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1.
Light-induced release/uptake of both protons and other ions cause transient changes in conductivity in suspensions of purple membrane (PM) fragments (Marinetti, Tim, and David Mauzerall, 1983, Proc. Natl. Acad. Sci. USA, 80:178-180). We find that the release/uptake of nonproton ions with quantum yield greater than 1 is observed at most pHs and ionic strengths. Only at both low pH and low ionic strength is the conductivity transient mostly due to protons. Our hypothesis is that during the photocycle, changes occur in the PM's dense surface charge distribution that result in changes in the number of counterions bound or condensed at the membrane surface. To test this, the PM structure was perturbed with the nonionic detergent Triton X-100. Immediately after addition, Triton does not abolish the nonproton ion movements; in fact at low detergent concentrations (0.02% vol/vol) the signal amplitudes increased considerably. However, when PM is completely solubilized into monomers in Triton, the conductivity transients are due to protons alone, though at lower quantum yield compared with native PM. These results suggest that changes in the surface charge distribution in native PM's photocycle could contribute to proton transfer between the aqueous phase and bR itself. 相似文献
2.
Light-induced conductivity transients have been observed in preparations of bacteriorhodopsin (bR) in phospholipid vesicles at high lipid/protein molar ratios. Under these conditions, bR is known to be dissolved as monomers in the lipid bilayer. The conductivity transients are due mostly to proton movements, including a trans-membrane component. Kinetic resolution of the conductance change due to proton ionophore-induced leakage through the vesicle membrane provides a novel method to quantitate the number of protons pumped, even in heavily buffered solutions. Some of the transient signal seen on the timescale of the bR photocycle is due to nonproton ions but is smaller than that observed in native purple membranes at pH 7 in low salt. Furthermore, when the pH is raised to 8, the very large transient nonproton ion release seen in purple membranes is not seen in the vesicles. This correlates well with previous results (Marinetti, T., and D. Mauzerall, 1986, Biophys. J., 50:405-415), in which the nonproton ion movements observed with native purple membranes were abolished by solubilization in Triton X-100. Thus, the nonproton ion release appears to be a property of bR in the native aggregated state. 相似文献
3.
The amplitudes of the conductivity transients in photoexcited purple membranes were studied as a function of the energy of the actinic flash to determine the optical cross section of the process giving rise to the conductivity transient. Heating of the solution by the absorbed light causes an additional conductivity change and serves as an internal actinometer; the experiment directly yields the ratio of the cross section of ion release/uptake to that for light absorption. In effect, this counts the number of bacteriorhodopsin (bR) molecules involved in the conductivity transient per photon absorbed. At pH 7 in 0.4-0.5 M NaCl, where the conductivity signals are dominated by nonproton ions, the ratio is between 3 and 4, i.e., excitation of any one of several chromophores generates the same ion release signal. The simplest interpretation is that at pH 7 cooperative conformational changes cause a transient change in the surface charge distribution near all the affected bR molecules, resulting in the transient release of numerous counterions. As a comparison, at pH 4 where the signals are due to protons alone, the cross section data indicate that only a single bR molecule is involved in the proton movements. In this case, the results also show that the sum of the primary forward and reverse quantum yields (for the reactions: bR----K) is 0.88 +/- 0.09. 相似文献
4.
The actinic light effect on the bacteriorhodopsin (BR) photocycle kinetics led to the assumption of a cooperative interaction between the photocycling BR molecules. In this paper we report the results of the actinic light effect and pH on the proton release and uptake kinetics. An electrical method is applied to detect proton release and uptake during the photocycle [E. Papp, G. Fricsovszky, J. Photochem. Photobiol. B: Biol. 5 (1990) 321]. The BR photocycle kinetics was also studied by absorption kinetics measurements at 410 nm and the data were analyzed by the local analysis of the M state kinetics [E. Papp, V.H. Ha, Biophys. Chem. 57 (1996) 155]. While at high pH and ionic strength, we found a similar behavior as reported earlier, at low ionic strength the light effect proved to be more complex. The main conclusions are the following: Though the number of BR excited to the photocycle (fraction cycling, fc) goes to saturation with increasing laser pulse energy, the absorbed energy by BR increases linearly with pulse energy. From the local analysis we conclude that the light effect changes the kinetics much earlier, already at the L intermediate state decay. The transient electric signal, caused by proton release and uptake, can be decomposed into two components similarly to the absorption kinetic data of the M intermediate state. The actinic light energy affects mainly the ratio of the two components and the proton movements inside BR while pH has an effect on the kinetics of the proton release and uptake groups at the membrane surface. 相似文献
5.
