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1.
It has been shown previously that the proton-pumping activity of bacteriorhodopsin from Halobacterium salinarium can transmit an attractant signal to the bacterial flagella upon an increase in light intensity over a wide range of wavelengths. Here, we studied the effect of blue light on phototactic responses by the mutant strain Pho8l-B4, which lacks both sensory rhodopsins but has the ability to synthesize bacteriorhodopsin. Under conditions in which bacteriorhodopsin was largely accumulated as the M412 bacteriorhodopsin photocycle intermediate, halobacterial cells responded to blue light as a repellent. This response was pronounced when the membrane electric potential level was high in the presence of arginine, active oxygen consumption, or high-background long-wavelength light intensity but was inhibited by an uncoupler of oxidative phosphorylation (carbonyl cyanide 3-chlorophenylhydrazone) and was inverted in a background of low long-wavelength light intensity. The response to changes in the intensity of blue light under high background light was asymmetric, since removal of blue light did not produce an expected suppression of reversals. Addition of ammonium acetate, which is known to reduce the pH gradient changes across the membrane, did not inhibit the repellent effect of blue light, while the discharge of the membrane electric potential by tetraphenylphosphonium ions inhibited this sensory reaction. We conclude that the primary signal from bacteriorhodopsin to the sensory pathway involves changes in membrane potential.  相似文献   

2.
《BBA》1986,850(1):162-169
Oriented purple-membrane preparations from Halobacterium halobium were obtained by electrophoretic sedimentation of a purple-membrane suspension on a transparent current-conducting surface. Light exposure of orderly oriented purple-membrane films causes the generation of a photopotential amounting to several volts. The effects of external electric field on the dark state and photocycle of bacteriorhodopsin is studied in dry orderly oriented purple-membrane films. In contrast to nonuniformly oriented preparations (Borisevich, G.P., Lakashev, E.P., Kononenko, A.A. and Rubin, A.B. (1979) Biochim. Biophys. Acta 546, 171–174 and Lukashev, E.P., Vozary, E., Kononenko, A.A. and Rubin, A.B. (1980) Biochim. Biophys. Acta 590, 258–266), a specific feature of the field-induced phenomena observed in orderly oriented films is their vectorial character. The field-induced bathochromic shift of the maximum absorbance of bacteriorhodopsin is observed in an electric field, directed from the periplasmatic to cytoplasmatic side of the purple membrane and the field-induced rise of the photo-stationary M412 concentration in a field of opposite sign. This field-induced rise is a result of slowering of M412 decay. The observed effects seem likely to reflect the existence of the potential-dependent regulation of the bacteriorhodopsin photocycle in intact purple membranes.  相似文献   

3.
Melittin differentially slowed down the fast (M412f) and the slow (M412s) decay components of the photocyde intermediate M of trimeric bacteriorhodopsin in purple membrane while it accelerated the M412s of Triton X-100-solubilized bacteriorhodopsin monomers. Raising the bulk pH could enhance the effect of melittin on the M412s of bacteriorhodopsin in these two states. From pH 5.5 to 8.8, melittin slightly influenced the yield of intermediate M in purple membrane, whereas the yield of M412s decreased and subsequently reversed with the addition of melittin. Moreover, the monomeric bacteriorhodopsin bleached more readily in the presence of melittin and the higher pH made the bleaching effect of melittin more intensive as well. These results re-certify our former suggestions that there was electrostatic interaction between melittin and bacteriorhodopsin, and indicate that the biphasic M decay may not result from the well-known linear kinetic scheme (M→N →BR). At last the mechanisms underlying the interact  相似文献   

4.
The photochemical cycle and the proton-pumping function of bacteriorhodopsin modified with lanthanum and formaldehyde has been studied. In both preparations, the M412 leads to BR570 transition time has been found to increase considerably. The deceleration of the photochemical cycle has been shown to be accompanied by inhibition of the millisecond phase of the photoelectrical response of bacteriorhodopsin membranes associated with phospholipid-impregnated collodion film. Photoelectrogenic activity measured with permeable ion probe in proteoliposomes was also inhibited. Effects of lanthanum were reversed by EDTA. Formation of M412 was slightly accelerated and the microsecond electrogenic phase was not affected by lanthanum and by formaldehyde. It is concluded that lanthanum, but not formaldehyde, can be used as a specific reversible inhibitor of the second half of the bacteriorhodopsin photocycle and of the associated H+ uptake on the cytoplasmic side of the halobacterial membrane. Possible mechanisms of these effects are discussed.  相似文献   

