine and other calmodulin antagonists on phosphatidylcholine-induced vesiculation of platelet plasma membranes.

Dilauroylglycerophosphocholine (C12:0PC)-induced vesiculation of platelet plasma membranes (Kobayashi, T., Okamoto, H., Yamada, J.-I., Setaka, M. and Kwan, T. (1984) Biochim. Biophys. Acta 778, 210-218; Kobayashi, T., Yamada, J.-I., Satoh, N., Setaka, M. and Kwan, T. (1985) Biochim. Biophys. Acta 817, 307-312) was inhibited by chlorpromazine. Preincubation of platelets with chlorpromazine was required for inhibition but incorporation of chlorpromazine into C12:0PC liposomes was not necessary for it, indicating that the observed inhibition of vesiculation was mainly due to the effect of chlorpromazine on platelets and not that on liposomes. The change in platelet membrane fluidity caused by chlorpromazine was not the cause of inhibition of vesiculation. The inhibition of vesiculation by various other calmodulin antagonists was also observed. The inhibitory activities of these calmodulin antagonists and chlorpromazine correspond very well to their abilities to bind to calmodulin. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited vesiculation but a structural analogue of it, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had no inhibitory activity. These results suggest the involvement of calmodulin in membrane vesiculation.

Animals↗

PubMed1986

Light-induced potential and current across a large bacteriorhodopsin-asolectin planar membrane stabilized on a polyacrylamide gel surface.

A phospholipid bilayer membrane was spread from an organic solvent solution between a polyacrylamide gel surface and an aqueous buffer solution. The membrane was quite similar to the conventional black lipid membrane, but was of a large size and was stable since it was supported on the gel surface. Bacteriorhodopsin, impregnated into the membrane, generated membrane potential and current upon illumination. The induced current was large, and this was attributed to the large area of the present membrane. Remarkable responses of the light-induced potential and current were also observed with a thick layer of organic solvent containing phospholipids. The effects of applied membrane potential, carbonylcyanide-m-chlorophenyl hydrazone (CCCP) and gramicidin were examined on these photoresponses. Steady-state current, which is due to protons flowing through the membrane, was enormously enhanced by applying membrane potential opposite to the photopotential or by adding gramicidin to the membrane-forming solution.

Acrylic Resins↗

PubMed1985

Inhibition of platelet aggregation by synthetic phosphatidylcholines: possible involvement of vesiculation of platelet plasma membranes.

Synthetic phosphatidylcholines inhibited thrombin-induced aggregation of rabbit platelets. The inhibitory effect of the phosphatidylcholines increased with an increase in the chain-length of the constituent fatty acids up to 12, and then decreased, and C14:0PC and C16:0PC did not inhibit platelet aggregation. The activity of synthetic phosphatidylcholines as to induction of vesiculation of platelet plasma membranes (Kobayashi, T., Okamoto, H., Yamada, J.-I., Setaka, M. and Kwan, T. (1984) Biochim. Biophys. Acta 778, 210-218) and the inhibitory effect of these phosphatidylcholines on platelet aggregation showed the same dependence on the constituent fatty acids of the phosphatidylcholines. The amounts of phosphatidylcholines required for 50% inhibition of platelet aggregation correspond very well to those required for 15% exfoliation of acetylcholinesterase activity, suggesting that there is a close relationship between platelet aggregation and vesiculation of the platelet plasma membrane. The possible mechanism of inhibition of platelet aggregation by synthetic phosphatidylcholines is discussed.

Acetylcholinesterase↗

PubMed1985

Spin label study of the effect of ticlopidine on platelets.

The effect of ticlopidine on platelet membrane fluidity was investigated using a spin label technique. Ticlopidine, when orally administered to rats, increased both the order parameter and the motion parameter, indicating a decrease in the membrane fluidity of platelets. On the other hand, the order parameter and the motion parameter decreased markedly when the platelets were aggregated by thrombin. Ticlopidine inhibited the thrombin-induced aggregation of platelets and caused a slight increase in order parameter and motion parameter in thrombin-aggregated platelets. Judging from sodium dodecyl sulfate polyacrylamide gel electrophoresis, ticlopidine did not modify the electrophoretic pattern of platelet proteins appreciably. Ticlopidine decreased cholesterol/phospholipids molar ratio and increased slightly total amounts of proteins of the platelets. These results indicate that the inhibitory action by ticlopidine was accompanied by changes in membrane fluidity, and these changes were due to a perturbation of the membrane phospholipid core of the platelets by ticlopidine and/or its metabolites.

