Structural changes in bacteriorhodopsin induced by electric impulses

Electric impulses of high field intensity (2 × 10⁵ to 3 × 10⁶ Vm^?1, 1 to 20 μs duration) cause transient changes in the optical absorbance of suspended purple membranes of Halobacterium halobium. The electric dichroism at 1 mm NaCL, pH ≈ 6 and at 293K is dependent on field strength, pulse duration and wavelength of the monitoring, plane-polarized light in the range 400 to 650 nm. The optically detected processes are, however, independent of bacteriorhodopsin concentration, of ionic strenght and of the intensity of the monitoring light. These data together with the analysis of time course ands steady state of the reduced dichroism, suggest electric field-sensitive, intramemembraneous structural changes which lead to restricted orientation changes of the chromophore. A thoretical analysis of restricted orientation is developed and applied to the electro-optic data. As a result, it is found that the electric dichroism of purple membrane is associated with a large polarizability anisotropy of 2.4 × 10^?30 Fm² (2.2 × 10^?14 cm³); the electric permanent dipole moment which is involved amounts to 4.7 × 10^?28 Cm(140 Debye). The kinetic data suggest a cyclic reaction scheme with at least five different conformations. The high polarizability is probably due to displaceable ionic groups within the cooperative lattice of bacteriorhodopsin molecules in purple membranes.     

Electric birefringence study of the purple membrane of Halobacterium halobium

An electric birefringence study was carried out on aqueous suspensions of the purple membrane of Halobacterium halobium. In addition to the characterization of both native and modified membrane samples, the dependence of electric birefringence on pH and ionic strength was also investigated. The results indicate that purple membrane shows electric birefringence at a field strength as low as 200 V/cm. The permanent dipole moment and polarizability ranged from 20,500 debyes and 1.01 × 10^?14 cm³ for a purple membrane concentration of 0.40 mg/mL to 41,000 debyes and 2.05 × 10^?14 cm³ for a concentration of 0.80 mg/mL. It was also found that removal of the retinyl group of bacteriorhodopsin substantially decreases but does not eliminate the electric birefringence of the membrane. The solubilization of the membrane by Triton X-100, however, completely abolishes the electric birefringence. These experiments indicate that there is an interaction between adjacent bacteriorhodopsin molecules within the purple membrane via the retinyl chromophore moiety that builds up the permanent dipole moment. They also suggest that there are two types of response when purple membrane suspensions are placed in an electric field. One is an alignment of the disk-shaped particles with the field. The other is a stacking of the particles following their alignment by the electric field, which is promoted by the induced dipole moment.     

Effects of electric field on the photocycle of bacteriorhodopsin

The photoconversion of bacteriorhodopsin and the effects of an applied electric field (5 · 10⁷ V · m^?1) were studied in dry films of purple membranes from Halobacterium halobium. The electric field was found to cause at least two different effects: (1) it blocks in part the formation of the batho-bacteriorhodopsin (K), most probably due to electrically-induced dark transition of some bacteriorhodopsin molecules into the photochemically inactive form; (2) it decreases the rate of the intermediate M decay, the rise time of the M formation being unaffected by electric field. The observed phenomena may suggest a feedback control mechanism for the regulation of the bacteriorhodopsin photocycle in purple membranes.     

Electric field promotion of the bacteriorhodopsin BR570 to BR412 photoconversion in films of Halobacterium halobium purple membranes

The combined action of electric field (10⁵–10⁷ V · m^?1) and light (380–580 nm, 80 W · m^?2) activating the photoenergetic reaction of bacteriorhodopsin (BR) in dry films of purple membranes from Halobacterium halobium was studied. A new stimulating effect of the field on the BR₄₁₂ intermediate accumulation in the normal photochromic cycle of BR₅₇₀ has been observed. The formation of the product BR₄₁₂ is supposed to be accompanied by specific rearrangements of certain charged, polar and polarizable groups in the BR pigment-protein matrix. Such an intrinsic polarization could be promoted by an external electric field, the displacement vector of those groups being oriented in the direction of the field. The dielectric polarization properties of the purple membranes have been demonstrated by electret-thermal analysis.     

