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T Ohyashiki

Publications and source records attributed to T Ohyashiki.

At least 55 records · Page 3Linked to original sources
PubMed1984

Studies on cation-induced membrane vesicle aggregation of porcine intestinal brush borders.

Cation specificity and effects of pH and temperature on membrane aggregation of porcine intestinal brush borders were studied in terms of the turbidity change of the membrane suspensions due to the addition of polyvalent cations. Monovalent cations (K+ and Na+) did not induce the membrane aggregation at 5 mM or below. Divalent and trivalent cations induced membrane aggregation even below 0.5 mM in the following orders of effectiveness: Hg2+ much greater than Cd2+ greater than Zn2+ greater than Mn2+ greater than Ca2+ greater than or equal to Ba2+ greater than Sr2+ greater than or equal to Mg2+ and Lu3+ greater than Yb3+ greater than Nd3+ = Er3+ = Tb3+ greater than Ce3+ greater than La3+, respectively. The aggregation of membranes induced by these polyvalent cations was reversible. Membrane aggregation induced by either divalent or trivalent cations followed saturation kinetics with increasing cation concentration and the apparent dissociation constants of these cations for the membranes were estimated to be at 4-6 mM for divalent cations (Ca2+, Mn2+, and Sr2+) and 1-2 mM for trivalent cations (Lu3+, Tb3+, and La3+). Cd2+ and Hg2+ had peculiarly much lower dissociation constants than any other divalent cations tested, being rather comparable to trivalent ones. The membrane aggregation induced by Ca2+, Mn2+, or Sr2+ was independent of pH in the range of 6.6 to 8.0, whereas that induced by Tb3+ or Cd2+ was linearly enhanced with increasing pH, very steeply above pH 7.0. The membrane aggregation induced by Cd2+, Hg2+, or Tb3+ was enhanced steadily with increasing temperature between 15 and 45 degrees C. On the other hand, Ca2+-induced aggregation showed a peak at around 30 degrees C in the temperature dependence profile, still being enhanced above 40 degrees C.

Animals↗
PubMed1984

Stoichiometry and location of terbium and calcium bindings to porcine intestinal calcium-binding protein.

Quantitative analyses were carried out on Tb3+ binding to porcine intestinal calcium-binding protein (CaBP). Tb3+ (emission at 547 nm) and intrinsic tyrosine (emission at 303 nm) fluorescences upon excitation at 260 nm increase almost in parallel with increasing Tb3+ concentration up to a molar ratio of 2 against the protein in the CaBP solution. The pH dependence profile of Tb3+ fluorescence of the Tb3+-CaBP complex suggests that some free carboxylate groups are involved in the binding, as also suggested for Ca2+ binding. The results of fluorometric titration of Tb3+ and intrinsic tyrosine fluorescences of the CaBP complex with Tb3+ or Ca2+ led us to conclude that Tb3+ and Ca2+ have two common binding sites for each CaBP molecule. An equilibrium dialysis experiment showed that the dissociation constants of the two Tb3+-binding sites are 0.29 and 3.51 microM. Tb3+ strongly inhibits 45Ca binding to one of the two Ca2+-binding sites in the CaBP. All of these and previous results indicate that each Tb3+ ion can bind to either of two high-affinity Ca2+-binding sites in porcine intestinal CaBP with an affinity different from that for Ca2+ ion. We discuss the localization of the Ca2+- and Tb3+-binding sites in the CaBP.

Animals↗
PubMed1984

Effect of valinomycin on membrane vesicle aggregation of porcine intestinal brush borders.

It has been found in the brush border membrane vesicles from porcine small intestine that valinomycin causes vesicle aggregation and that Ca2+-induced aggregation of the vesicles is saturably stimulated in the presence of valinomycin. The apparent half-maximal concentration of valinomycin required to enhance the Ca2+ effect on the membrane aggregation is approximately 40 microM. Results of a fluorometric study using 1-anilino-8-naphthalene sulfonate (ANS) showed that the addition of valinomycin to the membranes induces slight decreases in the ANS-binding affinities for the membranes in the presence and absence of Ca2+. On the other hand, measurements of the incorporation rate of 1,6-diphenyl-1,3,5-hexatriene (DPH) into the vesicles and of anisotropies of DPH-labeled membranes suggested that the lipid organization of the membranes was not altered upon the addition of valinomycin. From these results, it was suggested that the valinomycin effect on the membrane aggregation is mainly related to the nature of the membrane surface charge.