Animals↗

PubMed1984

Vesiculation of platelet plasma membranes. Dilauroylglycerophosphocholine-induced shedding of a platelet plasma membrane fraction enriched in acetylcholinesterase activity.

Incubation of washed rabbit platelets with suspensions of dilauroylglycerophosphocholine resulted in the shedding of vesicles without causing any appreciable leakage of cytoplasmic marker (lactate dehydrogenase) or organelle marker [( 14C]serotonin). The response was dependent on incubation time, concentration of dilauroylglycerophosphocholine and reaction temperature. Vesicles were separated from platelets and exogenous dilauroylglycerophosphocholine by a series of centrifugation steps. An average diameter of vesicles was 100-200 nm on scanning electron microscopy. Vesicles were enriched 5-fold in plasma membrane marker enzyme, acetylcholinesterase, whereas specific activities of lactate dehydrogenase and intracellular membrane marker enzyme, NADH-cytochrome c reductase were decreased in vesicles. Protein analysis by SDS-polyacrylamide gel electrophoresis showed that actin and actin-binding protein were present, while myosin was barely detectable in vesicles. Vesicles contained all phospholipid species of intact platelets and cholesterol but almost 50% of phospholipids in vesicles was dilauroylglycerophosphocholine. The phospholipid to protein ratio in vesicles was about 6.5-times higher than in intact platelets.

Acetylcholinesterase↗

PubMed1983

Spontaneous formation of a monolayer membrane from sarcoplasmic reticulum at an air-water interface.

Surface pressure was found to be produced spontaneously at the interface between air and a suspension containing fragmented sarcoplasmic reticulum (FSR) from rabbit white muscle. Large and stable surface pressure was formed only in a limited concentration range of FSR in the suspension and the pressure formation was proved to be an irreversible phenomenon, suggesting the formation of a monolayer membrane resulting from the disruption of FSR vesicles. Monolayer formation was directly confirmed by analyzing the components included in the membrane and by calculating the surface area occupied by these components. The monolayer included phospholipids, cholesterol and proteins, and appeared to originate from FSR vesicles since the molecular ratios of these components as well as the results of the SDS polyacrylamide gel electrophoresis were similar in both membranes. This phenomenon can be utilized as a method of monolayer preparation from biological membrane vesicles, and should be very useful for the reconstitution of planar biological membranes.

Air↗

PubMed1983

Spin label measurement of platelet alignment in a clot induced by isometric tension.

A spin label technique was applied to examine the alignment of platelet membranes in the platelet-fibrin clot. The ESR spectrum of the clot in the control experiment was typical of isotropically oriented membranes, while the ESR spectrum of the clot which was kept isometric was quite different from that of the control experiment. These spectra were simulated with a computer and explained to indicate that the shape of platelets in the clot was transformed to a cylindrical form under the isometric tension so that axes of all membrane cylinders were directed parallel to the generated force.

Animals↗

PubMed1982

A stable planar bilayer membrane of phospholipid supported by cellulose sheets.

A new method is reported for preparing a thin planar membrane of 1,2-distearoylsn-glycero-3-phosphocholine and egg yolk lecithin-cholesterol (molar ratio of 1:1) between a pair of cellulose sheets. This technique, developed from the method of the multilayer planar membrane preparation (Setaka, M., et al. (1979) J. Biochem. 86, 355-362; 1619-1622; (1980) J. Biochem. 88, 1819-1829), consisted of three experimental processes. First, a phospholipid monolayer was prepared at an air-water interface, then taken up on a stretched cellulose sheet. A thin lipid membrane, supported from both sides by cellulose sheets, was constructed by combining two of these lipid monolayer-cellulose sheets. The permeability coefficient of the thin lipid membrane was estimated by removing the effect of two outer cellulose sheets, and this permeability was found to be larger than those of other model membranes of a lipid bilayer, indicating that the present lipid membrane is not a perfect single lipid bilayer. However, certain experimental evidence suggests that the bulk of the phospholipids formed a bilayer between the two cellulose sheets. Since this lipid membrane is particularily stable, larger membranes can be prepared by the present method than other planar bilayer membranes of lipid, which are usually constructed inside a pin hole in a thin teflon sheet.

