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At least 19 recordsLinked to original sources
PubMed1979

Association of bacteriorhodopsin with lipid-impregnated filters. Evidence for fusion of bacteriorhodopsin-containing vesicles with the lipid phase of the filter.

Bacteriorhodopsin vesicles were associated with cellulose-nitrate filters impregnated with a solution of phospholipids in hexadecane. The generation of (photo)potentials upon illumination of the filter was studied in the absence and presence of ionophores, phospholipase A2, EDTA or polyene antibiotics. From these experiments the following conclusions are drawn. 1. Upon illumination of the filter, bacteriorhodopsin pumps protons into aqueous compartments located in the filter. 2. These aqueous compartments possibly do not originate from the compartments enclosed by the bacteriorhodopsin vesicles. Evidence is obtained that aqueous compartments are present in the surface layers of the lipid-impregnated filters. 3. The results are explained most easily by a mechanism, whereby fusion occurs between the vesicles and the lipids of the filter.

Amphotericin B↗
PubMed1978

Flash kinetic study of the last steps in the photoinduced reaction cycle of bacteriorhodopsin.

The reaction cycle of light adapted bacteriorhodopsin (BR) in aqueous purple membrane suspensions was studied by laser flash photolysis at different temperatures (2--49 degrees C) and pH values (3--10). The activation energy for several reaction steps was determined at pH 7.6. The kinetics of O-bacteriorhodopsin (one of the last intermediates in the cycle) were analyzed in some detail and it was found that the simple consecutive reaction scheme M-BR leads to O-BR leads to BR may explain the kinetics of O-bacteriorhodopsin as measured at 680 nm. Since the pH change in neutral aqueous suspensions of purple membrane follows similar kinetics as O-bacteriorhodopsin it is suggested that protons are released during the reaction M-BR leads to O-BR and taken up again during the reacton O-BR leads to BR. Another long-lived intermediate, which absorbs to a greater extent than bacteriorhodopsin at 570 nm and less than bacteriorhodopsin at 420 nm, was identified with the strongly fluorescing species, pseudo- or P-bacteriorhodopsin. The decay of P-bacteriorhodopsin in bacteriohodopsin had an activation energy of only approx. 1.2 kcal/mol, which suggests that the last step of the photocycle is a relaxation around a single bond. At pH 9--10, the simple first-order kinetics of all the intermediates were changed into a kinetics consisting of two first-order decays. This change of kinetics was accompanied by a drastic decrease in the rotational diffusion relaxation time. To explain the results obtained in this work and those of others, a model involving proton uptake and release by the Schiff base nitrogen combined with an isomerization reaction is finally proposed.

Bacteriorhodopsins↗
PubMed1976

Reconstitution of Biological Molecular generators of electric current. Bacteriorhodopsin.

1. Photoinduced generation of electric current by bacteriorhodopsin, incorporated into the planar phospholipid membrane, has been directly measured with conventional electrometer techniques. 2. Two methods for bacteriorhodopsin incorporation have been developed: (a) formation of planar membrane from a mixture of decane solution of phospholipids and of the fraction of violet fragments of the Halobacterium halobium membrane (bacteriorhodopsin sheets), and (b) adhesion of bacteriorhodopsin-containing reconstituted spherical membranes (proteoliposomes) to the planar membrane in the presence of Ca2+ or some other cations. In both cases, illumination was found to induce electric current generation directed across the planar membrane, an effect which was measured by macroelectrodes immersed into electrolyte solutions on both sides of the membrane. 3. The maximal values of the transmembrane electric potential were of about 150 mV at a current of about 10(-11) A. The electromotive force measured by means of counterbalancing the photoeffect by an external battery, was found to reach the value of 300 mV. 4. The action spectrum of the photoeffect coincides with the bacteriorhodopsin absorption spectrum (maximum about 570 nm). 5. Both components of the electrochemical potential of H+ ions (electric potential and delta pH) across the planar membrane affect the bacteriorhodopsin photoelectric response in a fashion which could be expected if bacteriorhodopsin were a light-dependent electrogenic proton pump. 6. La3+ ions were shown to inhibit operation of those bacteriorhodopsin which pump out H+ ions from the La3+-containing compartment. 7. The photoeffect, mediated by proteoliposomes associated with thick planar membrane, is decreased by gramicidin A at concentrations which do not influence the planar membrane resistance in the light. On the contrary, a protonophorous uncoupler, trichlorocarbonylcyanidephenylhydrazone, decreases the photoeffect only if it is added at a concentration lowering the light resistance. The dark resistance is shown to be higher than the light one, and decreases to the light level by gramicidin. 8. A simple equivalent electric scheme consistent with the above results has been proposed.