Transient electric birefringence of T-even bacteriophages. I. T4B in the absence of tryptophan and fiberless T4 particles

Measurements of the electric birefringence of suspensions of T4B in the absence of tryptophan and of fiberless T4 particles show that both kinds of particles are hydrodynamically equivalent. Their rotational diffusion coefficients corrected to 25°C and water viscosity (D_25,w) are 280 ± 9 sec^?1 and 295 ± 10 sec^?1, respectively. These corrected rotational diffusion coefficients are almost independent of buffer concentration and temperature. The sedimentation coefficient (s_20,w) of T4 B is equal to 1023 ± 12 S, a value which is likewise independent of buffer concentration. By analysis of the field strength dependence of the steady-state birefringence and by reversing pulse experiments it could be shown that the orientation in an electric field is largely due to a permanent dipole moment. This dipole moment is somewhat dependent on buffer concentration and amounts to about 24,000 debye for T4B and 95,000 debye for fiberless T4. An approximate calculation shows that the difference in dipole moment may be ascribed to positive charges on the fiber tip (at least ten per fiber), to negative charges along the fiber or (and) positive charges on the fiberless particle at those places where the fibers are attached in normal particles.     

Transient and linear dichroism studies on bacteriorhodopsin: determination of the orientation of the 568 nm all-trans retinal chromophore

The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways.In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a time-independent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78 ° ± 3 ° is determined.In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of the light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient dichroism method, linear dichroism measurements on oriented samples lead to an angle of 71 ° ± 4 °.No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substantially smaller than the recently suggested value of 20 nm (Ebrey et al., 1977).The orientation of the 568 nm transition dipole moment, which coincides with the direction of the all-trans polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.     

Surface Charge Movements of Purple Membrane During Light-Dark Adaptation

The difference in the surface charge distribution between light-adapted and dark-adapted purple membranes was investigated with electric dichroism measurements from approximately pH 5 to pH 11. Purple membrane sheets in solution are oriented in a weak electric field by their permanent dipole moment, which is due to the charge distribution of the membrane surfaces and/or within the membrane. The degree of orientation of purple membrane sheets was obtained from the measurement of “electrical anisotropy” of retinal chromophore in the membranes. At about pH 7, there was no difference in the “electric anisotropy” between light- and dark-adapted purple membranes. At about pH 9, the electric anisotropy of dark-adapted purple membrane was larger than that of light-adapted purple membrane. But at around pH 6 the difference was opposite. Linear dichroism experiments did not show any change of retinal tilt angle with respect to the membrane normal between the two forms from approximately pH 5 to pH 10. This result indicates that the changes in the “electric anisotropy” are not due to the change of retinal tilt angle, but due to the change in the permanent dipole moment of the membrane. To estimate the change in surface charges from the permanent dipole moment, we investigated the difference of the permanent dipole moment between the native purple membrane and papain-treated purple membrane in which negative charges in the cytoplasmic-terminal part are removed. This estimation suggests that this light-dark difference at around pH 9 can be accounted for by a change of ~0.5 electric charge per bacteriorhodopsin (bR) molecule at either of the two surfaces of the membrane. We also found from pH electrode measurements that at about pH 8 or 9 light adaptation was accompanied by an uptake of ~0.1 protons per bR. A possible movement of protons during light-dark adaptation is discussed. The direction of the permanent dipole moment does not change with papain treatment. The permanent dipole moment in papain-treated purple membrane is estimated to be 27 ±2 debye/bR.     

Hydrophobic ion probe studies of membrane dipole potentials

Hydrophobic anions of dipicrylamine and of sodium tetraphenylborate have been employed as probes of interfacial dipole potential variations in lipid bilayer membranes. Systematic variation of dipole potentials has been achieved by introduction of compounds incorporating N⁺ and B^? charge centers. Distribution of hydrophilic and and hydrophobic groups relative to these charge centers has been shown to control the orientation in the membrane/solution interface of the electric dipole moment formed by these centers. Thus triphenyl-[4-trimethylphenylammonium] borate orients with the B^? center, surrounded by phenyl groups, embedded in the membrane, while the smaller methylated N⁺ center is directed toward the aqueous phases. This orientation has been confirmed using dipicrylamine probe ions. Results obtained in this system have been interpreted quantitatively using a previously developed model incorporating discrete charge effects. A second class of compounds, $\text{tri}\text{-n-}\text{alkylamine}$ borane (T_nAB) complexes having the generic formula (C_nH_{$\text{2n+1}$})₃N⁺B^?H₃, have also been synthesized for this study, using even-carbon alkyls ranging from ethyl to decyl. Molecular orientation of the complex is with the N⁺ center and its associated alkyl groups directed into the membranes, while the protonated B^? center is directed toward the aqueous phases, as confirmed by use of tetraphenylborate ions as probes.     