嵌入紫膜碎片的BLM系统在不同离子强度的介质中产生不同的光电位信号。本文通过在介质中加入离子载体或离子通道剂对紫膜的光电响应信号进行了系统的研究。结果表明:上述光电位信号的差异主要是由紫膜的非质子离子的迁移引起的。与紫膜的质子跨膜迁移相比,这种非质子离子迁移也是由光驱动的,随介质的离子强度的增强而显著增强,但它的迁移速率更快,且产生反向的光电位。用通常方法所获得的光电位信号主要是这两种迁移信号的迭加。三价阳离子(La~(3+))可使紫膜质子泵反向并能抑制质子的吸收,但它似乎并不参与紫膜的非质子离子迁移。 相似文献
6.
Bacteriorhodopsin, a light-driven proton pump found in the purple membrane of Halobacterium salinarum, exhibits purple at neutral pH but its color is sensitive to pH. Here, structures are reported for an acid blue form and an alkaline purple form of wild-type bacteriorhodopsin. When the P622 crystal prepared at pH 5.2 was acidified with sulfuric acid, its color turned to blue with a pKa of 3.5 and a Hill coefficient of 2. Diffraction data at pH 2-5 indicated that the purple-to-blue transition accompanies a large structural change in the proton release channel; i.e. the extracellular half of helix C moves towards helix G, narrowing the proton release channel and expelling a water molecule from a micro-cavity in the vicinity of the retinal Schiff base. In this respect, the acid-induced structural change resembles the structural change observed upon formation of the M intermediate. But, the acid blue form contains a sulfate ion in a site(s) near Arg82 that is created by re-orientations of the carboxyl groups of Glu194 and Glu204, residues comprising the proton release complex. This result suggests that proton uptake by the proton release complex evokes the anion binding, which in turn induces protonation of Asp85, a key residue regulating the absorption spectrum of the chromophore. Interestingly, a pronounced structural change in the proton release complex was also observed at high pH; i.e. re-orientation of Glu194 towards Tyr83 was found to take place at around pH 10. This alkaline transition is suggested to be accompanied by proton release from the proton release complex and responsible for rapid formation of the M intermediate at high pH. 相似文献
7.
The sign of B2, the micro-second component of the photocurrent from oriented purple membrane, is that of positive charge moving away from the purple membrane in the direction of proton release. B2 could be due to internal dipole or proton movement, proton release, or metal cation release. We found that the waveform of B2 is virtually insensitive to changes in the salt concentration as long as it is >40 mM KCl, >5 mM CaCl 2, or >0.5 mM LaCl 3. However, below these limits, B2's apparent rate of decay increases as the salt concentration decreases without any change in the initial amplitude. This salt dependence suggests that B2 is due to a positive charge, either a metal cation or a proton, moving from the membrane into the solution. That the positive charge is not a metal cation is suggested by the waveform of B2 remaining unchanged upon replacing the cations both in solution and in the binding sites of the purple membrane. Direct evidence that the positive charge movement is due to protons was obtained by examining the correlation of B2 with the proton dependent processes of bacteriorhodopsin in buffers and dyes. Based on these observations, we suggest that most, if not all, of the intrinsic B2 component of the photocurrent at moderate salt concentration is due to proton release. The photocurrents from purple membranes whose surface potential has been reduced by delipidation or chemical modification of carboxyl groups with methyl esters were found to be only modestly changed. This suggests that the salt effect is not through its modulation of the surface potential. Rather, we propose that in low salt B2 represents the sum of a proton release from the surface of the purple membrane and a second current component, due to cations moving back towards the membrane, which is only important in low salt. The cation counter current is induced by proton release which creates a transient uncompensated negative charge on the membrane. 相似文献
8.
