Tsunehisa Araiso

Membrane Viscosity Correlates with α1-Adrenergic Signal Transduction of the Aged Rat Cerebral Cortex

Abstract: We investigated, using adult (2‐month‐old) and senescent (12‐ and 24‐month‐old) rats, the effects of aging on the relationship between the α1‐adrenergic coupling system and the membrane viscosity of the cerebral cortex. There was no age‐related difference in the KD values of [3H]prazosin binding on the membranes. The Bmax values of [3H]prazosin binding were reduced with advanced age. Norepinephrine induced formation of 3H‐labeled inositol phosphates (3H‐IPs) in the slices increased with advanced age. The EC50 values for norepinephrine to stimulate the formation of 3H‐IPs at advanced age were lower than that at adult age. The cholesterol content in membranes increased with advanced age. No changes in the phospholipid content in membranes were observed with advanced age. Concomitantly, an increase of the molar ratio of cholesterol to phospholipids was observed with advanced age. The membrane viscosity as measured by 1,6 diphenyl‐1,3,5‐hexatriene increased with advanced age. These results indicate that the altered cholesterol content and/or viscosity in cortical membranes of the aged rat may account for the loss of α1‐adrenergic receptor density and/or compensatory changes in the receptor‐phospholipase C coupling system.

AZIDE ACCELERATES THE DECAY OF M-INTERMEDIATE OF PHARAONIS PHOBORHODOPSIN

Natronobacterium pharaonis has retinal proteins, one of which is pharaonis phoborhodopsin, abbreviated as ppR (or called pharaonis sensory rhodopsin II, psR-II). This pigment protein functions as a photoreceptor of the negative phototaxis of this bacterium. On photoexcitation ppR undergoes photocycling; the photoexcited state relaxes in the dark and returns to the original state via several intermediates. The photocycle of ppR resembles that of bR except in wavelengths and rate. The cycle of bR is completed in 10 ms while that of ppR takes seconds. The Arrhenius analysis of M-intermediate (ppR(M)) decay which is rate-limiting revealed that the slow decay is due to the large negative activation entropy of ppR. The addition of azide increases the decay rate 300-fold (at pH 7); Arrhenius analysis revealed decreases in the activation energy (activation enthalpy) and a further decrease in the activation entropy.

Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of lambda(max) upon Cl(-) addition, from which the dissociation constants of Cl(-) were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degrees C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding.

Viscosity and order in erythrocyte membranes studied with nanosecond fluorometry

The viscosity and the order in the interior of human erythrocyte membranes were investigated by the fluorescence depolarization technique in the nanosecond region with 1,6-diphenyl-1,3,5-hexatriene (DPH). After pulsed excitation with a polarized light, the fluorescence anisotropy ratio of DPH in membranes rapidly decreased and gave a final value (r infinity). The rate of initial decrease and the value of r infinity related to the viscosity in the interior of the membranes and a wobbling angle of DPH which reflects a size of range for the phospholipid motion relating to the order of membrane structure. For normal human erythrocyte membranes the viscosity and the wobbling angle were obtained to be 0.82 poise and 42 degrees, at 37 degrees C. Similar values were obtained for spectrin-free membranes. Hardened membranes by the cross-linking of the cytoskeletal proteins with glutaraldehyde showed a small wobbling angle of 37 degrees, but the viscosity of them was unchanged.

Effect of far-infrared light irradiation on water as observed by X-ray diffraction measurements

X-ray diffraction measurements were made on water irradiated with far-infrared (FIR) light. It was found that the X-ray diffraction intensity at a 2θ angle of 30° increased by about 2 times with the irradiation. The increase in the X-ray diffraction intensity was interpreted in terms of the destruction of water clusters.

Change of Motion and Localization of Cholesterol Molecule during Lα-HII Transition

Abstract Formation of the inverted hexagonal (H II ) phase from the lamellar (L α ) phase of bovine brain-extracted phosphatidylcholine (BBPC) and phosphatidylethanolamine (BBPE) was investigated using 31 P-NMR with or without cholesterol. When the ratio of BBPC to BBPE was 1:1, the H II formation was observed in the presence of 33mol% cholesterol (i.e., BBPC:BBPE:cholesterol=1:1:1) at 47°C. The fraction of the H II phase in the BBPC/BBPE/cholesterol system could be controlled by the addition of dioleoylglycerol. The change of molecular motion of cholesterol affected by the H II formation was measured at various ratios of the L α to H II phase with the time-resolved fluorescence depolarization method, using dehydroergosterol as a fluorescent probe. It is observed that the motion of cholesterol became vigorous in the mixture state of the L α and the H II phases compared to that in the L α or the H II phase only. These facts show that cholesterol has the strong ability to induce the H II phase, probably by special molecular motion, which includes change of its location from the headgroup area to the acyl-chain area.