5.
Methylation of the nonactive site lysines of bacteriorhodopsin to form permethylated bacteriorhodopsin does not interfere with the formation of the short wavelength intermediate M412 or light-induced proton release/uptake. The absorption spectrum is similar to that of the native bacteriorhodopsin. However, additional monomethylation of the active site lysine of bacteriorhodopsin causes a red shift of the absorption maximum from 568 nm in light-adapted bacteriorhodopsin [BR] to 630 nm. The photochemistry of active-site methylated BR does not proceed beyond the L-photointermediate. In particular, the photointermediate corresponding to M412 does not form, and there is no proton pumping. Moreover, there is no tyrosine deprotonation. Thus, the formation of an M-type photointermediate is required for proton pumping by BR.  相似文献   

6.
C Ganea  C Gergely  K Ludmann    G Váró 《Biophysical journal》1997,73(5):2718-2725
The changes in the photocycle of the wild type and several mutant bacteriorhodopsin (D96N, E204Q, and D212N) were studied on dried samples, at relative humidities of 100% and 50%. Samples were prepared from suspensions at pH approximately 5 and at pH approximately 9. Intermediate M with unprotonated Schiff base was observed at the lower humidity, even in the case where the photocycle in suspension did not contain this intermediate (mutant D212N, high pH). The photocycle of the dried sample stopped at intermediate M1 in the extracellular conformation; conformation change, switching the accessibility of the Schiff base to the cytoplasmic side, and proton transport did not occur. The photocycle decayed slowly by dissipating the absorbed energy of the photon, and the protein returned to its initial bacteriorhodopsin state, through several M1-like substates. These substates presumably reflect different paths of the proton back to the Schiff base, as a consequence of the bacteriorhodopsin adopting different conformations by stiffening on dehydration. All intermediates requiring conformational change were hindered in the dried form. The concentration of intermediate L, which appears after isomerization of the retinal from all-trans to 13-cis, during local relaxation of the protein, was unusually low in dried samples. The lack of intermediates N and O demonstrated that the M state did not undergo a change from the extracellular to the cytoplasmic conformation (M1 to M2 transition), as already indicated by Fourier transform infrared spectroscopy, quasielastic incoherent neutron scattering, and electric signal measurements described in the literature.  相似文献   

7.
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.  相似文献   

8.
The pH dependence of the subpicosecond decay of the retinal photoexcited state in bacteriorhodopsin (bR) is determined in the pH range 6.8-11.3. A rapid change in the decay rate of the retinal photoexcited state is observed in the pH range 9-10, the same pH range in which a rapid change in the M412 formation kinetics was observed. This observation supports the previously proposed heterogeneity model in which parallel photocycles contribute to the observed pH dependence of the M412 formation kinetics in bR.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
The photoinduced electric response of oriented purple membranes associated with processes before the K-intermediate decay of bacteriorhodopsin was measured in the 180-300 K temperature range. These response signals consist of two kinetically distinct components (both temperature dependent). The experimental data show a correlation between the time constants of the rise of the signal and solution resistance. A model is proposed to assign these components to two diffusion-limited processes of charge displacement in the solution. The displacement is caused by the electric field of the photoinduced transient dipole which is formed in the primary act of the bacteriorhodopsin photocycle. The two processes are assigned as: (a) the conduction of electrical current through H-bonds (time resolved only in the temperature range 180-200 K) and (b) the diffusion of charges through the interfacial layer.  相似文献   

12.
The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.  相似文献   

13.
The effects of pressure and temperature on the decay kinetics of the M412 (M) intermediate in the photocycle of bacteriorhodopsin were studied to provide information about the phase transitions of the purple membrane lipids. The activation volume (delta V++) for the decay of M is expected to be different below and above a phase transition. However, no abrupt change in delta V++ was found from 3.5 degrees to 60 degrees C. But a sharp break was observed in a plot of the logarithm of the rate of M decay vs. pressure. Extrapolation of this break point to standard atmospheric pressure gives a temperature of -42 degrees C, which probably corresponds to the phase transition of the purple membrane lipids. This conclusion is supported by studies of the effect of pressure on the M kinetics of bacteriorhodopsin incorporated into dimyristoylphosphatidylcholine vesicles, whose phase transition has previously been characterized.  相似文献   

14.
C Gergely  C Ganea  G Groma    G Váró 《Biophysical journal》1993,65(6):2478-2483
Absorption kinetic and electric measurements were performed on oriented purple membranes of D96N bacteriorhodopsin mutant embedded in polyacrylamide gel and the kinetic parameters of the photointermediates determined. The rate constants, obtained from fits to time-dependent concentrations, were used to calculate the relative electrogenicity of the intermediates. The signals were analyzed on the basis of different photocycle models. The preferred model is the sequential one with reversible reaction. To improve the quality of the fits the necessity of introducing a second L intermediate arose. We also attempted to interpret our data in the view of reversible reactions containing two parallel photocycles, but the pH dependencies of the rate constants and electrogenicities favored the model containing sequential reversible transitions. A fast equilibrium for the L2<==>M1 transition and a strong pH dependence of the M2 electrogenicity was found, indicating that the M1 to M2 transition involves complex charge motions, as is expected in a conformational change of the protein.  相似文献   

