EFFECT OF TRYPTOPHAN RESIDUES ON ADSORPTION OF A CATIONIC ARGININE RICH POLYPEPTIDE BY HYDROXYAPATITE NANOPARTICLES AND FOLLOWING TRANSLOCATION OF THE POLYPEPTIDE THROUGH A LIPID VESICLE MEMBRANE


Formation of Poly-L-lysine - hydroxyapatite Nanoparticle Complexs and Interactions between These Complexes and Lipid Bilayer Membranes


Poly-L-arginine-Hydroxyapatite Nanoparticle Complexes Translocate Through Lipidbilayer Membranes

Certain molecules, which are able to directly translocate across phospholipid bilayer membranes (cell or endosormal membrane), can be useful as carriers (vectors) for drags (especially polymeric drags). We have studied the translocationability of the hydroxyapatite nanoparticle-poly-L-arginine complex through the negatively charged phospholipid bilayer membranes by using several instruments. It was confirmed by means of a confocal laser scanning microscopy (CLSM) not only the fact that the complex can translocate through the membranes but also the fact that the complexes were still retained in the inner water layer of the liposome even after the translocation.


Formation of Poly-L-Arginine - Hydroxyapatite Nanoparticle Complexes and Interactions between Complexes and Lipidbilayer Membranes


Solubility of monoalkyl phosphate in water in the presence of arginine and Triton, and solubilization of methyl yellow through the mixed micelle

モノアルキルリン酸およびヂセチルリン酸は水には殆ど溶解しない．70℃の高温でTriton-XあるいはTriton-Nの存在で前者はやや溶解度が上がるが，後者はそれでも殆ど溶解しないことを指摘した．アルギニンが存在すると室温でも溶解することを確認したのち，モノアルキルリン酸ーTXあるいはTN-アルギニン混合ミセルによるメチルイエロウの可溶化量を測定した．モノアルキルリン酸の溶解機構を検討し，KOHで中和する場合とアルギニンで中和する場合の相違点について考察した．


Effects of ionic surfactants on the adsorption of bovine serum albumin to the surface of hydroxyapatite


Effects of Phosphate Ions on the Polypeptide-Liposome Membrane Interaction


Polyethyleneimine can permeate through a lipid bilayer of a giant liposome

Journal of Japanese Medical Society for Biological Interface


Effect of Dodecyltrimethylammonium Chloride on the Adsorption of Bovine Serum Albumin to the Surface of Hydroxyapatite


Preparation and adsorption properties of hydroxyapatite surface-modified by cetylphosphate

セチル燐酸共存でヒドロキシアパタイトを合成した．その表面は疎水化されており有機化合物を容易に吸着するようになった．


Translocation of positively charged copoly(Lys/Tyr) across phospholipid membranes

Much attention has recently been paid to the study of positively charged polypeptides as a possible carrier for therapeutic protein or DNA delivery to cells. In this study, we have investigated the translocation of positively charged copoly(Lys/Tyr) (MW=72000, DP=385) across lipid membranes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), as well as soybean phospholipids (SBPL) using zeta potential method, circular dichroism spectroscopy (CD), electrophysiology technique, fluorescence spectroscopy, and confocal laser scanning microscopy. Results of zeta potentials show that copoly(Lys/Tyr) associate with lipid membranes and become gradually saturated on the membranes either hydrophobically or electrostatically or both. CD studies demonstrate that the copoly(Lys/Tyr) takes and remains beta-sheet conformation during its interaction with liposome membranes, indicating that the translocation process should be carpet-mode like. Data from the electrophysiology technique reveal that positively charged copoly(Lys/Tyr) can cause transmembrane currents under an applied voltage, confirming its transfer across lipid membranes. Fluorescence spectroscopy results display a three-step mechanism of translocation across membrane: adsorption, transportation, and desorption, which has been verified by results from confocal laser scanning microscopy. We provided the first direct observation that the positively charged polypeptides, copoly(Lys/Tyr), can translocate through SBPL and EPC/DOPE lipid bilayer membranes. In addition, we found that the translocation efficiency of copoly(Lys/Tyr) was higher on the EPC/DOPE lipid membrane than on the SBPL lipid membrane.

Biochemical and Biophysical Research Communications


Local anesthetics facilitate ion transport across lipid planar bilayer membranes under an electric field: Dependence on type of lipid bilayer

In order to elucidate the role of structural change of lipid membrane bilayer in the mode of action of local anesthetic, we studied the effects of local anesthetics, charged tetracaine and uncharged benzocaine, on ion permeability across various lipid planar bilayers (PC, mixed PC/PS (4/1, mol/mol); mixed PC/PE (1/1, mol/mol); mixed PC/SM (4/1, mol/mol)) under a constant applied voltage. The membrane conductances increased in the order of PC<PC/PS<or=PC/SM<PC/PE. When the constant voltage of -100 or -70 mV was applied through the lipid bilayer membranes in the presence of positively charged tetracaine, the fluctuating current pulses with the large amplitude generated, but not appeared in the absence of tetracaine. The addition of uncharged benzocaine generated the fluctuating currents with the small amplitude. Both charged tetracaine and uncharged benzocaine facilitated electrophoretically the transport of small ions such as KCl in the buffer solution through the fluctuating pores in the lipid bilayer membranes formed by interaction with the local anesthetic under the negative applied membrane potential. The current pulses also contained actual transport of charged tetracaine together with the transport of the small ions. The amplitude and the duration time of the electrical current generated by adding the local anesthetics were dependent on the type of the lipid, the applied voltage and its voltage polarity.


Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning alpha-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of "the cation" in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca2+, Mg2+ and Mn2+) and monovalent metal cations (Li+, Na+, K+ and Cs+), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.

Biochimica et Biophysica Acta (BBA) - Biomembranes


Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.


The local anesthetics facilitate the transport of small ions across phospholipid bilayer

局所麻酔薬は小イオンの脂質二分子膜透過を促進する


Ability of ferric nitrilotriacetate complex with three pH-dependent conformations to induce lipid peroxidation.


Enzymatic and non-enzymatic formation of reactive oxygen species from 6-anilino-5,8-quinolineequinone

The nonenzymatic and enzymatic formation of reactive oxygen species (ROS) from LY83583 (6-anilino-5,8-quinolinequinone) was investigated by electron paramagnetic resonance (EPR) spectroscopy. In the presence of thiol compounds such as glutathione and L-cysteine, LY83583 underwent a one-electron reduction due to low redox potential (-0.3+/-0.01 V vs. SCE), followed by formation of LY83583 semiquinone anion radical. This species was characterized by EPR spectroscopy under an argon atmosphere at neutral pH. Under an aerobic condition, this species interacts with molecular oxygen to form a superoxide anion radical. GSH-conjugated LY83583 was also identified by NMR and FAB-MS. When LY83583 was applied to PC12 cells, ROS formation was completely inhibited by both the flavoenzyme inhibitor DPI and the DT-diaphorase inhibitor dicumarol. On the other hand, ROS generation occurred independent of intracellular GSH level. These results indicate that LY83583 can generate ROS both enzymatically and nonenzymatically, although the enzymatic formation is dominant over the nonenzymatic system in PC12 cells.

Biochimica et Biophysica Acta (BBA) - General Subjects


Synthetic copoly(Lys/Phe) and poly(Lys) translocate through lipid bilayer membranes

Several membrane-transporting peptides (MTP) containing basic amino acid residues such as Lys and Arg that carry macromolecules such as DNA and proteins across cell plasma membranes by an unknown mechanism have been actively studied. On the basis of these results, we have been investigating the translocation ability of synthetic polypeptides, copoly(Lys/Phe) and poly(Lys), through negatively charged phospholipid (soybean phospholipid (SBPL)) bilayer membranes by zeta potential analysis, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, an electrophysiology technique, and confocal laser scanning microscopy (CLSM). The binding of these polypeptides to the membrane, which is the first step for translocation across the membrane, resulted in the conformational transition of the polypeptide from a random coil form or helix-poor form to a helix-rich form. The fluorescence studies demonstrated that the time-dependent decrease in the fluorescence intensities of the FITC-labeled polypeptides bound to the SBPL liposome reflected translocation of the polypeptide across the lipid bilayer with the low dielectric constant. Both the rate constant and the efficiency of the polypeptide translocation across the lipid bilayer were greater for copoly(Lys/Phe) than for poly(Lys). These results suggest that the random incorporation of the hydrophobic Phe residue into the positively charged Lys chain results in a lowering of the potential barrier for passage of the polypeptide in the hydrophobic core portion of the lipid bilayer. We presented the first direct observation that the positively charged polypeptides, copoly(Lys/Phe) (MW: 41,500) and poly(Lys) (MW: 23,400), could translocate across the lipid bilayer membrane.


Redox potentials of the oriented film of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsin

The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were -470 mV for the 13-cis configuration of the retinal Shiff base in bR and -757 mV for the all-trans configuration in H(2)O, and -433 mV for the 13-cis configuration and -742 mV for the all-trans configuration in D(2)O. The solvent isotope effect (DeltaV=V(D(2)O)-V(H(2)O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated C=N part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were -507 mV for the 13-cis configuration and -788 mV for the all-trans configuration; and for the E204Q mutant they were -491 mV for the 13-cis configuration and -769 mV for the all-trans configuration. Replacement of the Glu(194) or Glu(204) residues by Gln weakened the electron withdrawing interaction to the protonated C=N bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were -471 mV for the 13-cis configuration and -760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the C=N part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.