The membraneless bioelectrochemical reactor stimulates hydrogen fermentation by inhibiting methanogenic archaea

The membraneless bioelectrochemical reactor (Ml-BER) is useful for dark hydrogen fermentation. The effect of the electrochemical reaction on microorganisms in the Ml-BER was investigated using glucose as the substrate and compared with organisms in a membraneless non-bioelectrochemical reactor (Ml-NBER) and bioelectrochemical reactor (BER) with a proton exchange membrane. The potentials on the working electrode of the Ml-BER and BER with membrane were regulated to ?0.9 V (versus Ag/AgCl) to avoid water electrolysis with a carbon electrode. The Ml-BER showed suppressed methane production (19.8?±?9.1 mg-C·L^?1·day^?1) and increased hydrogen production (12.6?±?3.1 mg-H·L^?1·day^?1) at pH_out 6.2?±?0.1, and the major intermediate was butyrate (24.9?±?2.4 mM), suggesting efficient hydrogen fermentation. In contrast, the Ml-NBER showed high methane production (239.3?±?17.9 mg-C·L^?1·day^?1) and low hydrogen production (0.2?±?0.0 mg-H·L^?1·day^?1) at pH_out 6.3?±?0.1. In the cathodic chamber of the BER with membrane, methane production was high (276.3?±?20.4 mg-C·L^?1·day^?1) (pH_out, 7.2?±?0.1). In the anodic chamber of the BER with membrane (anode-BER), gas production was low because of high lactate production (43.6?±?1.7 mM) at pH_out 5.0?±?0.1. Methanogenic archaea were not detected in the Ml-BER and anode-BER. However, Methanosarcina sp. and Methanobacterium sp. were found in Ml-NBER. Prokaryotic copy numbers in the Ml-BER and Ml-NBER were similar, as were the bacterial community structures. Thus, the electrochemical reaction in the Ml-BER affected hydrogenotrophic and acetoclastic methanogens, but not the bacterial community.     

Magneto-electro-fusion of human erythrocytes

In inhomogeneous (static) magnetic fields close contact between ‘magnetic’ human erythrocytes was established. The cells were made magnetic by incubating them in a medium containing small Fe₃O₄-particles which adsorbed to the outer membrane surface. Fusion was induced by applying two electric field pulses (field strength: 8.5 kV · cm^?1; duration: 60 μs) to the magnetically collected cells. This procedure allowed the use of electrically conductive media (3 · 10^?1 Ω^?1 · cm^?1). Fusion of red blood cells occured very often. If cell suspensions of high density were used fusion resulted in the formation of giant red blood cells with osmotically intact membranes.     

Bacteriorhodopsin (BR570) bathochromic band shift in an external electric field

In dry films of bacteriorhodopsin-containing purple membranes from Halobacterium halobium the external electric field (10⁴–10⁵ V · cm^?1) induces the appearance of a product spectrally close to the initial intermediate of bacteriorhodopsin (BR) photochromic cycle (batho form, K). This result and also preliminary data of the electret-thermal analysis of the preparations suggest that the dielectric polarization in chromophore-protein-lipid complexes might be an essential step of the primary stabilization of light energy in photo-bioenergetic processes.     

紫膜碎片的电二色性研究

悬浮在水中的嗜盐菌紫膜碎片,在外电场作用下产主定向排列.在20℃时,568nm的电二色性研究表明:外加电场为2kV/m时取向程度可达60%以上;大于5.5kV/m时,取向作用趋于饱和状态;饱和时简约电二色性为-0.437左右,视黄醛生色团的跃迁矩方向与电偶极矩方向形成60.9°夹角;紫膜的永久偶极短为9.2×10~(-24)C、M,剩余电极化率为3.0×10~(-27)m~2;紫膜的旋转扩散常数为0.53秒~(-1).曲线拟合分析表明,感应偶极对紫膜碎片的定向的贡献应予考虑.本文对紫膜碎片的定向机理进行了讨论.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

Bimodal oxygen uptake in a freshwater air-breathing fish,Notopterus chitala

Measurements of bimodal oxygen uptake have been made in a freshwater air-breathing fish,Notopterus chitala at 29.0±1(S.D.)°C. xhe mean oxygen uptake from continuously flowing water without any access to air, was found to be 3.58±0.37 (S.E.) ml O₂ · h^?1 and 56.84+4.29 (S.E.) ml O₂ · kg^?1 · h^?1 for a fish weighing 66.92 + 11.27 (S.E.) g body weight. In still water with access to air, the mean oxygen uptake through the gills were recorded to be 2.49 ± 0.31 (S.E.) ml O₂ · h^?1 and 38.78 ± 1.92 (S.E.) ml O₂ · kg^?1 · h^?1 and through the accessory respiratory organs (swim-bladder) 6.04±0.87 (S.E.) ml O₂ · h^?1 and 92.32±2.91 (S.E.) ml O₂ · kg^?1 · h^?1 for a fish averaging 66.92±11.27 (S.E.) g. Out of the total oxygen uptake (131.10 ml O₂ · kg^?1 · h^?1), about 70% was obtained through the aerial route and the remainder 30% through the gills.     

