The molecular motion of bacteriorhodopsin mutant D96N in the purple membrane.

We measured the flash-induced absorption anisotropies of mutant bacteriorhodopsin (bR), D96N, in the purple membrane suspension. The measured anisotropy decay at 410 nm differed from that at 570 nm. These wavelength-dependent anisotropies show that the motion of absorption dipole of non-excited bR is faster than that of M-intermediate. The motion of non-excited bR is considered as the rotational motion of whole protein in the purple membrane. This fact suggests that the photo-excitation induces the conformational change of the protein and/or the inter-protein interaction within the membrane, which prevents the motion of M-intermediate.

Dynamics of the bilayer-water interface of phospholipid vesicles and the effect of cholesterol: a picosecond fluorescence anisotropy study.

The motion of the head group of phospholipid molecules in the bilayer structure was investigated by a picosecond fluorescence anisotropy technique using a newly synthesized fluorescent phospholipid, dipalmitoyl-L-alpha-phosphatidyl-(3-p-methoxyphenyl)umbelliferone (DPPU). In this phospholipid, a coumarin derivative is attached covalently to the phosphate moiety. The motion of the acyl chain of the phospholipid was also investigated by the same method using 1-palmitoyl-2-(3-diphenylhexatrienyl)-propanoyl-L-alpha-phospha tid ylcholine (DPHpPC). From fluorescence anisotropy decay the wobbling diffusion rate (Dw) of DPPU and DPHpPC in DPPC vesicles at 45 degrees C was calculated to be 2.7 x 10(9) s-1 and 5.1 x 10(7) s-1 using the wobbling-in-cone-model. The range of the motion was calculated as the cone angle (theta c), which is half of the angle of the cone in which the fluorophore can diffuse. The cone angle of the coumarin skeleton of DPPU in DPPC vesicles at 45 degrees C was 64 degrees, which was larger than that of the DPH skeleton of DPHpPC, 40 degrees. These results indicate that the motion of the head group is much faster and wider than that of the acyl chain. When cholesterol was added to the DPPC vesicles, the range of motion of the acyl chain decreased, but that of the head group increased. These facts show that cholesterol restricts the motion of the acyl chain but enhances that of the head group in the phospholipid bilayer.

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.

Adrenoceptor coupling mechanisms which regulate salivary secretion during aging.

In parotid slices and membranes from Wistar rats 2, 12 and 24 months old, changes are noted in adrenoceptor-stimulated K+ fluxes, formation of [3H]inositol phosphates ([3H]IPs), cAMP production, and membrane environment. Norepinephrine-stimulated K+ efflux and formation of [3H]IPs in the slices proceed through an alpha 1-adrenergic mechanism and are reduced 20% and 40% during aging, respectively. In beta-adrenoceptor stimulation with isoproterenol, no age changes were observed in K+ influx and cAMP production. The cholesterol content in membranes was reduced with age; concomitantly, the membrane viscosity decreased with age. These results indicate that the alterations in the membrane environment may provide age-dependent modulation of alpha 1-adrenoceptor coupling mechanisms and their functions.

Fluidity of glycerol skeletal region in phospholipid bilayers: a time-resolved fluorescence depolarization study.

The fluidity of glycerol skeletal region in phospholipid bilayer was investigated by the time-resolved fluorescence depolarization technique with L-alpha-dihexadecanoyl-sn-glycero-3-phospho-[N-(4-nitrobenzo-2-oxa-1,3- diazole)]ethanolamine (NBD-PE) as a fluorescent probe. In this probe, the fluorescent moiety, 4-nitrobenz-2-oxa-1,3-diazole (NBD), is attached to a nitrogen atom at the polar head group of phosphatidylethanolamine molecule. When this probe is embedded in a lipid bilayer, the NBD moiety locates near the glycerol skeletal region. The time courses of fluorescence anisotropy of NBD-PE in dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) bilayers were analyzed using a wobbling-in-cone model, in which the molecular motion is characterized by a half cone angle (theta c) and a wobbling diffusion rate (Dw). Values of Dw of NBD moiety in phospholipid bilayers were found to be on the order of 10(7) s-1 at the physiological temperatures, which is almost the same value as that of the hydrocarbon chain in lipid bilayers. This fact indicates that the fluidity in the glycerol skeletal region is similar to that in the hydrocarbon layer.

