Abrupt onset of large scale nonproton ion release in purple membranes caused by increasing pH or ionic strength.

The abrupt onset of large scale nonproton ion release by photo-excited purple membrane suspensions has been observed near neutral pH using transient conductivity measurements. At pH 7 and low ionic strength, the conductivity transients due to proton and nonproton ions are of comparable magnitude but of opposite sign: fast proton release and ion uptake, followed by slow proton uptake and ion release. By increasing either the pH or the NaCl concentration, the amplitude of the conductivity transient increases sharply and the signal is then dominated by nonproton ion release. These results can be understood in terms of light-induced changes in the population of counterions condensed at the purple membrane surface caused by changes in the surface charge density. The critical charge density required for condensation to occur is evidently achieved near neutral pH by ionizing dissociable groups on the membrane by either titration (increasing the pH) or shifting their pKs (increasing the ionic strength).     

Large scale nonproton ion release and bacteriorhodopsin's state of aggregation in lipid vesicles. I. Monomers.

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.     

Nonproton ion release by purple membranes exhibits cooperativity as shown by determination of the optical cross-section.

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.     

Actinic light and pH effect on the proton pumping of bacteriorhodopsin

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.     

The quantum efficiency of proton pumping by the purple membrane of Halobacterium halobium.

The quantum yield of H+ release in purple membrane (PM) sheets, and H+ uptake in phospholipid (egg phosphatidylcholine, PC) vesicles containing PM, was measured in single turnover light flashes using a pH-sensitive dye, p-nitrophenol, with rhodopsin as an actinometer. We have also calculated the ratio of H+ released per M412 formed (an unprotonated Shiff-base intermediate formed during the photocycle). In PM sheets, the quantum yield of H+ release depends on the medium. The quantum yield of M412 is independent of salt concentration. The ratio H+/M412 is approximately 1.8 M KC; and approximately 0.64 in 10 mM KCl. Direct measurements of the quantum yield of H+ give approximately 0.7 when the PM is suspended in 0.5 M KC; and 0.25 in 10 mM KCl. Using a quantum yield for M412 formation of 0.3 (Becher and Ebrey, 1977 Biophys J. 17:185.), these measurements also give a H+/M412 approximately 2 at high salt. In PM/PC vesicles, the H+/M412 is approximately 2 at all salt concentrations. The M412 decay is biphasic and the dye absorption change is monophasic. The dissipation of the proton gradient is very slow, taking on the order of seconds. Addition of nigericin (H+/K+ antiporter) drastically reduces the pH changes observed in PM/PC vesicles. This and the observation that the proton relaxation time is much longer than the photochemical cycling time suggest that the protons are pumped across the membrane and there is no contribution as a result of reversible binding and release of protons on just one side of the membrane.     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.     

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin activity of S. aureus cells under nongrowing conditions, and this lytic response was markedly reduced in energy-poisoned cells. In contrast, the detergent had no effect on the activity of autolysins in cell-free systems, and growth in the presence of Triton X-100 did not alter either the cellular autolysin activity or the susceptibility of cell walls to exogenous lytic enzymes. Treatment with either Triton X-100 or penicillin G in the growth medium stimulated release of predominantly acylated intracellular lipoteichoic acid and sensitized staphylococci to Triton X-100-induced autolysis. There was no significant difference in the cell wall and membrane compositions or Triton X-100 binding between the parental strains and the resistant mutants. The resistant mutant TXR1, derived from S. aureus H, had a higher level of L-alpha-glycerophosphate dehydrogenase activity, and its oxygen uptake was more resistant to inhibition by a submicellar concentration (0.008%) of Triton X-100. Growth in the presence of subinhibitory concentrations of Triton X-100 rendered S. aureus H cells phenotypically resistant to the detergent and greatly stimulated the level of oxygen uptake. Membranes isolated from such cells exhibited enhanced activity of the respiratory enzymes succinic dehydrogenase and L-alpha-glycerophosphate dehydrogenase.     

Triton X-100对细菌视紫红质中间产物M_(412)的效应

本文研究用非离子表面活性剂Triton X-100处理后的细菌视紫红质(BR Bacteriorhodo-psin)光循环中间产物M_(412)动力学过程的变化.实验结果表明,用不同浓度的Triton处理pH=6.5的BR体系时,其中间产物M_(412).快衰减成分的半衰期(τ_(1/2)~f)在Triton浓度为0.05%(w/w)附近突然变慢,随着Triton浓度的加大,τ_(1/2)~f又逐渐加快;慢衰减部分的半衰期(τ_(1/2)~s)则随Tri-ton浓度的增加逐渐变慢.BR的生色团峰发生蓝移.说明不同浓度的Triton在水溶液中聚集状态不同,可不同程度地破坏膜脂的液晶态结构,从而导致镶嵌在其中的BR发生构象的变化,使转运质子的氢键通道受到不同程度的影响,故质子泵转运通道发生改变、致使M_(412)的衰减速率改变.     