Bacteriorhodopsins↗
PubMed1977

Isomeric composition of retinal chromophore in dark-adapted bacteriorhodopsin.

1. Retinal isomers extracted from the acid-hydrolysate of cetyltrimethylammonium bromide-treated dark-adapted bacteriorhodopsin (bRD) were analyzed in a high performance liquid chromatograph (HPLC) system. The extract from bRD contains almost equal molar amounts of both 13-cis retinal and all-trans retinal isomers. The extent of isomerization and the yield of both isomers during the isolation process were investigated by the application of the same extraction procedure to artificial bacteriorhodopsin reconstituted with 13-cis retinal isomer (13-cis bacteriorhodopsin) and also to light-adapted bacteriorhodopsin (bRL) which has been shown to contain only the all-trans isomer (all-trans bacteriorhodopsin). 2. A reconstituted bacteriorhodopsin, which had been prepared from apo-bacteriorhodopsin and an equimolar mixture of both 13-cis retinal and all-trans retinal isomers, showed an absorption spectrum having the same maximum wavelength as that of bRD even at the beginning of the reconstitution process. 3. Analysis of the photosteady states of bRD at -190 degrees C revealed that it was composed of two different species, one having 13-cis retinal and the other having all-trans retinal isomers in approximately equal molar amounts. These two also gave their respective photoproducts. 4. From these results it can be concluded that bRD contains both 13-cis retinal and all-trans retinal isomers in nearly equal molar amounts as its chromophore.

Bacteriorhodopsins↗
PubMed1976

Bacteriorhodopsin formation in Halobacterium halobium.

Systematic examinations were made of factors influencing bacteriorhodopsin formation during the growth of Halobacterium halobium. Light-induced adenosine triphosphate (ATP) production and [14C]proline uptake were used as measures of functional ability of the purple membrane. Maximum bacteriorhodopsin formation occurred under growth conditions of illumination and limited aeration. The purple membrane -ATP production system did not confer an appreciable growth advantage. Growth in the dark or with adequate aeration partially suppressed bacteriorhodopsin formation and the effects were additive. Nicotine effectively inhibited bacteriorhodopsin formation. A rapid synthesis of functional pigment occurred when washed suspensions of cells which had been grown under illumination with nicotine present were incubated under dark, aerobic conditions. The alleviation of this nicotine inhibition was not blocked by chloramphenicol or bacitracin. Bacteriorhodopsin formation was negligible when washed suspensions of cells from dark, limited aeration or light, adequate aeration cultures were incubated in the light with limited aeration. A nutritionally complex medium was needed to elicit appreciable bacteriorhodopsin formation by the cells from the dark or adequately aerated cultures. Bacitracin partially inhibited this bacteriorhodopsin formation by cells form the light, adequately aerated culture.

Adenosine Triphosphate↗
PubMedDate not supplied

[Temporal characteristics of bacteriorhodopsin as a molecular biological generator of current].

Generation of electric potential difference by bacteriorhodopsin proteoliposomes incorporated into the phospholipid-impregnated collodion film has been studied. It is shown that illumination of this film by continuous light gives rise to the generation of an electric potential difference across the film (plus on the bacteriorhodopsin-free side), which can be as high as 300 mV. Short unsaturating flash inducing single turn-over of bacteriorhodopsin generates the potential difference which is a function of the flash intensity (70 mV at 3 mjoule light). The flash-induced photoelectric response consists of four phases. (1) Very fast (tau less than 1 microsec) generation of a potential difference (minus in the bacteriorhodopsin-free compartment). The amplitude of this phase is rather small (1--5 mV). (2) Fast phase of positive charging of the bacteriorhodopsin-free compartment (tau = 25--50 microsec). (3) Slow phase of positive charging of this compartment (tau = 6--12 msec) Amplitude of the second phase is to that of the third as 1 : 2. (4) A very slow phase of discharge of the flash-induced potential difference (tau = 1 sec at 10(8) ohm X cm2 film resistance). The third phase was specifically inhibited by La3+. Both the second and the third phases are decelerated by substitution of D2O in 4.5--5 and 2 times, respectively, while the amplitude of the first phase increases. Prolonged storage of the system in the dark (tua = 20--25 min) causes the decrease in the amplitudes of the second and the third phases as if the amount of active bacteriorhodopsin molecules were increased by factor 2. Such an inhibition was reversed by 30--60 sec illumination of the system. The dark adaptation is accompanied by some increase in the first phase amplitude. Comparison of these data with results of other studies on bacteriorhodopsin suggests that (1) the first phase is due to the photoinduced change in the retinal dipole; (2) the second phase corresponds to H+ transfer from Schiff base to the water solution in the proteoliposome interior; 3) the third phase represents H+ transfer from the incubation mixture to Schiff base; (4) the dark adaptation is a result of transition of photoelectrochemically active all-trans-retinal to the inactive 13-cis-retinal.