Microdynamics of outer and inner membranes of mitochondria from bullfrog myocardium.

Outer and inner mitochondrial membranes were separated from bullfrog myocardium. Membrane viscosity and wobbling angle of phospholipids were measured with a nanosecond time-resolved fluorometer using a fluorophore, DPH, in each membrane. Measurements were also made on liposomes prepared from lipids extracted from each membrane. The anisotropy decay curve for DPH fluorescence was assumed to represent a mean value of decays in several microenvironments in membranes. Phospholipid constituents in membranes were analyzed by HPLC. A high proportion of PE and CL, both of which contain large amounts of unsaturated acyl chain, were found in the inner membrane. The low viscosity and large wobbling angle of phospholipids in the liposome from the inner membrane were consistent with the probable high content of unsaturated acyl chains and the low content of cholesterol in the inner membrane. Measurements of the dynamic microstructure of mitochondrial membranes suggested multifactorial characteristics probably resulting from the lipid-protein interactions. The average viscosity was found to be 0.39 +/- 0.08 P in the outer membrane and 0.58 +/- 0.01 P in the inner membrane. The wobbling angle of phospholipids in the outer and the inner membrane was, respectively, 47 and 49 degrees (non-significant difference). The liposomes prepared from lipid extracts of the membranes showed a lower viscosity and/or higher wobbling angle of phospholipids compared with the membranes themselves. The difference in the viscosity and wobbling angle between the mitochondrial membrane and its respective liposome was large in the inner membrane. These results suggest that the motion of phospholipids is limited by membrane proteins and the limitation of molecular motion of phospholipids results in an increase in the average viscosity. The results also suggest that the microdynamic values obtained in the inner membrane fraction reflect the interaction between phospholipid and protein which is abundant in the inner membrane.

Effects of Ca2+ and Mg2+ on dynamics of the polar head group of phosphatidylserine bilayers.

The effects of Ca2+ and Mg2+ on the molecular motion of the polar head group in phosphatidylserine (PS) bilayers were measured by the time-resolved fluorescence depolarization method probed by 1,2-dihexadecanoyl-sn-glycero-3-phospho-[N-(4-nitrobenzo- 2-oxa-1,3-diazole)]ethanolamine [formula: see text] (NBD-PE). By this method, the rate and width of the molecular motion at the fluorescent moieties in the probe molecules could be evaluated as the wobbling diffusion rate (Dw, s-1) and the half cone angle of the wobbling cone (theta c, degree). The values of Dw and theta c measured for NBD-PE embedded in bovine brain phosphatidylserine bilayers were 3.7 x 10(7) s-1 and 46 degrees in the absence of divalent cations at 25 degrees C. When 3 mM of Ca2+ was added, both Dw and theta c distinctly dropped to 1.7 x 10(7) s-1 and 38 degrees, respectively. By the addition of 3 mM of Mg2+, however, only Dw decreased to 2.7 x 10(7) s-1 and theta c remained unchanged. These results show that both Ca2+ and Mg2+ decrease the rate of motion at the head part in PS molecules, but only Ca2+ narrows the distance between the neighboring head groups. Since Mg2+ does not promote vesicle fusion, it appears that the deformation at the head group region in the bilayer structure induced by Ca2+ is an important step in the membrane fusion process.

Effects of temperature and transfer from seawater to freshwater on blood microrheology in Pacific salmon.