Roles of functional lipids in bacteriorhodopsin photocycle in various delipidated purple membranes

Purple membrane (PM) is composed of several native lipids and the transmembrane protein bacteriorhodopsin (bR) in trimeric configuration. The delipidated PM (dPM) samples can be prepared by treating PM with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) to partially remove native lipids while maintaining bR in the trimeric configuration. By correlating the photocycle kinetics of bR and the exact lipid compositions of the various dPM samples, one can reveal the roles of native PM lipids. However, it is challenging to compare the lipid compositions of the various dPM samples quantitatively. Here, we utilize the absorbances of extracted retinal at 382 nm to normalize the concentrations of the remaining lipids in each dPM sample, which were then quantified by mass spectrometry, allowing us to compare the lipid compositions of different samples in a quantitative manner. The corresponding photocycle kinetics of bR were probed by transient difference absorption spectroscopy. We found that the removal rate of the polar lipids follows the order of BPG ≈ GlyC < S-TGD-1 ≈ PG < PGP-Me ≈ PGS. Since BPG and GlyC have more nonpolar phytanyl groups than other lipids at the hydrophobic tail, causing a higher affinity with the hydrophobic surface of bR, the corresponding removal rates are slowest. In addition, as the reaction period of PM and CHAPS increases, the residual amounts of PGS and PGP-Me significantly decrease, in concomitance with the decelerated rates of the recovery of ground state and the decay of intermediate M, and the reduced transient population of intermediate O. PGS and PGP-Me are the lipids with the highest correlation to the photocycle activity among the six polar lipids of PM. From a practical viewpoint, combining optical spectroscopy and mass spectrometry appears a promising approach to simultaneously track the functions and the concomitant active components in a given biological system.     

Factors influencing capacitance-based monitoring of microbial growth.

Microbiological impedance devices are routinely used by food and manufacturing industries, and public health agencies to measure microbiological growth. Factors contributing to increases and decreases in capacitance at the culture medium-electrode interface are poorly understood. To objectively evaluate the effects of temperature, cell density and medium conductivity on capacitance, admittance values from an impedance device were standardized; capacitance was converted to susceptance to allow unit comparisons with conductance. Although increases in temperature increased susceptance, a linear relationship could not be established between the change of susceptance with temperature and conductance of the medium. Cell density by itself had no measureable effect on susceptance or conductance, indicating that cells did not impede the movement of ions in the medium or around the electrode. In a low conductivity medium, increases in conductance by the addition of ions resulted in a concomitant increase of susceptance values. However, in a high conductivity medium, increases in conductance resulted in little or no increase of susceptance values because ions saturated the electrode surface. Susceptance increased when Escherichia coli, Pseudomonas aeruginosa, Alcaligenes faecalis and Staphylococcus aureus were grown in high conductivity media because protons produced by metabolically active bacteria balance more charge on the electrode than other ions. Increases in susceptance due to bacterial growth and metabolism in low conductivity media were attributed to both increases in protons and ionic metabolites. These results indicate that capacitance may provide a better measure of microbial growth and metabolism than conductance.     

Effects of detergent environments on the photocycle of purified monomeric bacteriorhodopsin

Time-resolved difference spectra have been obtained for the photocycle of delipidated bacteriorhodopsin monomers (d-BR) in six different detergent micelle environments that were prepared by two new detergent-exchange techniques. A global kinetic analysis of the photocycle spectra for d-BR in each detergent environment was performed. Comparison of these results with those obtained for the photocycle of bacteriorhodopsin in purple membrane (PM) shows that there is one fewer kinetically distinguishable process for monomeric BR between the decay of the K intermediate and the rise of the M intermediate. Assuming a sequential pathway occurs in the photocycle, it appears that the equilibrium between the L and M intermediates is reached much more rapidly in the detergent micelles. This is attributed to a more direct interaction between Asp-85 and the proton on the nitrogen of the Schiff base of retinal for BR in the detergents. Equilibrium concentrations of late photocycle intermediates are also altered in detergents. The later steps of the photocycle, including the decay of the M intermediate, are slowed in detergents with rings in their hydrocarbon region. This is attributed to effects on conformational changes occurring during the decay of M and/or other later photocycle intermediates. The lifetime of dark adaptation of light-adapted d-BR in different detergent environments increases in environments where the lifetime of the M intermediate increases. These results suggest that the high percentage of either unsaturated or methyl-branched lipids in PM and the membranes of other retinal proteins may be important for their effective functioning.     