Bacteriorhodopsins↗
PubMed1977

Structural studies on Halobacterium halobium bacteriorhodopsin.

Bacteriorhodopsin, isolated from Halobacterium halobium, is an intrinsic membrane protein containing covalently attached retinal and driven by a cyclic light-driven protonmotive force. In order to extend three-dimensional analyses already in progress and to define the covalent features of such proteins, the determination of the primary structure of bacteriorhodopsin has been initiated, using techniques that can be applied to the sequence analyses of other intrinsic membrane proteins as well. The amino acid composition of bacteriorhodopsin prepared by new procedures compares well to that previously published. Various delipidation procedures have been examined for their effectiveness in yielding protein in a physical state that is susceptible to fragmentation by a number of chemical and proteolytic agents. Organic extraction, particularly in acetone:ammonium hydroxide, has proved most useful. Detergent extraction was effective in delipidating but rendered the protein inert to attack by various cleavage reagents. Sequence information was obtained from fragments isolated from several hydrolytic preparations of low percent cleavage. The most complete fragmentation was obtained from hydrolysis of bacteriorhodopsin previously modified with succinic or maleic anhydride. Both solubility and fragmentation susceptibility were enhanced by these reactions and several other derivatives of bacteriorhodopsin are currently being investigated. The behavior of bacteriorhodopsin under the conditions examined, as well as the sequence information obtained, suggest that bacteriorhodopsin is mostly contained within the lipid milieu of the membrane and may represent a class of membrane proteins that do not contain appreciable hydrophilic domains.

Amino Acids↗
PubMed1977

Proton transport by bacteriorhodopsin through an interface film.

Interface films of purple membrane and lipid containing spectroscopically intact and oriented bacteriorhodopsin have been used as a model system to study the function of this protein. Small positive charges in surface potential (less than 1 mV) are detected upon illumination of these films at the air-water interface. These photopotentials are not affected by overlaying the interface film with a thin layer (0.3 mm) of decane. However, they are dramatically increased when lipid soluble proton carriers FCCP or DNP are added to the decane. The polarity of the photopotential indicates that, in the light, positive charges are transported through the interface from the aqueous to the organic phase. The action spectrum of the photopotential is identical to the absorption spectrum of bacteriorhodopsin. Since bacteriorhodopsin molecules are oriented with their intracellular surface towards the aqueous subphase, the characteristics of the photopotential indicate that in the light bacteriorhodopsin translocates protons from its intracellular to its extracellular surface. The kinetics of the photopotential reveal that the rate and extent of proton transport are proportional both to the fraction of bacteriorhodopsin molecules excited and to the concentration of proton acceptor. The photopotentials result from changes in the ionic distribution across the decane-water interface and can be cancelled by lipid soluble anions.

Air↗
PubMed1978

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane.

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M412) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.

Bacteriorhodopsins↗
PubMed1978

Transient photovoltages in purple membrane multilayers. Charge displacement in bacteriorhodopsin and its photointermediates.

The photovoltaic properties of bacteriorhodopsin molecules and their photochemical intermediates have been investigated in an experimental cell consisting of multilayered films of highly oriented, dry fragments of purple membrane and lipid sandwiched between two metal (Pd) electrodes. The electrical time constant of these sandwich cells containing between 5 and 30 layers is less than 10(-5) S. Bright illumination of these cells with actinic flashes of approximately 1 ms duration generates transient photovoltages. These photovoltages, which make the extracellular surface of purple membrane positive with respect to the intracellular surface, follow the time course of the flash with no detectable latency. The amplitude of the photovoltages increases linearly with light intensity and their action spectrum matches the absorption spectrum of the light-adapted state of bacteriorhodopsin, BR570. In these dry multilayer cells, the slow photointermediates of bacteriorhodopsin, M412, N520 and O640 are long lived. Illumination of the sandwich cells with long duration (200 ms) pulses of light results, therefore, in the formation of photomixtures containing all these slow photointermediates. Flash illumination of the sandwich cells immediately following the conditioning pulse produces photovoltages whose action spectra match the absorption spectra of the M412 and N520 photointermediates. The M412 photovoltages, like the BR570 photovoltages, follow the time course of the actinic flash with no detectable latency and increase in amplitude linearly with light intensity. But, unlike the BR570 photovoltage, the M412, N520 and O640 photovoltages make the extracellular surface of purple membrane negative with respect to the intracellular surface. Through the of their specific photovoltaic signals, M412 and N520 are shown to be kinetically distinct photointermediates of bacteriorhodopsin. Detection of fast photovoltages with these characteristics in the absence of any ionic solution, and in parallel with spectrophotometric changes, suggest that they arise from charge displacements in the bacteriorhodopsin molecules and their photointermediates as they undergo photochemical conversion in response to the absorption of photons.

