Yoshikazu Tahara

A new method to measure bilayer thickness: Cryo-electron microscopy of frozen hydrated liposomes and image simulation

A new method to measure the bilayer thickness of liposomes is described. Frozen hydrated liposomes composed of either phosphatidylcholine (PC) or PC containing 30 mol% cholesterol were observed in vitreous ice with a cryo-electron microscope, which enables high-resolution observation of frozen-hydrated unstained biological specimens. The examined PCs were di-(saturated acyl) PCs (di-C12:0 (DL), diC14:0 (DM), di-C16:0 (DP)), unsaturated acyl PCs (diC18:1 (DO), C16:0-C18:1 (PO)) and PC from a natural source (egg-yolk (EY) PC). Every liposome image displayed a pair of concentric circles indicating a different bilayer structure due to the acyl-chain species. The observation of the liposomes showed the following results; (1) liposomes including unsaturated acyl PC (DOPC, POPC and EYPC) displayed a smooth and regular image of the bilayer and sharp distributions of the bilayer thickness, while the images of those composed of di-(saturated acyl) PC (DLPC, DMPC and DPPC) showed irregular shapes and wide distributions of the bilayer thickness, (2) addition of 30 mol% cholesterol to the di-(saturated acyl) PCs dispelled the irregularity in the shape of the membrane and the distribution of the bilayer thickness, and (3) the mean bilayer thickness of the liposome composed of di-(saturated acyl) PCs increased corresponding to the carbon numbers of the acyl chains. The reliability of the observations and measurements was confirmed by coupling of the cryo-electron microscopy with image simulation using the MULTI SLICE computer program and the error estimation of +/- 0.63 A using images of crystalline chlorinated copper phthalocyanine.

A pH induced two-dimensional crystal of membrane-bound Na+,K+-ATPase of dog kidney

Two‐dimensional crystals of membrane‐bound Na+,K+‐ATPase were formed in acidic media and their qualities were investigated by electron cryo‐microscopy as well as by conventional electron microscopy. At pH 4.8 in sodium citrate buffer, the best crystallization condition, more than 80% of membranes formed crystals. The high ratio allowed high‐resolution images to be taken by electron cryo‐microscopy. Image processing revealed that they had unique lattice constants (a = 108.7 Å, b = 66.2 Å, γ = 104.2°) and had few defects in the crystalline arrays. The reconstituted Fourier map of the ice‐embedded crystal showed that there are two high contrast parts in one unit cell.

Isolation of Stable (αβ)4-Tetraprotomer from Na+/K+-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

Previously, it was demonstrated that acetate anions increase the higher oligomer (H), consuming (alphabeta) 2-diprotomer (D) and alphabeta-protomer (P) of solubilized dog kidney Na (+)/K (+)-ATPase [ Kobayashi, T. et al. (2007) J. Biochem. 142, 157-173 ]. Presently, short-chain fatty acids, such as propionate (Prop) and butyrate, have been substituted effectively for acetate. The molecular weight of 6.01 x 10 (5) for H and quantitative Na (+)/K (+)-dependent interconversion among H, D, and P showed that H was an (alphabeta) 4-tetraprotomer (T). T was optimally isolated from the enzyme solubilized in aqueous 40 mM K (+)Prop at pH 5.6 by gel chromatography performed at 0 degrees C with elution buffer containing synthetic dioleoyl phosphatidylserine (PS). K 0.5 values of K (+)-congeners constituting K (+)Prop for the maximal amount of T were NH 4 (+) >> Rb (+) congruent with K (+) > Tl (+), while Na (+) had no effect. The oligomers of T, D, and P were simultaneously assayed for ATPase upon elution from the gel column, resulting in a specific activity ratio of 1:2:2. The activity of the chromatographically isolated T increased with an increasing dioleoyl PS, giving a saturated activity of 2.38 units/mg at pH 5.6 and 25 degrees C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 degrees C. On the basis of these data, the specific ATPase activities of T, D, and P were concluded to be 32, 65, and 65 units/mg, respectively, under the conventionally optimal conditions of pH 7.3 and 37 degrees C, suggesting an equivalence to a fully active enzyme for D and P but half activity for T. The physiological significance of the stable form of T remains to be investigated.