Changes in lipid mobility associated with alamethicin incorporation into membranes.

The binding state of the antibiotic peptide alamethicin with phospholipid bilayers was investigated in terms of the changes induced in lipid mobility. Fluorescence anisotropy was used for the study. It was found that an increase in peptide concentration induced different changes in lipid mobility above and below a critical peptide concentration. This concentration was also critical for an increase in the cooperative binding of the peptide, as detected by circular dichroism. Above the critical peptide concentration, the mobility of both lipid regions, around the polar head and hydrocarbon chain, became restricted with an increased peptide concentration. Below the critical level, however, an increased peptide concentration induced a wobbling of the lipid hydrocarbon chain. These results show that an increase in the cooperative binding of the peptide is accompanied by a change in the dominant configuration of the binding peptide. When the binding peptide increases, the dominant configuration appears to shift from surface association to deep incorporation within the membrane. This shift in configuration means that in the formation of ion-conductive pores, voltage-driven insertion of the peptide is a prominent step below a critical peptide concentration.

Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol

The molecular motion of retinal within the purple membrane was investigated by flash-induced absorption anisotropies with or without ethanol. In the absence of ethanol, the measured anisotropies at several wavelengths exhibited almost the same slow decay. This slow decay was attributed to only the rotation of purple membrane sheet itself in the aqueous suspension. In the presence of ethanol, however, we observed the wavelength-dependent anisotropies. The fluidity of the purple membrane, investigated with a fluorescence anisotropy method, was increased by the addition of ethanol. These facts indicated that the characteristic motion of bacteriorhodopsin is induced in perturbed purple membrane with ethanol. The data analysis was performed, taking account of the overlapping of absorption from ground-state bacteriorhodopsin and photointermediates. The results showed that the rotational motion of photointermediates within the membrane was more restricted than that of nonexcited bacteriorhodopsin. The addition of ethanol facilitated the rotation of nonexcited protein, whereas it did not significantly affect the motion of photointermediates. The restricted motion of photointermediates is probably caused by a conformational change in them, which may hinder the rotation of monomer protein and/or induce the interaction between photointermediate and neighboring proteins.

Fluidity and osmotic sensitivity changes of phospholipase A2-treated liposomes.

Membrane fluidity and osmotic sensitivity were examined in DPPC liposomes treated with phospholipase A2 (PL.A2) in the presence of Ca2+ or Mg2+. The amount of liposome phospholipid hydrolyzed differed with the two ions. Embedded DPH, a rod-like fluorescent probe, was employed in the determination of membrane fluidity. Membrane fluidity decreased according to the degree of phospholipid hydrolization in liposomes by PL.A2. The reciprocal value of absorption at 450 nm was measured as the index of osmotic sensitivity of liposomes. Intact sonicated liposomes showed osmotic insensitivity. PL.A2-treated liposomes in which about 40% of total phospholipid was hydrolyzed showed osmotic sensitivity. No change in the membrane fluidity was obtained when PL.A2-treated liposomes were exposed to hypertonic or hypotonic solution. These results suggested that the motion of the acyl-chain of phospholipids and free fatty acids was resisted in PL.A2-treated liposomes. The resistance may be due to a phase separation between phospholipids and free fatty acids. The pore for water permeation might be induced in the border between phase-separated domains in PL.A2-treated liposomes.

Diffusion Pathway of Oxygen in OX Lung

The diffusion coefficients of cell membranes of pneumocytes plus endothelial cells, cytosol plus blood plasma, erythrocyte membranes, and hemoglobin solution in erythrocytes were estimated from the fluorometrically measured membrane viscosity. The time course of oxygen partial pressure distribution was numerically calculated in a model for the pathway of oxygen in the lung. The high viscosity of the phospholipid bilayers seems to cause a reduction in the rate of oxygenation of the hemoglobin solution.