Cell Membrane Permeability↗

PubMed1982

Orientation of platelets on the surfaces of glass and Teflon plates as studied by a spin label technique.

Platelets were found by a spin label technique to orient on flat surfaces of glass as well as of Teflon. This kind of platelet orientation was not caused by centrifugation or partial dehydration of the membrane preparation as employed usually to make oriented planar multilayers of biological membranes on the surface of a supporting plate (1-8), but was considered to be closely related to the adhesion or the aggregation properties of platelets. The amount of oriented platelets varied depending on the platelet treatment and was estimated from a computer simulation of the observed ESR spectra to be about one-half of that of the non-oriented ones in the case of thrombin-treated platelets. This technique may be useful as a new tool to explore the adhesion or aggregation properties of platelets.

Animals↗

PubMed1980

A spin-label study of the effects of drugs on calcium release from isolated sarcoplasmic reticulum vesicles.

The effects of caffeine, thymol, and procaine on calcium release from fragmented sarcoplasmic reticulum (FSR) from rabbit skeletal white muscle were investigated by the spin label method at the organellar level. Two thiol-directed spin labels, 4-maleimide-2,2,6,6-tetramethylpiperidinooxyl and 4-(2-iodoacetamide)-2,2,6,6-tetramethylpiperidinooxyl, were used for the labeling of SR proteins. The ratio (W/S) of the weakly (W) and strongly (S) immobilized ESR signals was measured for the maleimide and iodoacetamide labeled FSR. The two labels gave different W/S values, which means that the two labels report conformational changes at different loci of SR proteins. The dependences of the W/S ratios on the concentration of the drugs showed that conformational changes of SR proteins induced by these drugs are not the same. From measurements of the distribution of 5-doxyldecanoic acid methylester between the lipid and water phases, it was found that the conformational changes of the SR proteins caused by thymol or procaine induced a disorder in local regions of the phospholipid bilayers of FSR, while such disordering was not induced by caffeine. On the other hand, caffeine and thymol showed definite effects on calcium release from FSR, while procaine did not. These results indicate that the effects of the drugs on the protein conformations can be well characterized at the organellar level by means of the spin label technique and that some specific changes in the conformations of SR proteins are necessary for calcium release from FSR.

Animals↗

PubMed1980

Permeability properties of a multilayer planar membrane.

The permeability properties of multilayer planar membranes of uniformly oriented lipids between a pair of cellulose sheets were investigated. The effect of the two cellulose sheets supporting the lipid membrane on the glucose or the Ca permeation was subtracted empirically, and values of 5 X 10(-6) and 8 X 10(-6) cm/s were thus obtained for the permeability coefficients of an egg yolk lecithin (egg PC)-cholesterol membrane of about 200 bilayers to Ca2+ and glucose, respectively. These values are discussed as compared with the permeability coefficients of other model membranes. The membrane permeability was moderately affected by the addition of chemical substances to egg PC membranes. It was reduced by the presence of cholesterol, but enhanced by the presence of isopropanol, n-butanol, or thymol in the same solution above a critical concentration of each compound. These and previous observations (8) suggest that a close correlation may exist between the permeability of the membrane and the orientation of the membrane lipids. The glucose permeation was drastically suppressed by the presence of Ca2+ (10 mM) in the sample solution with the membrane containing phosphatidylserine, but was not at all suppressed with the membrane of the egg PC-cholesterol mixture.

1-Propanol↗

PubMed1979

On the overshoot of calcium accumulation in fragmented sarcoplasmic reticulum induced by thymol.