Blood of Pacific salmon was studied with particular interest in red blood cell (RBC) deformability in relation to migration. Blood samples were taken via cardiac puncture or chronic cannula placed in the dorsal aorta and heparinized. As an index of RBC deformability the mean passage time of single RBCs through micropores of 8 micron in diameter and 10 micron in length was determined under a pressure difference of 10 cmH2O. Despite about 100 mOsmol/l difference in plasma osmolality, there was no marked difference in RBC passage time between fish in seawater and those well acclimatized to freshwater. However, it seemed probable that a transient decrease in RBC passage time, i.e., an increase in RBC deformability, occurred immediately following transfer from seawater to freshwater. Plasma osmolality decreased to about 300 mOsmol/l within 1 hr after the transfer and showed no fluctuations thereafter. The temperature dependence of RBC deformability was much smaller in comparison with those previously observed in yellowtail and carp; salmon RBCs were still highly deformable even at 5 degrees C, a possible temperature of cold river water.

Dynamic microstructure of plasma and mitochondrial membranes from bullfrog myocardium--a nanosecond time-resolved fluorometric study.

The viscosity of the phospholipid bilayer and the wobbling angle of the phospholipid molecules of mitochondrial membranes and plasma membranes from bullfrog myocardium were measured with a nanosecond time-resolved fluorometer using pulsed excitation of a hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). The mean +/- S.D. in the viscosity of the mitochondrial and plasma membranes was 0.54 +/- 0.9 and 0.37 +/- 0.03 P, respectively, at 30 degrees C. The wobbling angle of phospholipid molecules was 42 +/- 1 and 47 +/- 1 degree, respectively. Cholesterol content was lower in mitochondria (6.4 micrograms/mg protein) than in plasma membranes (43.7 micrograms/mg protein) but phosphatidylethanolamine concentration was higher in mitochondria (31.8%) than in plasma membranes (27.3%). Cardiolipin was contained only in mitochondria. The results of these lipid analyses appear consistent with the measurements of membrane viscosity and phospholipid wobbling angle. When the results are compared with those from a previous study on the erythrocyte membranes from bullfrogs, viscosity is found to increase in the order mitochondrial membranes less than plasma membranes less than erythrocyte membranes. The complex requirements of biomembranes of organelles performing different functions appear to be met by the particular dynamic microstructure of the biomembrane. The effect of membrane viscosity on oxygen diffusion through membranes is discussed.

Dynamic microstructure and hydration of peroxidized membrane of rat cardiac mitochondria and effects of adriamycin.

Nanosecond time-resolved fluorometry of diphenyl hexatriene, DPH, fluorescence was used to study the effects of lipid peroxidation caused by NADH or adriamycin treatment on the dynamic microstructure of mitochondrial membranes from rat myocardium. Isolated mitochondria were incubated with NADH, FeCl3, and ADP, or with adriamycin. Parameters for microdynamics were calculated from the fluorescence intensity and anisotropy decay curves for DPH fluorescence. Peroxidized lipids were measured as malondialdehyde (MDA) resulting from the thiobarbiturate reaction. As peroxidized lipids accumulated, the membrane viscosity increased and the wobbling angle of the phospholipids decreased. The structural changes induced in unsaturated phospholipids by peroxidation probably increased the friction of neighboring phospholipids and restricted the range of their wobbling motion. The fluorescence intensity and fluorescence lifetimes decreased significantly when MDA was higher than 10 nmol/mg protein. These alterations in the behavior of DPH fluorescence strongly suggest that the hydration of the phospholipid layer of the mitochondria is occurring as a consequence of lipid peroxidation, since the fluorophore, DPH, is hydrophobic and its fluorescence is known to be quenched by increasing the dielectric constant of the surrounding media. The present results provide experimental supports to the hypothesis of membrane hydration induced by lipid peroxidation.

Dynamic structure of phospholipid bilayers on the path for oxygen diffusion in the ox lung.

The dynamic properties of the phospholipid bilayer of cell membranes were studied with a nanosecond fluorometer to obtain information on the microstructure of the path for oxygen diffusion in the ox lung. The viscosity in membranes of pneumocytes, endothelial cells from pulmonary artery, and erythrocytes was 47, 54 and 162 mPa . sec respectively. The wobbling angle representing the oscillation of phospholipid molecules was 47, 44 and 38 degrees, respectively.