Anilino Naphthalenesulfonates↗
PubMed1983

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes. A fluorescence probe study.

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles (out greater than in) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47 degrees C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.

Anilino Naphthalenesulfonates↗
PubMed1983

Quantitative analysis of calcium binding to porcine intestinal calcium-binding protein.

An equilibrium dialysis study has revealed that porcine intestinal calcium-binding protein (CaBP) has two binding sites for Ca2+ whose dissociation constants (Kd) are the same, 0.56 microM. The intrinsic fluorescence spectrum of the CaBP shows a peak (at 303 nm) in tyrosine band. Ca2+ binding to the CaBP induces a monophasic increase in the intensity of intrinsic fluorescence without any shift to either excitation or emission maximum. The change in the fluorescence intensity induced by Ca2+ binding is complete at a bound Ca2+/CaBP molar ratio of about 2, and the apparent Kd value is 0.51 microM. The same bound Ca2+/CaBP molar ratio has been obtained from the maximal changes in UV absorption and CD spectrum of the CaBP upon addition of Ca2+. A CD study has shown an about 5% increase in alpha-helix content in the CaBP at the maximal binding of Ca2+. All these results indicate that the porcine intestinal CaBP has two high-affinity binding sites with equal affinity for Ca2+ and suggest that it undergoes a quantitative conformation change accompanying Ca2+ binding in the vicinity of single tyrosine and phenylalanine residues of the protein molecule.

Animals↗
PubMed1983

Fluorescence probe studies on the interaction of phlorizin with rabbit intestinal brush border membranes.

Spectrofluorometric studies on the interaction of phlorizin with rabbit intestinal brush border membranes under various conditions were carried out using 1-anilino-8-naphthalene sulfonate (ANS) and N-(1-anilinonaphthyl-4)maleimide (ANM). The fluorescence intensity of membrane-bound ANS was markedly decreased upon addition of phlorizin. The apparent dissociation constant of phlorizin and the membranes at pH 7.4 was determined to be approximately 24 microM from the slope of a plot of the fluorescence change of membrane-bound ANS against phlorizin concentration. Addition of D-glucose diminished the quenching effect of phlorizin on the ANS fluorescence by lowering the binding affinity of phlorizin for the membranes. The interaction between phlorizin and the membranes was influenced by temperature, ionic strength and divalent cations. The binding affinity of phlorizin for the membranes is markedly reduced at higher temperature, showing a transition between 25 and 30 degrees C. Imposition of an ionic strength gradient across the membrane vesicles (out greater than in) increased the binding affinity. On the other hand, divalent cations decreased the affinity; Mg2+ was more effective than Ca2+. The binding affinity of ANS for the membranes was evidently decreased, and the reactivity of SH groups of the membrane proteins with ANM was apparently reduced, upon binding of phlorizin to the membranes. Based on these results, the relationships among modification of membrane structures, the extent of phlorizin binding and phlorizin-induced conformational changes in the membrane proteins are discussed.

Anilino Naphthalenesulfonates↗
PubMed1983

Leucine transport coupled to proton movement in membrane vesicles from Chang liver cells.

Leucine transport into membrane vesicles obtained from Chang liver cells was stimulated by an inward H+ gradient. The stimulatory effect of the proton gradient on the rate of leucine uptake (1 min) was inhibited by the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. When the vesicles had been preloaded with a high concentration of KCl, addition of valinomycin stimulated leucine uptake by the vesicles, showing that the leucine transport is dependent on potential gradient. Leucine-coupled H+ accumulation inside the vesicles was confirmed by measuring leucine dependent quenching of the fluorescence of 9-aminoacridine added to medium. These results imply that electrochemical gradient of proton can serve as a driving force for leucine transport across the cell membrane and proton movement is coupled to leucine transport.

Animals↗
PubMed1982

Temperature-induced transitions of porcine intestinal brush border membranes.

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33 degrees C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35 degrees C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35 degrees C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35 degrees C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33 degrees C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.