Ventilation and oxygen consumption in the hagfish, Myxine glutinosa L.

Ventilation was measured directly in the hagfish, Myxine glutinosa L., by means of an electro-magnetic blood flowmeter. Ventilatory flow and frequency increased from 0.86 ± 0.27 ml·min^?, and 18.2 ± 5.1·min^?, respectively, at 7°C to 1.70 ± 0.20 ml·min^?, and 70.1 ± 9.5·min^? at 15 ·C.Standard oxygen consumption,V?O₂, was measured in non-buried hagfish. V?O₂ was 0.57 ± 0.17μl O₂·g^?1·min^?1 at 7°C, and 0.85 ± 0.12μl O₂·g^?1·min^?1 at 15°C.     

Osmotic permeability of Novikoff hepatoma cells

The osmotic permeability coefficient (P_f) for water movement across Novikoff hepatoma cells was found to be 82 ± 3 (S.E.) · 10^?5 cm · s^?1 at 20°C. The corresponding diffusional permeability coefficient for ³HHO (P_d) was 97 ± 10 (S.E.) · 10^?5 cm · s^?1, therefore the ratio $\text{P}{}_{\mathrm{<mtext>f</mtext>}}\text{P}{}_{\mathrm{<mtext>d</mtext>}}$ is close to unity. The apparent activation energy for water filtration was 10.4 ± 0.4 (S.E.) kcal · mol^?1. This value is significantly greater than the activation energy for the self diffusion of water. The product of the hydraulic permeability coefficient and the viscosity coefficient for water was temperature-dependent. However, the product of the hydraulic permeability coefficient and the viscosity coefficient for membrane lipid did not vary with temperature. These data are interpreted as evidence for water movement across a lipid membrane barrier rather than through aqueous channels.     

Analysis of potassium conductance in squid giant axons

Assuming a model of facilitated ionic transport across axonal membranes proposed by McIlroy (1975) and extended by McIlroy and Hahn (1978), it is shown that if the selectivity coefficient, π_K, of the potassium conducting system ?59 the permeabilityP _Ks, of the periaxonal barrier of the squid giant axon for K⁺ ions?(1.2±0.44)×10^?4 cm sec^?1 and the thickness of the periaxonal space ?477±168 Å. Using a value (10^?4 cm sec^?1) ofP _Ks in the foregoing range the experimental curves for the steady state membrane ionic conductance versus measured membrane potential difference (p.d.), ?, of Gilbert and Ehrenstein (1969) are corrected for the effect of accumulation of K⁺ in the periaxonal space. This correction is most marked for the axon immersed in a natural ionic environment, whose conductance curve is shifted ?70mV along the voltage axis in the hyperpolarization direction. By assuming that the physico-chemical connection between a depolarization of the axonal membrane and the consequent membrane conductance changes is a Wien dissociative effect of the membrane's electric field on a weak electrolyte situated in the axolemma, the position of the peaks of the corrected conductance versus ? curves can be identified with zero membrane electric field and hence with zero p.d.across the axolemma. A set of values for the double-layer p.d.s at the axonal membrane interfaces with the external electrolytes in the vicinity of the K⁺ conducting pores can therefore be deduced for the various external electrolytes employed by Gilbert and Ehrenstein. A model of these double-layer p.d.s in which the membrane interfaces are assumed to possess fixed monovalent negatively charged sites, at least in the neighbourhood of the K⁺ conducting pores, is constructed. It is shown that, using the previously deduced values for the doublelayer p.d.s, such a model has a consistent, physically realistic solution for the distance between the fixed charged sites and for the dissociation constants of these sites in their interaction with the ions of the extramembrane electrolytes.     