We have used x-ray diffraction to study the interactions between myelin membranes in the sciatic nerve (PNS) and optic nerve (CNS) as a function of pH (2-10) and ionic strength (0-0.18). The period of myelin was found to change in a systematic manner with pH and ionic strength. PNS periods ranged from 165 to 250 A or more, while CNS periods ranged from 150 to 230 A. The native periods were observed only near physiological ionic strength at neutral or alkaline pH. The smallest periods were observed in the pH range 2.5-4 for PNS myelin and pH 2.5-5 for CNS myelin. The minimum period was also observed for PNS myelin after prolonged incubation in distilled water. At pH 4, within these acidic pH ranges, myelin period increased slightly with ionic strength; however, above these ranges, the period increased with pH and decreased with ionic strength. Electron density profiles calculated at different pH and ionic strength showed that the major structural alteration underlying the changes in period was in the width of the aqueous space at the extracellular apposition of membranes; the width of the cytoplasmic space was virtually constant. Assuming that the equilibrium myelin periods are determined by a balance of nonspecific forces/i.e., the electrostatic repulsion force and the van der Walls attractive force, as well as the short-range repulsion force (hydration force, or steric stabilization), then values in the period-dependency curve can be used to define the isoelectric pH and exclusion length of the membrane. The exclusion length, which is related to the minimum period at isoelectric pH, was used to calculate the electrostatic repulsion force given the other forces. The electrostatic repulsion was then used to calculate the surface potential, which in turn was used to calculate the surface charge density (at different pH and ionic strength). We found the negative surface charge increases with pH at constant ionic strength and with ionic strength at constant pH. We suggest that the former is due to deprotonation of the ionizable groups on the surface while the latter is due to ion binding. Interpretation of our data in terms of the chemical composition of myelin is given in the accompanying paper (Inouye and Kirschner, 1988). We also calculated the total potential energy functions for the different equilibrium periods and found that the energy minima became shallower and broader with increasing membrane separation. Finally, it was difficult to account directly for certain structural transitions from a balance of nonspecific forces.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
9.
We have developed a surface model of purple membrane and applied it in an analysis of the purple-to-blue color change of bacteriorhodopsin which is induced by acidification or deionization. The model is based on dissociation and double layer theory and the known membrane structure. We calculated surface pH, ion concentrations, charge density, and potential as a function of bulk pH and concentration of mono- and divalent cations. At low salt concentrations, the surface pH is significantly lower than the bulk pH and it becomes independent of bulk pH in the deionized membrane suspension. Using an experimental acid titration curve for neutral, lipid-depleted membrane, we converted surface pH into absorption values. The calculated bacteriohodopsin color changes for acidification of purple, and titrations of deionized blue membrane with cations or base agree well with experimental results. No chemical binding is required to reproduce the experimental curves. Surface charge and potential changes in acid, base and cation titrations are calculated and their relation to the color change is discussed. Consistent with structural data, 10 primary phosphate and two basic surface groups per bacteriorhodopsin are sufficient to obtain good agreement between all calculated and experimental curves. The results provide a theoretical basis for our earlier conclusion that the purple-to-blue transition must be attributed to surface phenomena and not to cation binding at specific sites in the protein. 相似文献
10.
本文研究了中性红与紫膜结合,并用直接滴定法和测定结合量法,求得了结合于膜上之中性红本征PK和在不同离子强度时的表观PKa值.从它们的差值中计算得到紫膜表面电位.并得不同盐浓度的表面电位和质子泵效率作了比较.结果说明.阳离子对质子泵效率的影响不能完全用表面电位的变化来说明.这一结果暗示了一定的阳离子对于紫膜质子泵功能的完成有着特殊作用. 相似文献
11.
This paper investigates the effects that surface dipole layers and surface charge layers along the pore mouth-water interface can have on the electrical properties of a transmembrane channel. Three specific molecular sources are considered: dipole layers formed by membrane phospholipids, dipole layers lining the mouth of a channel-forming protein, and charged groups in the mouth of a channel-forming protein. We find, consistent with previous work, that changing the lipid-water potential difference only influences channel conduction if the rate-limiting step takes place well inside the channel constriction. We find that either mouth dipoles or mouth charges can act as powerful ion attractors increasing either cation or anion concentration near the channel entrance to many times its bulk value, especially at low ionic strengths. The effects are sufficient to reconcile the apparently contradictory properties of high selectivity and high conductivity, observed for a number of K+ channel systems. We find that localizing the electrical sources closer to the constriction entrance substantially increases their effectiveness as ion attractors; this phenomenon is especially marked for dipolar distributions. An approximate treatment of electrolyte shielding is used to discriminate between the various mechanisms for increasing ionic concentration near the constriction entrance. Dipolar potentials are far less sensitive to ionic strength variation than potentials due to fixed charges. We suggest that the K+ channel from sarcoplasmic reticulum does not have a fixed negative charge near the constriction entrance; we suggest further that the Ca+2-activated K+ channel from transverse tubule does have such a charge. 相似文献
12.