Isolation and properties of the native chromoprotein halorhodopsin

The native chromoprotein of the light-driven chloride pump halorhodopsin (HR) was isolated from Halobacterium halobium strain L-33 which lacks bacteriorhodopsin but contains 'slow cycling rhodopsin-like pigment' (SR). A membrane fraction was prepared in low salt and dissolved in a high salt medium by the detergents Lubrol PX or octylglucoside. These conditions destroyed the chromophore of SR but not the HR pigment. Chromatography on phenyl-Sepharose and hydroxylapatite produced, in 60% yield, a 230-fold enriched monomeric chromoprotein with an apparent mol. wt. of 20,000. The chromoprotein was stable in 1 M NaCl and 1% octylglucoside and remained stable upon removal of detergent. It reacted with borohydride in the dark and with hydroxylamine in the light. The absorption maximum of the light-adapted state is at 580 + 2 nm and its molar extinction approximately 50,000/M/cm. Upon illumination in the presence of detergent it was converted into a 410 nm absorbing species with concomitant release of protons. A thermal reconversion to the 580 nm species occurred with a half time of 76 s at -6 degrees C. Blue light absorbed by the photoproduct accelerated the re-conversion as well as the re-uptake of protons. Removal of the detergent prevented the light-induced formation of the 410 nm species. Under these conditions a photochemical behaviour similar to that in intact cells and cell vesicles, i.e., a photocycle in the 10-20 ms range was observed. These findings form the basis for functional reconstitution of HR.     

Kinetics of proton release and uptake by channelrhodopsin-2

Electrophysiological experiments showed that the light-activated cation channel channelrhodopsin-2 (ChR2) pumps protons in the absence of a membrane potential. We determined here the kinetics of transient pH change using a water-soluble pH-indicator. It is shown that ChR2 released protons prior to uptake with a stoichiometry of 0.3 protons per ChR2. Comparison to the photocycle kinetics revealed that proton release and uptake match rise and decay of the P(3)(520) intermediate. As the P(3)(520) state also represents the conductive state of cation channeling, the concurrence of proton pumping and channel gating implies an intimate mechanistic link of the two functional modes. Studies on the E123T and S245E mutants show that these residues are not critically involved in proton translocation.     

Sensitivity of Escherichia coli cell membranes to various classes of detergents

The mechanisms of interaction between non-ionic or cationic surfactants with Escherichia coli K-12 cell membranes were studied using an approach based on the registration of changes in the membrane permeability to ethidium bromide, a fluorescent dye for nucleic acids. Triton X-100, a non-ionic detergent, was shown to exert no effect on the permeability of intact cell membranes. Triton X-100 interacted with the bacteria only after treatment with EDTA, a complexing agent for bivalent cations. Cetyltrimethyl ammonium bromide increased the permeability to ethidium bromide and the action of this cationic detergent did not require the pretreatment with the complexing agent. SDS, an anionic detergent, damaged E. coli K-12 and this could be registered by the lowering of intensity of light scattering by the bacterial suspension. The surface charge of E. coli K-12 cells was shown to influence the interaction of ionic detergents with bacterial cell membranes. Its variation by changing the pH of the incubation medium did not make E. coli K-12 sensitive to Triton X-100.     

Energization-induced redistribution of charge carriers near membranes

The electric field arising from proton pumping across a topologically closed biological membrane causes accumulation close to the membrane of ionic charges equivalent to the charge of the pumped protons, positive on the side towards which protons are pumped, negative on the other side. We shall call this the 'active surface charge'. We here use the Poisson-Boltzmann equation to evaluate the effects of zwitterionic buffer molecules and uncharged proteins in the aqueous phase bordering the membrane on the magnitude and ionic composition of the active surface charge. For the positive side of the membrane, the main results are: (1) If the membrane is freely accessible to bulk phase ions, pumped protons exchange with these ions, such that the active surface charge consists of salt cations. (2) If a significant fraction of the ions in bulk solution consists of buffer molecules, then some of the pumped protons will remain close to the membrane and constitute a major fraction of the active surface charge. (3) If a protein layer borders the membrane, a significant part of the transmembrane electric potential difference exists within that protein layer and protons inside this layer dominate the active surface charge. (4) On the negative side of the membrane the corresponding phenomena would occur. (5) All these effects are strictly dependent on the transmembrane electric potential difference arising from proton pumping and would come in addition to the well known effects of buffers and electrically charged proteins on the retention of scalar protons. (6) No additional proton diffusion barrier may be required to account for a deficit in number of protons observed in the aqueous bulk phase upon aeration-induced proton pumping.     