Animals↗
PubMed1982

Ca2+-induced conformation changes of intestinal brush border membranes. Studies with fluorescence probes and sulfhydryl reagent.

We have applied the fluorescent probes 1-anilino-8-naphthalene sulfonate (ANS) and pyrene, and an SH-directed reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in order to analyze Ca2+-induced conformational changes in porcine intestinal brush border membranes. Addition of Ca2+ to the ANS-membrane complex resulted in enhancement of the fluorescence intensity accompanied by an increase of the affinity of the dye for the membranes, shift of the transition temperature of the ANS fluorescence toward a higher temperature range, and disappearance of the slope in the thermal Perrin plot of the fluorescence polarization. These results suggest that the membrane structure becomes more rigid upon binding of Ca2+ to the membranes. This hypothesis was further confirmed by a marked decrease of the excimer formation efficiency of pyrene in the membranes with increasing Ca2+ concentration. Reaction of the membranes with DTNB suggested that there are at least two classes of SH groups with different pseudo-first-order that constants, 1.48 x 10(-3)s-1 (SHf) and 0.25 x 10(-3)s-1 (SHs) at pH 7.4 in the absence of Ca2+. Of the estimated 52.2 nmol of SH groups per mg protein in the presence of sodium dodecyl sulfate, about 23.8 nmol are reactive with DTNB under the same conditions. The reactivity of SH groups with DTNB is responsive to Ca2+ concentration and the maximum number of reactive SH groups was increased by the addition of Ca2+. Based on these results, the conformational changes of the membrane structure induced by Ca2+ are discussed.

Anilino Naphthalenesulfonates↗
PubMed1980

Change of the reactivity of sulfhydryl groups of the membrane proteins in response to amino acids in Chang liver cells.

Intensity of fluorescence induced by reaction of a SH-directed fluorogenic reagent with whole Chang liver cells or their plasma membrane preparations which were previously treated with 0.5 mM N-ethylmaleimide (NEM) plus 5 mM leucine was enhanced by 30% or more in the presence of 5 mM leucine or valine in assay medium consisting of Tris-Hepes buffer, pH 6.85, compared with the control. The dissociation constant for binding of leucine to the membrane protein(s) was estimated to be approximately 3 mM from the increments of fluorescence intensity with increasing leucine concentration. On the other hand, the amount of [14C]NEM bound to the membrane preparations pretreated with NEM plus leucine was significantly reduced in the presence of leucine in the reaction medium. A decrease of reactivity of SH groups near the binding sites of the membrane proteins and of mobility of the fluorescent adducts on binding leucine was suggested to explain all these results consistently.

Amino Acids↗
PubMed1979

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. IV. Conformational changes in the region containing fluorescence-labeled sulfhydryl group(s) of troponin.

Conformational changes associated with the functional states of the molecule of troponin were studied using SH-direct fluorogenic reagents, N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM) and N-(1-anilinonaphthyl-4) maleimide (ANM). 1. The fluorescence parameters of ANM-troponin, intensity, and polarization, did not change on combining it with tropomyosin alone, but markedly changed when F-actin was further added to the system. 2. The conformation around the dye-labeled sulfhydryl group(s) was shown to be susceptible to Ca2+ in terms of fluorescence intensity of the label, thermal transition of the conformation, and the microenvironment near the label. 3. On addition of Ca2+, the fluorescence characteristics of the two systems, ANM-troponin . tropomyosin and ANM-troponin . tropomyosin . F-actin complexes, were altered in opposite directions. When BIPM was used in place of ANM, similar changes were observed: a simple decrease in the intensity when pCa was decreased from 7.4 to 5.5 in the system without F-actin and a sigmoidal increase in the range from pCa 7 to 6 in the system with F-actin. Heavy meromyosin, when added to the latter complex (the reconstituted thin filaments), made the profile of its Ca2+ concentration dependence of fluorescence similar to that of the former complex. When tropomyosin was labeled in place of troponin, similar results were obtained. The data obtained imply that the Ca2+-induced conformational changes of troponin are markedly modified when detached from actin, and that heavy meromyosin weakens the interaction of the troponin . tropomyosin complex with F-actin.

Actins↗
PubMed1979

Fluorometric studies on conformational changes in tropomyosin associated with depolymerization.