Bacteriorhodopsins↗
PubMed1977

Formations of electrochemical proton gradient and adenosine triphosphate in proteoliposomes containing purified adenosine triphosphatase and bacteriorhodopsin.

Proteoliposome vesicles containing both bacteriorhodopsin of Halobacterium halobium and H+-translocating ATPase [EC 3.6,1.3] of a thermophilic bacterium, PS3, (TF0-F1) were reconstituted by either the dialysis method or the sonication method. Generation of the electrochemical proton gradient (deltamuH+) in these vesicles was measured using 9-aminoacridine for estimation of the chemical (deltapH) component and 8-anilinonaphthalene sulfonate for the electrical (deltaphi) component). In illuminated bacteriorhodopsin-vesicles the deltamuH+ reached 180-190 mV when reconstituted by the dialysis method and 210-220 mV when reconstituted by the sonication method. Vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method generated a deltapH+ of about 200 mV on addition of ATP, while vesicles prepared by the sonication method generated very little deltamuH+, if any. These vesicles generated similar deltamuH+ on illumination to that found in bacteriorhodopsin-vesicles. Using vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method, light dependent ATP synthesis was measured in relation to deltamuH+ formation. It was necessary to generate a deltamuH+ of above 170 mV for demonstration of appreciable formation of ATP and the greater the deltamuH+, the faster the rate of ATP synthesis.

Adenosine Triphosphatases↗
PubMed1975

Flash photometric experiments on the photochemical cycle of bacteriorhodopsin.

The photochemical reaction cycle of bacteriorhodopsin was investigated by means of flash photometric methods. Three different intermediates with absorption maxima at about 630 nm, 411 nm, and 646 nm could be detected. Kinetic data of the occurrence of these intermediates were obtained from isolated purple membrane in different mediums and from intact halobacteria. An activation energy of 14.1 +/- 0.4 kcal-mol-1 and of about 19 kcal-mol-1 for formation of bacteriorhodopsin 411 and of bacteriorhodopsin 565, resp., was calculated. pH-changes in the medium caused by the reaction cycle of bacteriorhodopsin were detected by use of the pH-indicator bromocresol green.

Bacteriorhodopsins↗
PubMed1977

Effect of light-adaptation on the photoreaction of bacteriorhodopsin from Halobacterium halobium.

Light-induced formation of the 410 nm intermediate was investigated on dark-and light-adapted bacteriorhodopsin. The amplitude of the light-induced absorption increase at 410 nm of the light-adapted bacteriorhodopsin was twice as large as that of the dark-adapted bacteriorhodopsin. The amount of protons released from bacteriorhodopsin in response to illumination was also enhanced by light-adaptation. The degree of the enhancement was independent of the temperature in the dark-adaptation. The relation between these photochemical events and the isomeric configurations of retinal is discussed.

Adaptation, Physiological↗
PubMed1979

A low temperature investigation of the intermediates of the photocycle of light-adapted bacteriorhodopsin. Optical absorption and fluorescence measurements.