The isoionic pH of Chromatium chromatophores is 5.2±0.1. At pH 7.7, the net charge on the chromatophore is approx. −1·10 4. If a change in this charge accompanies the oxidation of an electron carrier, the midpoint redox potential ( Em) of that carrier should be a function of the solution ionic strength ( I). of that carrier should be a function of the solution ionic strength ( I). The Em values of P870 and cytochrome c-555 increase strongly with increasing I at low values of I. The Em of cytochrome c-552 also increases with increasing I, though not so strongly. These effects probably cannot be attributed to an influence of I on the activity coefficient of a dissociable ion. We conclude that, when either P870 or cytochrome c-555 loses an electron, no specific ions (including protons) are bound or released in significant amounts, and the absolute value of the charge on the chromatophore decreases. The Em values of the primary and secondary electron acceptors, X and Y, do not depend on I. Because these Em values have been shown previously to depend on pH, we conclude that the uptake of a proton keeps the charge on the chromatophore constant when either X or Y accepts an electron. This means that the primary and secondary electron transfer reactions in Chromatium result in a net decrease in the charge on the photosynthetic membrane. They do not result in the translocation of protons across the membrane. The Em of the soluble flavocytochrome c-552 from Chromatium depends only weakly on I, but depends strongly on the pH. The uptake of a proton appears to keep the net charge on this cytochrome constant upon reduction. 相似文献
13.
The Poisson-Boltzmann equation is modified to consider charge ionogenicity, steric exclusion, and charge distribution in order to describe the perimembranous electrostatic potential profile in a manner consistent with the known morphology and biochemical composition of the cell's glycocalyx. Exact numerical and approximate analytical solutions are given for various charge distributions and for an extended form of the Donnan potential model. The interrelated effects of ionic conditions, bulk pH, ion binding, local dielectric, steric volume exclusion, and charge distribution on the local potential, pH, and charge density within the glycocalyx are examined. Local charge-induced, potential-mediated pH reductions cause glycocalyx charge neutralization. Under certain conditions, local potentials may be insensitive to ionic strength or may decrease in spite of increasing charge density. The volume exclusion of the glycocalyx reduces the local ion concentration, thereby increasing the local potential. With neutral lipid membranes, the Donnan and surface potential agree if the glycocalyx charge distribution is both uniform and several times thicker than the Debye length (approximately 20 A in thickness under physiological conditions). Model limitations in terms of application to microdomains or protein endo- and ectodomains are discussed. 相似文献
14.
The order of proton uptake and release in an aqueous suspension of purple membrane in response to a light flash has been investigated at lowered pH. pH indicator dyes and a flash spectrophotometer were used for the study. At pH 6.6 it was found that the release of protons from the purple membrane precedes uptake, as reported by other investigators. At pH 5.9, 4.9, and 4.1 it was also found that release precedes uptake. These results are not in agreement with those of previous investigators. 相似文献
15.
Replacement of the Arg residue at position 82 in bacteriorhodopsin by Gln or Ala was previously shown to slow the rate of proton release and raise the pK of Asp 85, indicating that R82 is involved both in the proton release reaction and in stabilizing the purple form of the chromophore. We now find that guanidinium chloride lowers the pK of D85, as monitored by the shift of the 587-nm absorbance maximum to 570 nm (blue to purple transition) and increased yield of photointermediate M. The absorbance shift follows a simple binding curve, with an apparent dissociation constant of 20 mM. When membrane surface charge is taken into account, an intrinsic dissociation constant of 0.3 M fits the data over a range of 0.2-1.0 M cation concentration (Na+ plus guanidinium) and pH 5.4-6.7. A chloride counterion is not involved in the observed spectral changes, as chloride up to 0.2 M has little effect on the R82Q chromophore at pH 6, whereas guanidinium sulfate has a similar effect to guanidinium chloride. Furthermore, guanidinium does not affect the chromophore of the double mutant R82Q/D85N. Taken together, these observations suggest that guanidinium binds to a specific site near D85 and restores the purple chromophore. Surprisingly, guanidinium does not restore rapid proton release in the photocycle of R82Q. This result suggests either that guanidinium dissociates during the pump cycle or that it binds with a different hydrogen-bonding geometry than the Arg side chain of the wild type. 相似文献
16.
Light-driven potassium ion uptake in Halobacterium halobium is mediated by bacteriorhodopsin. This uptake is charge-balanced by sodium ions and not by proton release. Light-induced shifts in concentrations of divalent cations were found to be negligible. The transient changes in extracellular pH (alkaline overshoot) can be understood by the concomitant processes of ATP synthesis, proton/sodium exchange and potassium uptake. The driving force of potassium ion uptake is the membrane potential, no ATP-dependent potassium transport process is found. Fluorescence measurements indicate a high permeability of the membrane to potassium ions compared to sodium ions. Therefore the potassium ion diffusion potential contributes to the membrane potential (about 30 mV/decade) and thereby influences the ATP level. Sudden enhancement of the diffusion potential by the potassium ionophore monactin leads to the expected transient increase in cellular ATP level. Due to the large size (up to 100-fold) of the potassium ion gradient and its high capacity (intracellular concentration up to 3 M) the potassium ion gradient can well serve the cell as a long term storage form of energy. 相似文献
17.