15.
Maximum of the M intermediate difference spectrum in the wild-type Halobacterium salinarium purple membrane is localized at 405-406 nm under conditions favoring accumulation of the M(N) intermediate (6 M guanidine chloride, pH 9.6), whereas immediately after laser flash the maximum is localized at 412 nm. The maximum is also localized at 412 nm 0.1 msec after the flash in the absence of guanidine chloride at pH 11.3. Within several milliseconds the maximum is shifted to short-wavelength region by 5-6 nm. This shift is similar to that in the D96N mutant which accompanies the M(N) (M(open)) intermediate formation. The main two differences are: 1) the rate of the shift is slower in the wild-type bacteriorhodopsin, and is similar to the rate of the M to N intermediate transition (t1/2 approximately 2 msec); 2) the shift in the wild-type bacteriorhodopsin is observed at alkaline pH values which are higher than pK of the Schiff base (approximately 10.8 at 1 M NaCl) in the N intermediate with the deprotonated Asp-96. Thus, the M(N) (M(open)) intermediate with open water-permeable inward proton channel is observed only at high pH, when the Schiff base and Asp-96 are deprotonated. The data confirmed our earlier conclusion that the M intermediate observed at lower pH has the closed inward proton channel.  相似文献   

16.
Photoacoustic spectroscopy was applied to study the energetics and the kinetics of the slow intermediates of the bacteriorhodopsin photocycle. An analysis of the modulation frequency dependence of the photoacoustic signal allowed us to estimate the enthalpy changes and the kinetic parameters associated with those intermediates. The effects of pH, salt concentration, and protein aggregation were studied. Three photoacoustic transitions were found. The two low frequency transitions were attributed to O660 and M412, respectively. The third transition was interpreted as resulting from a protein conformational change undetected spectrophotometrically. The frequency spectra were simulated between 5 and 180 Hz at pH's 5.1, 7.0, and 8.9 assuming a branching in the bacteriorhodopsin photocycle at the M412 level. The enthalpy changes associated with M412 and O660 were computed and compared with the experimental values.  相似文献   

17.
A gentle method for the lysis of oral streptococci.   总被引:41,自引:0,他引:41  
Black lipid planar membranes were prepared by incorporating polymers such as polystyrene in a membrane forming solution. The polymerincorporated planar membranes showed greater stability to applied electric fields and have longer lifertimes. Photopotentials were studied under several conditions; with bacteriorhodopsin in the planar membrane; with bacteriorhodopsin in liposomes; with bacteriorhodopsin fragments in suspension; and with bacteriorhodopsin both in the planar membrane and in liposomes. Skulachev's laboratory has reported that bacteriorhodopsin-liposomes develop potentials across a black lipid planar membrane. In our studies, because the polymer incorporated planar membranes are very stable, it was possible to obtain somewhat larger photopotentials in the presence of bacteriorhodopsin ranging between 30–500 mV. The enhancement of bacteriorhodopsin catalyzed photopotentials, found in the presence of Ca++ (or other bivalent cations) and/or applied electric fields, appeared to result from an orientation effect of bacteriorhodopsin at the membrane interface.  相似文献   

18.
The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X-ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH-values. The observed light-induced intensity changes of in-plane X-ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X-ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3-3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at -180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.  相似文献   

19.
Blue light effect on proton pumping by bacteriorhodopsin.   总被引:4,自引:1,他引:3       下载免费PDF全文
Proton pumping in closed vesicular systems containing bacteriorhodopsin that is initiated by an orange flash, is diminished by a subsequent blue flash. This blue light effect is due to light absorbed by the photocycle intermediate M412 (M), which was formed by the orange flash. A kinetic analysis of the blue-light-induced reduction of proton pumping shows that of the two components of M, only the slowly decaying component is involved in the reduction of proton movement. This may be the first correlation between a proton movement and a specific photochemical intermediate of bacteriorhodopsin. Furthermore, we report that blue light, acting on the slowly decaying intermediate, probably causes a movement of the protons in a direction opposite to that normally seen for light absorbed by bacteriorhodopsin.  相似文献   

20.
The interrelation was studied between the phototransient absorbing maximally at 412 nm (M412) and light-induced proton release under steady-state conditions in aqueous suspensions of 'purple membrane' derived from Halobacterium halobium. The decay of M412 was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M412 which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M412 was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M412 without affecting the level of M412 which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca2+ and their antagonists La3+ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed.  相似文献   

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