Proportional equilibration of K, Na ions, and sucrose molecules in pig lenses incubated in the presence of the non-ionic detergent Triton X-100

The release of sodium and potassium and the uptake of sucrose molecules was studied in pig lenses incubated in isosmotic sucrose solution in either the presence or absence of 1% Triton X-100 (a non-ionic detergent). This Triton X-100 treatment has been shown to cause severe disruptions of cell membrane integrity. If sodium and potassium were free in the lens fibers as in a dilute aqueous solution, they would be expected to diffuse three to four times faster than sucrose. However, measurements of sodium and potassium release and sucrose uptake in the Triton X-100 treated lenses show a 1:1 equilibration. When pig lenses were incubated in the same solution without detergent, the sucrose uptake was significantly less than the potassium and sodium release. It is postulated that a slow, detergent mediated collapse of protein-water-ion interactions within the lens is the rate-limiting step of the observed equilibration of monovalent cations and sucrose molecules.     

Late events in the photocycle of bacteriorhodopsin mutant L93A

In the photocycle of bacteriorhodopsin (bR) from Halobacterium salinarum mutant L93A, the O-intermediate accumulates and the cycling time is increased approximately 200 times. Nevertheless, under continuous illumination, the protein pumps protons at near wild-type rates. We excited the mutant L93A in purple membrane with single or triple laser flashes and quasicontinuous illumination, (i.e., light for a few seconds) and recorded proton release and uptake, electric signals, and absorbance changes. We found long-living, correlated, kinetic components in all three measurements, which-with exception of the absorbance changes-had not been seen in earlier investigations. At room temperature, the O-intermediate decays to bR in two transitions with rate constants of 350 and 1800 ms. Proton uptake from the cytoplasmic surface continues with similar kinetics until the bR state is reestablished. An analysis of the data from quasicontinuous illumination and multiple flash excitation led to the conclusion that acceleration of the photocycle in continuous light is due to excitation of the N-component in the fast N<-->O equilibrium, which is established at the beginning of the severe cycle slowdown. This conclusion was confirmed by an action spectrum.     

Effects of gamma-irradiation on the electrokinetic properties of purple membranes

The effect of gamma-irradiation (5, 10 and 15 Gy) on the kinetic surface charge of purple membranes (PM) was followed by means of particle microelectrophoresis. The changes in electrophoretic mobility (EPM) were examined at 2, 5 and 26 h, respectively, following irradiation of native PM, and at 2, 26, 50 h and 5 days following irradiation of delipidated PM. It was concluded that the high inhomogeneity of the suspension, even after sonication, largely affects the measured zeta-potential. The 15-Gy treatment significantly increased the net negative surface charge density at 5 and 26 h after irradiation of native PM. However, the opposite effect of approximately twofold reduction of EPM values was derived from simultaneous studies concerning their delipidated form. Low irradiation doses clearly induced an enhancement of negative surface charge density at 2 h post-exposure as well as the formation of unstable structures of delipidated PM. The changes in electrokinetic properties might reflect the specific aggregate formation in both native and delipidated PM. It was suggested that the effect observed of both types of PM was mainly a structural phenomenon possibly related to the modification of functionally active residues. Received: 15 May 1997 / Accepted in revised form: 8 January 1998     

Effect of iodination of the purple membrane on the photocycle of bacteriorhodopsin

Bacteriorhodopsin in the purple membrane of Halobacterium halobium is coupled to a photocycle that results in the release and uptake of protons. The role of tyrosyl residues in the photocycle of bacteriorhodopsin has been investigated by the technique of chemical modifications of these residues by iodination and nitration. The studies indicate that modification of a tyrosyl residue accelerates M₄₁₂ formation, whereas modification of another type of tyrosine residue(s) accessible from the cytoplasmic surface of the purple membrane inhibits M₄₁₂ decay. The results support the hypothesis that a reversible deprotonation of tyrosine residues prior to and after M₄₁₂ formation in the photocycle are steps in the light-driven pathway of H⁺ translocation by bacteriorhodopsin.