Epigallocatechin-3-gallate is an inhibitor of Na+,K+-ATPase by favoring the E1 conformation

Four catechins, epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate, and epicatechin, inhibited activity of the Na(+),K(+)-ATPase. The two galloyl-type catechins were more potent inhibitors, with IC(50) values of about 1 microM, than were the other two catechins. Inhibition by epigallocatechin-3-gallate was noncompetitive with respect to ATP. Epigallocatechin-3-gallate reduced the affinity of vanadate, shifted the equilibrium of E1P and E2P toward E(1)P, and reduced the rate of the E1P to E2P transition. Epigallocatechin-3-gallate potently inhibited membrane-embedded P-type ATPases (gastric H+, K(+)-ATPase and sarcoplasmic reticulum Ca(2+)-ATPase) as well as the Na(+),K(+)-ATPase, whereas soluble ATPases (bacterial F(1)-ATPase and myosin ATPase) were weakly inhibited. Solubilization of the Na(+),K(+)-ATPase with a nonionic detergent reduced sensitivity to epigallocatechin-3-gallate with an elevation of IC50 to 10 microM. These results suggest that epigallocatechin-3-gallate exerts its inhibitory effect through interaction with plasma membrane phospholipid.

A method for observing cross-sectional views of biomembranes.

Cross-sectional views of intact biomembranes and synthetic lipid bilayer membranes were observed by electron cryomicroscopy using spontaneous orientation of disk-shaped membranes; purple membrane, thylakoid membrane, synthetic phospholipid membrane and a microcrystalline sheet made of Er-binding polypeptide were observed. The membranes were observed fully hydrated, embedded in vitreous ice, and their self-orientation was most probably caused by repulsion between their hydrophobic edges and their hydrophilic environment which forced their edges to be exposed to the air-water interface. The cross-sectional profiles of the native biomembranes were asymmetric and characteristic, whilst those of the synthetic membranes were symmetric and predictable by a simple model. Simple Fourier analysis showed that the cross-sectional images retained structural information up to a medium resolution.

Fusion of dioleoylphosphatidylcholine vesicles induced by an amphiphilic cationic peptide and oligophosphates at neutral pH.

Peptide E5 is an analogue of the fusion peptide of influenza virus hemagglutinin and K5 is a cationic peptide which has an arrangement of electric charges complementary to that of E5. We reported that a stoichiometric mixture of E5 and K5 caused fusion of large unilamellar vesicles (LUV) of neutral phospholipids (Murata, M., Kagiwada, S., Takahashi, S. and Ohnishi, S. (1991) J. Biol. Chem. 266, 14353-14358). K5 caused fusion of LUV composed of dioleoylphosphatidylcholine (DOPC) at pH > 10, but not at neutral pH. In the presence of oligophosphates, such as 1 mM ATP, GTP, or polyphosphate, K5 caused rapid and efficient fusion of DOPC LUV at neutral pH without hydrolysis of oligophosphate groups, but another anions such as citrate, acetate, AMP, phosphate, or EDTA were ineffective. The peptide/oligophosphate-induced fusion behaviors have been investigated by a fluorescence resonance energy transfer assay for lipid mixing of LUV and negative staining electron microscopy. At higher ionic strengths ( > 0.3 M KCl) or in the presence of 5.0 mM MgCl2, the fusion was inhibited. Even at the inhibitory conditions, the association of K5 with lipid vesicles at neutral pH was directly confirmed by the Ficoll gradient assay method and by blue shifts of the tryptophan fluorescence of the peptide. A nonhydrolyzable GTP analogue, GTP gamma S, also induced fusion. These observations suggested that the electrostatic interactions between the positive and negative charges of K5 and oligophosphate, respectively, induced complex formation, triggering membrane fusion.