Characterization of the uptake of sucrose and glucose by isolated seed coat halves of developing pea seeds. Evidence that a sugar facilitator with diffusional kinetics is involved in seed coat unloading

Uptake of ¹⁴C-labelled sucrose and glucose by isolated seed coat halves of pea (Pisum sativum L. cv. Marzia) seeds was measured in the concentration range <0.1 μM to 100 mM. The initial influx of sucrose was strictly proportional to the external concentration, with a coefficient of proportionality (k) of 6.2 μmol·(g FW)^?1·min^?1·M^?1. Sucrose influx was not affected by 10 μM carbonylcyanide m-chlorophenylhydrazone (CCCP), but it was inhibited by 40% in the presence of 2.5 mM p-chloromercuribenzenesulfonic acid (PCMBS). Influx with diffusional kinetics was also observed for glucose (k = 4.8 μmol·(g FW)^?1·min ^?1·M ^?1) and mannitol (k = 5.1 μmol·(g FW)^?1·min^?1·M^?1). For glucose an additional saturable system was found (K_m = 0.26 mM, V _max = 4.2 nmol·(g FW)^?1·min^?1), which appeared to be completely inhibited by CCCP and partly by PCMBS. In contrast to the diffusional pathway, uptake by this saturable system was slightly pH-dependent, with an optimum at pH 5.5. The influx of sucrose appears to be by the same pathway as the efflux of endogenous sucrose, which was inhibited by 36% in the presence of 2.5 mM PCMBS (De Jong A, Wolswinkel P, 1995, Physiol Plant 94: 78–86). It is argued that passive transport may be the only mechanism for sucrose transport through the plasma membrane of seed coat parenchyma cells. The estimated permeability coefficient of the plasma membrane for sucrose (P = 3.5·10^?7 cm·s^?1) is more than 1 × 10⁶-fold higher than that reported for artificial lipid membranes. This relatively high permeability is hypothesized to result from pore-forming proteins that allow the diffusion of sucrose. Furthermore, it is shown that a sucrose gradient across the plasma membrane of the seed coat parenchyma of only 22 mM will suffice to result in the net efflux of sucrose which is required to feed the embryo.     

Electro-optic scattering studies on deoxyribonucleic acid

Measurements have been made of the intensity of light scattered from aqueous solutions of calf thymus DNA with and without the application of electric fields. For fields approaching 150 V/cm and frequencies below 2.5 KHz, changes (ΔI) of up to 10% in the residual scattered intensity were observed. In agreement with previous dielectric and electric birefringence measurements, a low frequency dispersion of ΔI was observed, from which a rotary diffusion constant (D) of 1200 s^?1 was determined. Interpreting the electric field data in terms of the classical dipolar orientation theory led to values of 2.4 × 10^?25 cm (7.4 × 10^?14 esu) and 4.3 × 10^?25 cm (13 × 10^?14 esu) for the permanent dipole moment and the anisotropy of the electric polarisabilities respectively. Furthermore the permanent dipole moment was along the major molecular axis and the particles orientated in the field as rigid entities. The zero field data indicated a molecular shape which was not rodlike but corresponded to the Kratky-Porod “stiffness” parameter of x = 24 for the wormlike coil model. Although curved, the molecules appeared to orientate in low-intensity electric fields as rigid, but not rodlike molecules. The implications of this on recent discrepancies in D determined by two or more dynamic relaxation methods is briefly discussed.     

Consequences of electroshock‐induced narcosis in fish muscle: from mitochondria to swim performance

Adult zebrafish Danio rerio were exposed to an electric shock of 3 V and 1A for 5 s delivered by field backpack electrofishing gear, to induce a taxis followed by a narcosis. The effect of such electric shock was investigated on both the individual performances (swimming capacities and costs of transport) and at cellular and mitochondrial levels (oxygen consumption and oxidative balance). The observed survival rate was very high (96·8%) independent of swimming speed (up to 10 body length s^?1). The results showed no effect of the treatment on the metabolism and cost of transport of the fish. Nor did the electroshock trigger any changes on muscular oxidative balance and bioenergetics even if red muscle fibres were more oxidative than white muscle. Phosphorylating respiration rates rose between (mean 1 s.e. ) 11·16 ± 1·36 pmol O₂ s^?1 mg^?1 and 15·63 ± 1·60 pmol O₂ s^?1 mg^?1 for red muscle fibres whereas phosphorylating respiration rates only reached 8·73 ± 1·27 pmol O₂ s^?1 mg^?1 in white muscle. Such an absence of detectable physiological consequences after electro‐induced narcosis both at organismal and cellular scales indicate that this capture method has no apparent negative post‐shock performance under the conditions of this study.