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.

Oxygen diffusion coefficient of cell membranes.

The microviscosity was measured in erythrocyte membranes of different animal species and in lung cells and myocytes of bull frogs by means of the nanosecond fluorescence depolarization technique with a rod-like fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). The diffusion coefficient of oxygen molecules (DO2) was estimated from the microviscosity and an equation for the relation between viscosity and diffusion coefficient. The DO2 value of erythrocyte ghosts was remarkably different among species; 0.84 in sheep, 1.29 in human, 1.49 in rat and 1.92 X 10(-7) cm2/sec in rabbit, while the wobbling diameter Wd was 10.7, 11.5, 12.1 and 10.7A in respective species. The DO2 value in frogs was different among organs; 1.07 in erythrocyte ghosts, 2.18 in lung cells, 2.64 X 10(-7) cm2/sec in myocytes. The Wd value was 12.2, 12.6 and 13.4A in respective organs at 30 C. A comparison between sizes of moving area of phospholipid molecules in membrane and in ideal fluid state suggested that the actual DO2 values in cell membranes may probably be ten times larger than the present values.

Oxygen diffusion through mitochondrial membranes.

The effect of the mitochondrial membrane on the oxygen supply to the interior of mitochondria was analyzed with a cylinder model of diffusion. This estimation is based on the assumption that cytochrome a,a3 is distributed only on the inner surface of the mitochondrial inner membrane. The diffusion coefficient in the mitochondrial membrane was approximated from the fluorescently-determined viscosity of rat mitochondrial membrane. A pico-second time-resolved fluorometer at 37 degrees C gave values of 43.8 cp for intact mitochondria and 51.4 cp after phospholipase A2 treatment. Using the mean oxygen consumption rate of 10 ml O2/100 g tissue/sec in beating heart, oxygen gradients of 3.9 and 4.6 nmol was predicted across the intact and phospholipase-A2 treated mitochondrial membranes, respectively. The increased oxygen consumption during systole will yield oxygen gradients of 11.6 and 13.7 nmol. These gradients were much larger than the values estimated in a hypothetical case using the diffusion coefficient for the mitochondrial membrane of 1.5 x 10(-5) cm2/sec. The predicted oxygen gradient suggests a non-uniform distribution of oxygen in the myocardial cell and may be of importance in understanding the relationship between oxygen supply and myocardial function in hypoxia. Phospholipase A2, which is known to be activated in ischemia, destroys the microstructure of myocardial cells, seems deleterious to oxygen transport to cytochrome a,a3.

Hypoxic reduction in blood flow velocity in pulmonary arterioles and capillaries.

A small ring chamber (I.D. = 6 mm) was placed on the exposed lung of anaesthetized bullfrogs. A localized hypoxia was induced in the ring chamber by introducing nitrogen in it. Blood flow velocity in pulmonary microvessels was measured by means of a laser Doppler microscope. The mean blood flow velocity was 1.98 +/- 0.45 and 1.52 +/- 0.10 mm/sec during the control condition in arterioles and capillaries, respectively. It was then reduced by the localized hypoxia to 1.63 +/- 0.32 and 1.33 +/- 0.08 mm/sec in arterioles and capillaries, respectively. The reduction, when expressed in the percentage ratio to the control flow velocity in each blood vessel group, was significantly larger in arterioles than in capillaries. A phase delay in the pulsation of the flow velocity contour was detected only in arterioles. These differences between pulmonary arterioles and capillaries in response to the localized hypoxia may be attributed to the dense interconnection of capillary network extending beyond the localized hypoxic area to the normoxic area.

Biosynthesis of B2-integrin, intracellular calcium signalling and functional responses of normal and CD18-deficient bovine neutrophils.