Redox potential of bacteriorhodopsin in oriented purple membrane films


Photocurrent of purple membrane adsorbed onto a thin polymer film --- action characteristics of the local anesthetics


膜蛋白質のプロトンポンプ活性に対するアミン形局所麻酔薬の二相性効果

膜蛋白質のプロトンポンプ活性に対するアミン形局所麻酔薬の二相性効果


Alkane derivative-bacteriorhodopsin interaction: proton transport and protein structure

The effects of alkane derivatives, 1-alcohols (ROH), aliphatic amine hydrochlorides (RNH(2).HCl) and sodium aliphatic carboxylates (ROONa), on the proton pumping activity of bacteriorhodopsin (bR) in a purple membrane have been examined. Photocurrents in bR in the purple membrane adsorbed onto polyester thin film were recorded before and after exposure to these test substances. The peak photocurrent in bR was reversibly suppressed by each substance. From the dose-response curve, the concentrations required to reduce the peak capacitive current by 50% were determined for each homolog and then the standard free energies per CH(2) for the adsorption of the alkane derivatives to the site of action were estimated: -3.13 kJ mol(-1) for ROH, -3.05 kJ mol(-1) for RNH(3)(+), and -2.95 kJ mol(-1) for ROO(-). The proton pumping activity of bR was mainly suppressed by the hydrophobic interaction with the additive. The relative potencies of the functional groups of the alkane derivatives were almost comparable between 1-octanol (C(8)OH) and octylamine hydrochloride (C(8)NH(3)(+)) and about 10 times less effective for sodium octanoate (C(8)OO(-)) than for others. The addition of C(8)OH or C(8)OO(-) changed the absorption spectra of bR with a maximum at 560 nm to the spectra of the intermediate state with a maximum at 480 nm, while the C(8)NH(3)(+) decreased the intensity of the 560 nm band only with no blue-shift by the 480 nm band. We conclude that the action of the alkane derivatives is nonspecific and directed to all organized purple membrane structures and that the binding sites of the ROH and ROO(-) are different from that of RNH(3)(+).


A comparative study of the ability of ferric nitrilotriacetate and other iron chelators to assist membrane lipid peroxidation by superoxide radicals

This study examined some of the variables determining the efficiency of lipid peroxidation in egg yolk phosphatidylcholine liposomes and in microsomes exposed to enzymatically-generated superoxide radicals. The initiation of peroxidation required the presence of preformed lipid peroxides and a chelated metal catalyst. Comparison of the relative effectiveness of four iron chelating agents showed that the chelate must bind to the membrane by coulombic attraction between the charged membrane and a chelate carrying an opposite net charge. Of the chelates tested, only the carcinogenic ferric nitrilotriacetate [corrected] (Fe(3+)-NTA) was an effective catalyst of oxidation of all membranes, whether carrying a net charge, or not. We postulate that the unique catalytic capacity of the ferric nitrilotriacetate [corrected] (Fe(3+)-NTA) can be explained by its existence in two forms at neutral pH, each binding to oppositely charged membranes and initiating their peroxidation. This gives the complex the unique ability to bind to any membrane, which may be a factor in its carcinogenicity.


Photocurrent of purple membrane adsorbed onto a thin solid film: effects of monovalent and divalent ions

Studies in Surface Science and Catalysis


膜蛋白質 - 麻酔薬相互作用:界面の水の効果

膜蛋白質 - 麻酔薬相互作用:界面の水の効果


揮発性麻酔薬と紫膜の相互作用:セボフルランの作用特性

揮発性麻酔薬と紫膜の相互作用:セボフルランの作用特性


好塩菌紫膜に対する電荷型および非電荷型テトラカインの作用部位

好塩菌紫膜に対する電荷型および非電荷型テトラカインの作用部位


バクテリオロドプシンのプロトンポンプ阻害:電荷形及び非電荷形三級アミンの効果

バクテリオロドプシンのプロトンポンプ阻害:電荷形及び非電荷形三級アミンの効果


Monolayer of Poly(L-glutamate) with an n-Dodecyl Group in the Side Chain on the Water Surface and Its Multilayers


Redox potential of bacteriorhodopsin in purple membrane determined by differential pulse voltammetry


揮発性麻酔薬と紫膜の相互作用における水の役剤

揮発性麻酔薬と紫膜の相互作用における水の役剤


Effect of water activity on unfolding of adsorbed protein at the interface


Interaction of neutral and charged local anesthetic with purple membrane


Anesthetic-bacteriorhodopsin interaction;alcohol induced biphasic effects


Monovalent cations depress the unfolding of the α-helical bacteriorhodopsin at the air-water interface


Protein surface denaturation and the surface-activated water molecules


Monovalent anions stabilize the structure of bacteriorhodopsin at the air-water interface


Preparation and characterization of liposomes incorporating hydrophobic poly(amino acid)s with different secondary structure


Dispersion polar surface free energies of polypeptide films


A synthetic analog for the active site of plant-type ferrodoxin: two different coordination isomers by a four-cys-containing [20]-peptide.


Polymerization and conformation of N-carboxy anhydrides of a L-glutamic acid derivative in monolayer


Synthesis, crystal structure and electronic properties of a 2Fe-2S complex with a bulky thiolato ligand, [NEt4]2[Fe2S2(SC6H2Me3-2,4,6)4]

Journal of the Chemical Society, Dalton Transactions


Penetration of 1-alkanols into monolayers of α-helical polypeptides with hydrophobic side chains


スーパーオキシド依存症の脂質過酸化を誘導する発癌性鉄錯体Fe-NTA(ニトリロ三酢酸)の特性