Optical absorption and emission measurements have been made on samples of light-adapted purple membrane of Halobacterium halobium at temperatures ranging from 77 K to room temperature. As a result of these experiments a set of equations is given which described thermal and photochemical reactions interrelating various intermediates of the reaction cycle of the chromophore of light-adapted bacteriorhodopsin (BR). Further some specific problems connected to these intermediates have been investigated. Thus the room temperature emission spectrum of bacteriorhodopsin has been found to exhibit a Stokes shift of 3430 cm-1 only, if low excitation intensities are used. The recently detected intermiediate P-BR can be shown to convert thermally into bacteriorhodopsin following a first-order decay with the activation energy delta E = 2.4 +/- 0.2 kcal/mol. The thermal decay of K-BR consists of two exponentials if measured on purple membrane suspensions in a mixture of H2O and glycerol (1 : 1, v/v). A simple procedure is given for trapping the intermediate L-BR at 170 K in a very pure form. M-BR is shown to consist of two species, MI-BR and MII-BR. They are characterized by similar optical absorption spectra but different thermal stability. Further the oscillator strengths corresponding to the long wavelength absorption bands of the intermediates bacteriorhodopsin, K-, L, MI- and MII-BR have been calculated. They have been discussed with respect to the question which of the corresponding absorption spectra show the characteristics of isomerism of the chromophore or simply solvatochromism.

Bacteriorhodopsins↗
PubMed1977

The preparation of lipid-depleted bacteriorhodopsin.

Bacteriorhodopsin, the protein of the purple membrane of Halobacterium halobium, was freed to the extent of 90-95% from the natural membrane lipids without loss of function. The residual lipid corresponded to less than 1 mol/mol of bacteriorhodopsin. Delipidation was achieved by treatment of the purple membrane with a mixture of the detergent dimethyldodecylamine oxide and sodium chloride. The detergent was removed by dialysis or by sucrose density gradient centrifugation. Analysis of the lipids removed and those still bound to bacteriorhodopsin was facilitated by the use of purple membrane preparations labelled with 35S, 32P, or 14C. The composition of the residual lipids associated with bacteriorhodopsin was similar to that of the total lipid in the purple membrane.

Bacteriorhodopsins↗
PubMed1978

Resonance Raman spectroscopy of the retinylidene chromophore in bacteriorhodopsin (bR570), bR560, M421, and other intermediates: structural conclusions based on kinetics, analogues, models, and isotopically labeled membranes.

Resonance Raman spectra of various intermediates in the bacteriorhodopsin proton pumping cycle have been obtained at physiological and low temperatures. To interpret these data, spectra of modél compounds, bacteriorhodopsin analogues, and isotopically labeled membranes have been measured. These results demonstrate that a protein group interacts with the Schiff base proton and, thus, the chromophore in protonated bacteriorhodopsin species is not a simple protonated Schiff base. This accounts for the abnormally low frequency of the C=N+H vibrational mode in bacteriorhodopsin and other failures to model the chromophore in bR570 with a simple butylamine protonated Schiff base of all-trans-retinal. To obtain the resonance Raman spectrum of M412 at physiological pH and temperatures, a dual beam kinetic technique was developed. We demonstrate that in the fingerprint region of the resonance Raman spectrum M412 is modeled accurately by a simple unprotonated butylamine Schiff base of all-trans-retinal. Spectral resolution and the solution environment of the membrane suspensions play important roles in this conclusion. Kinetic resonance Raman techniques are also used to monitor the time evolution of the M412 species and the intermediates which precede it. We find spectral features in our kinetic data which can be assigned to L550, and we present evidence for a new unprotonated species (X) which occurs before M412. Single pass flow resonance Raman spectra of bR560 also have been obtained, and, although bR570 and M412 appear to have all-trans chromophores, there are 13-cis-like features in the spectra of bR560, L550, and X.

Bacteriorhodopsins↗
PubMed1978

Bacteriorhodopsin: lipid environment and conformational changes.

The polar lipids of the purple membrane were exchanged for different phosphatidylcholine species. The resulting complexes had the same protein to lipid-phosphorus ratio as the natural membrane, but only about 0.5-1.0 mole of original lipid was still present per mole of bacteriorhodopsin. In such complexes the bacteriorhodopsin photocycle is slowed down 10-20 times, but the strong protein-protein interaction is not abolished. Due to the slow rate of the photocycle we were able to measure in the light the ratio between net proton release and net accumulation of the last intermediate of the photocycle, the unprotonated M412. This ratio was not constant and equal to 1.0, as expected for a single deprotonation reaction, but varied with pH from 1.5 to 0.4. The variable ratio suggests that light-induced conformational changes occur in the nonchromophore part of the protein, which shift the pKa values of unidentified groups so as to cause binding or release of additional protons. A similar conclusion was drawn from experiments on the kinetics of proton transfer by bacteriorhodopsin in subbacterial particles of Halobacterium halobium and in reconstituted bacteriorhodopsin proteoliposomes. However, in this case light-induced association and dissociation of additional protons occurs simultaneously on different sides of the membrane.

Bacteriorhodopsins↗