The mechanism was studied of the overshoot of calcium accumulation in fragmented sarcoplasmic reticulum (FSR) which is observed when the active transport of calcium into FSR is performed in the presence of thymol; the amount of calcium in FSR increases steeply during the first minute of the reaction and then decreases markedly. In contrast to this behavior, the amount of calcium in FSR increases monotonically and then reaches saturation in the absence of thymol. It is shown that the amount of calcium accumulated in FSR is determined by the balance between the rapid influx and efflux of calcium, and that both processes are depressed by thymol. The overshoot of calcium accumulation can be explained as follows: thymol so markedly depresses the efflux of calcium uncoupled with Ca2+-ATPase activity that the amount of calcium is increased in FSR in spite of partial deactivation of Ca2+-ATPase. However, the state of the FSR membrane is rapidly changed when the concentration of accumulated calcium exceeds a certain critical value. Concomitant with this change, the calcium permeability of the membrane is increased, leading to a decrease in the amount of accumulated calcium. The effects of magnesium and temperature on the overshoot of calcium accumulation can be accounted for by this proposed mechanism.

Animals↗

PubMed1979

Construction of a multilayer planar membrane applicable to ion-transport measurement.

Multilayer planar membranes applicable to ion-transport measurements were constructed from egg yolk lecithin, egg yolk lecithin-cholesterol mixture, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine between two tightly stretched cellulose sheets. While most of the phospholipids in the membranes were found by a spin label technique to be uniformly oriented with their long hydrocarbon chains perpendicular to the surfaces of the cellulose sheets, a small fraction of phospholipids were isotropically oriented in multilayer membranes. The amount of phospholipids with isotropic orientations decreased with increasing content of cholesterol in membranes and became zero in membranes of egg yolk lecithin-cholesterol mixture (molar ratio of 1: 0.67). The degree of orientation, S, of uniformly oriented phospholipids in membranes was also increased by adding cholesterol to the membranes. The orientation of phospholipids in membranes was rather stable in distilled water and in aqueous calcium chloride (1, 10, 100 mM), while a marked disordering of oriented phospholipids was induced in a aqueous solutions containing thymol, isopropanol, or butanol beyond certain specific concentrations. The membranes can be used for measurements of calcium permeation. An appreciable barrier function to calcium permeation was detected with these multilayer planar membranes as compared with control experiments using only cellulose sheets as membranes. A preliminary investigation suggested that changes in the orientational structure of phospholipids in the multilayer planar membranes are correlated with permeability properties of the membranes.

Biological Transport↗

PubMed1979

Multilayer planar membranes of sarcoplasmic reticulum.

Multilayer planar membranes were constructed between a pair of cellulose sheets from fragmented sarcoplasmic reticulum (FSR) as well as a mixture of egg yolk lecithin and the Ca2+-ATPase purified from FSR. Since sodium deoxycholate was used instead of organic solvents in order to dissolve phospholipids in the process of the membrane preparation, the total activity of the Ca2+-ATPase was still preserved in the planar membrane of FSR. It was also indicated using a spin label technique that the orientation of phospholipids in the planar membrane of FSR was considerably disturbed by the presence of proteins such as the Ca2+-ATPase included in FSR.

Animals↗

PubMed1979

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.

Animals↗

PubMed1978

Quantitative studies on competitive ligand binding to bovine serum albumin by use of the spin label 5-doxyl dodecanoic acid.

The binding of the spin label 5-doxyl dodecanoic acid to bovine serum albumin in phosphate buffer at pH 7.4 was studied by electron spin resonance spectroscopy. Free label and label bound to serum albumin could be quantitatively measured and evaluated from the superposition spectra of these two species with no previous separation. The efficiency relative to the spin label as competitors for binding to serum albumin was studied with salicylic acid and some fatty acids of medium length. The results were represented both by the stoichiometric model involving equilibrium constants Ki, by binding isotherms constructed from the Ki values, and by a purely graphical representation of the experimental data points without connection with any special binding model.

Binding, Competitive↗