1Biosynthesis of CD11/CD18 in bovine leucocytes, intracellular Ca2+ ([Ca2+]i) signalling, chemiluminescent responses and membrane fluidity of neutrophils and the effects of D-mannose on neutrophils from control heifers and a heifer with bovine leucocyte adhesion deficiency (BLAD) were measured. The synthesis of CD11/CD18 complex was clearly detected in leucocytes from a normal heifer, but not in a BLAD-affected heifer. The transient phase of increased [Ca2+]i was clearly detected in neutrophils from a heifer with BLAD stimulated with opsonised zymosan, aggregated bovine immunoglobulin G or concanavalin A, whereas the sustained phase was deficient or significantly decreased compared with control heifers. [Ca2+]i signalling of neutrophils from control heifers and a heifer with BLAD stimulated with phorbol myristate acetate via an 11b/CD18-independent pathway showed no transient phase, and the subsequent increase in [Ca2+]i was almost identical in neutrophils from affected and control heifers. [Ca2+]i concentration and chemiluminescent responses of neutrophils from a control heifer were clearly decreased by treatment with anti-CD18 and anti-IgG antibodies. No differences in membrane fluidity were detected between neutrophils derived from control and CD18-deficient cattle. D-mannose binds mainly to Fc rather than CD18 receptors, and decreased Agg-IgG induced [Ca2+]i and the chemiluminescent response of neutrophils. The [Ca2+]i responses and Agg-IgG induced chemiluminescent responses of neutrophils from control heifers and a BLAD-affected heifer were inhibited by D-mannose. The characteristic changes of [Ca2+]i signalling and functional responses of B2-integrin-deficient neutrophils were demonstrated.

A new analysis method for the membrane viscosity from steady-state fluorescence depolarization.

The absolute value of the viscosity in membrane lipid bilayers, which is different from the microviscosity advocated by Shinitzky, could be calculated from steady-state fluorescence depolarization of a hydrocarbon fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). This method was based on the theory of time-resolved fluorescence anisotropy and empirical relationships between fluorescence life time and the anisotropy parameters such as half cone angle in wobbling motion and wobbling diffusion rate of the fluorescent probe. Obtained viscosity values of various membranes from this method were consistent with those from time resolved method within experimental error.

Dynamics of membrane structure of frog erythrocyte ghosts measured with a nanosecond fluorometer.

The dynamics of membrane microstructure was studied as molecular motions of phospholipids for bullfrog erythrocyte ghosts by the DPH fluorescence depolarization technique with a nanosecond fluorometer. The bullfrog erythrocyte ghosts were obtained by hypotonic lysis and collagenase treatment. The constituents of membrane proteins were confirmed by the disk gel electrophoresis. The viscosity of erythrocyte membrane ghosts was estimated to be 3.3 +/- 1.0 at 10 degrees C, and 2.1 +/- 0.1 at 20 degrees C and 1.3 +/- 0.2 at 30 degrees C in the unit of poise and the wobbling angle of lipid molecule was 35 +/- 1, 41 +/- 1 and 43 +/- 1 degree at the respective temperatures on an average and +/- S.D. The viscosity is lower than that of human erythrocytes. The relatively low viscous phospholipid bilayer may be one of the factors for the deformability of bullfrog erythrocytes.

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.

Analysis for the molecular motion of phospholipid bilayer with picosecond fluorometry.

The viscosity and the molecular motion of phospholipid molecule in biological and artificial phospholipid bilayers were studied using picosecond fluorescence depolarization method with rod-like fluorophore, DPH. From the relationship between the viscosity in the lipid bilayer and the free space of phospholipid acyl-chain, it is concluded that the viscosity is determined mainly by the range of wobbling motion of the acyl-chain. Motion of polar head group was also measured by the same method with a newly synthesized fluorescent phospholipid, dipalmitoyl-phosphatidyl-umbelliferone. The rate and the range in the motion of head group were faster and larger than those of acyl-chain and gave the viscosity of head group layer to be 0.03 poise, which was about one tenth of that of acyl-chain layer in the liquid crystalline phase. This fact indicates that the head group layer would not resist the lateral diffusion of molecules in membrane and that the lateral diffusion rate of molecules could be estimated from the viscosity in the acyl-chain layer.