Spectrofluorometric studies on the conformational changes in tropomyosin associated with depolymerization of the molecules were carried out using 1-anilino-8-naphthalene sulfonate (ANS). When ANS-probed tropomyosin was depolymerized to its monomer, the fluorescence intensity markedly increased, with a decrease in fluorescence polarization. On the other hand, the emission maxima of the ANS-tropomyosin complexes of both forms were the same. The temperature dependence of the polarization of the complexes at various KCl concentrations suggested that the segmental motion of a moiety containing the fluorophore was considerably activated by depolymerization of tropomyosin. In the polymerized and oligomeric forms, a thermal transition in the polarization was observed with a transition temperature of 30 degrees C. Titration curves of tropomyosin with ANS showed simple saturation kinetics with both monomer and polymer, and the apparent dissociation constants were estimated to be 9.93 X 10(-5) M (monomer) and 7.43 X 10(-5) M (polymer). On the other hand, the number of the ANS-binding sites increased from 0.5 to 2.0 per tropomyosin monomer on depolymerization of the molecules. Based on these results, the conformational state of tropomyosin in the polymerized form is discussed.

Anilino Naphthalenesulfonates↗
PubMed1976

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. III. Changes in the conformation of tropomyosin associated with functional states.

The local conformational changes in the tropomyosin molecule under various conditions were studied by means of fluorimetry using SH-directed fluorescent dyes, N-(1-anilinonaphthyl-4)maleimide (ANM) and N-(3-pyrene)maleimide (PRM). 1. The fluorescence intensity, polarization and the emmission maximum of ANM-tropomyosin were found to be susceptible to ionic strength, but in different ways. The changes in these parameters suggest that the fluorescence-labeled sulfhydryl group or groups become more buried in a hydrophobic internal region by salt-induced depolymerization of aggregate and by adding F-actin to tropomyosin. 2. Titration of the labeled tropomyosin with F-actin revealed a cooperative nature in ANM labeling and a simple saturation kinetics in PRM labeling. The dissociation constant of F-actin to PRM-tropomyosin was calculated to be 5.8-10(-6) M. 3. Temperature dependence of the fluorescence polarization showed a thermal transition in the conformation of ANM- or PRM-tropomyosin at around 30 degrees C. Flexibility or segmental motion of the region containing the fluorophore was suppressed significantly on adding troponin and markedly on adding F-actin. 4. Measurements of the quantum yield and polarization of the ANM-tropomyosin-F-actin complex suggested that troponin strengthened the binding between the two proteins and that Ca2+ reversed this effect.

Actins↗
PubMedDate not supplied

Structure-toxicity relationship of monoketones: in vitro effects on beta-adrenergic receptor binding and Na(+)-K(+)-ATPase activity in mouse synaptosomes.

The structure-toxicity relationship of monoketones, a class of organic solvents widely used in industry, was investigated with respect to their in vitro effects on synaptosomal membrane proteins. The toxic parameters used were Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase), a well-known marker enzyme often used as a membrane toxicity model, and 3H-dihydroalprenolol (3H-DHA)-labeled beta-adrenergic receptor binding that has been shown to be vulnerable to solvent-induced changes in membrane fluidity. In vitro treatments with 12 kinds of monoketones (carbon chain length from 3-10) dose-dependently inhibited both 3H-DHA binding to mouse synaptosomes and Na(+)-K(+)-ATPase activity. The potency of inhibition (IC50) for both the two parameters was linearly related to n-octanol/water partition coefficient and synaptosome/buffer partition coefficient of the test compounds. Additions of monoketones did not significantly alter the number of 3H-DHA binding sites but markedly decreased their affinity. In each monoketone, the IC50 values for 3H-DHA binding and Na(+)-K(+)-ATPase activity were generally within the same range. The anisotropy of fluorescence probe 1,6-diphenyl-1,3,5-hexatriene-labeled synaptosomal membranes was dose-dependently decreased by the monoketones, implying increased membrane fluidity. These results indicate that increasing lipophilicity of monoketones results in increased solvent penetration of synaptic membrane preparations, leading to conformational changes in membrane structure and increased ability to inhibit both neuroreceptor binding and enzyme activity. The present data confirm the importance of the lipid micro-environment of membranes in maintaining the normal functions of membrane-bound proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