Several observations have already suggested that the carboxyl groups are involved in the association of divalent cations with bacteriorhodopsin (Chang et al., 1985). Here we show that at least part of the protons released from deionized purple membrane (`blue membrane') samples when salt is added are from carboxyl groups. We find that the apparent pK of magnesium binding to purple membrane in the presence of 0.5 mM buffer is 5.85. We suggest this is the pK of the carboxyl groups shifted from their usual pK because of the proton concentrating effect of the large negative surface potential of the purple membrane. Divalent cations may interact with negatively charged sites on the surface of purple membrane through the surface potential and/or through binding either by individual ligands or by conformation-dependent chelation. We find that divalent cations can be released from purple membrane by raising the temperature. Moreover, purple membrane binds only about half as many divalent cations after bleaching. Neither of these operations is expected to decrease the surface potential and thus these experiments suggest that some specific conformation in purple membrane is essential for the binding of a substantial fraction of the divalent cations. Divalent cations in purple membrane can be replaced by monovalent, (Na + and K +), or trivalent, (La +++) cations. Flash photolysis measurements show that the amplitude of the photointermediate, O, is affected by the replacement of the divalent cations by other ions, especially by La +++. The kinetics of the M photointermediate and light-induced H + uptake are not affected by Na + and K +, but they are drastically lengthened by La +++ substitution, especially at alkaline pHs. We suggest that the surface charge density and thus the surface potential is controlled by divalent cation binding. Removal of the cations (to make deionized blue membrane) or replacement of them (e.g. La +++-purple membrane) changes the surface potential and hence the proton concentration near the membrane surface. An increase in local proton concentration could cause the protonation of critical carboxyl groups, for example the counter-ion to the protonated Schiff's base, causing the red shift associated with the formation of both deionized and acid blue membrane. Similar explanations based on regulation of the surface proton concentration can explain many other effects associated with the association of different cations with bacteriorhodopsin. 相似文献
18.
Small light-induced changes in the conductivity of light-adapted purple membrane suspended in strong electrolyte solutions were detected. The method used involved modulated light and a phase sensitive detector and it allowed us to detect accurately changes as small as 0.0001% in the conductivity of the suspension. The light-induced conductivity changes turned out to be composed of at least two different event: a small fast increase in conductivity (tau approximately 2 ms) followed by a slower and larger decrease in this parameter (tau = 70 ms-80 ms). The effects of pH and temperature on these changes were studied. Both events reached maximal values around neutral pH and approached zero at both high and low pH's. Heating the suspension decreased the photoconductivity change and Arrhenius plots of the data showed breaks around 31 degrees C. It is suggested that the conductivity changes reflect changes in the surface charge of the membrane and can be used to follow the kinetics of the conformational changes occurring in the system. 相似文献
19.
The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively. 相似文献
20.
An alkaline suspension of light-adapted purple membrane exposed to continuous light showed a large absorption depletion at 580 nm and a small increase around 350 nm. We attribute this absorption change to an efficient photoconversion of bR570 into a photoproduct N (P,R350), which has a major absorption maximum between 550 and 560 nm but has lower absorbance than bR570. N was barely detectable at low pH, low ionic strength, and physiological temperature. However, when the thermal relaxation of N to bR570 was inhibited by increasing pH, increasing ionic strength, and decreasing temperature, its relaxation time could be as long as 10 s at room temperature. N is also photoactive; when it is present in significant concentrations, e.g., accumulated by background light, the flash-induced absorption changes of purple membrane suspensions were affected. Double-excitation experiments showed an M-like photoproduct of N,NM, with an absorption maximum near 410 nm and a much longer lifetime than M412. It may be in equilibrium with an L-like precursor NL. We suggest that N occurs after M412 in the photoreaction cycle and that its photoproduct NM decays into bR570. Thus, at high pH and high light intensity, the overall photoreaction of bR may be approximated by the two-photon cycle bR570----M412----N----(NL----NM)----bR570, whereas at neutral pH and low light intensity it can be described by the one-photon cycle bR570----M412----N----O640----bR570.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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