The effect of antibiotics on the photocycle and protoncycle of purple membrane suspensions.

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.     

Stimulation of ATP synthesis in Halobacterium halobium R1 by light-induced or artificially created proton electrochemical potential gradients across the cell membrane

The relationship between proton movement and phosphorylation in Halobacterium halobium R₁ has been investigated under anaerobic conditions. The light-induced changes in the bacteriorhodopsin are accompanied by proton movements across the cell membrane which result in pH changes in the suspending medium. The initial alkaline shift is shown to be closely paralleled by (and hence correlated with) ATP synthesis. Acidification of the medium in the presence of valinomycin, under conditions of low external potassium, brings about ATP synthesis in the dark.     

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane. The quenching effect of blue light

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.     

Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase

Light-mediated activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in intact spinach chloroplasts (Spinacia oleracea L.) is enhanced in the presence of 10⁻⁵ molar external free Ca²⁺. The most pronounced effect is observed during the first minutes of illumination. Ruthenium red, an inhibitor of light-induced Ca²⁺ influx, inhibits this Ca²⁺ stimulated activation. In isolated stromal preparations, the activation of fructose-1,6-bisphosphatase is already enhanced by 2 minutes of exposure to elevated Ca²⁺ concentrations in the presence of physiological concentrations of Mg²⁺ and fructose-1,6-bisphosphate. Maximal activation of the enzyme is achieved between 0.34 and 0.51 millimolar Ca²⁺. The Ca²⁺ mediated activation decreases with increasing fructose-1,6-bisphosphate concentration and with increasing pH. The data are consistent with the proposal that the illumination of chloroplasts leads to a transient increase of free stromal Ca²⁺. In dark-kept chloroplasts the steady-state concentration of free stromal Ca²⁺ is 2.4 to 6.3 micromolar as determined by null point titration. These observations support our previous proposal that light-induced Ca²⁺ influx into chloroplasts does not only influence the cytosolic concentration of free Ca²⁺ but also regulates enzymatic processes inside the chloroplast.     

Action of phenazine methyl sulfate,inhibitors, and uncouplers on the light-induced proton transport by cells ofRhodospirillum rubrum

Summary Suspensions of log phase cells ofRhodospirillum rubrum at pH 5.5 show a light-induced decrease in the pH of the medium which is reversed during the subsequent dark period. The velocity and magnitude of the pH change were the same whether the cells were bubbled with air, CO₂-free air or N₂ during experimentation. The pH response is temperature dependent. Phenazine methyl sulfate (PMS) at concentrations above 0.05mm stimulates the light-induced pH change. PMS at 1mm gives a 2-fold increase in the initial rate upon illumination and a 1.5-fold increase in the total change in pH after 2 min of illumination. The inhibition of the proton transport by 10 g/ml antimycin A or 20 m 2-n-heptyl-4-hydroxyquinoline-N-oxide can be partially relieved by PMS. However, inhibition of the light-induced proton transport with 0.5mm 2,4-dinitrophenol or 3 m carbonylcyanide-m-chlorophenylhydrazone (CCCP) cannot be overcome by addition of PMS. Valinomycin, at a concentration of 3 m, caused a slight stimulation of the light-induced proton transport in the presence of 200mm KCl. The inhibition of proton transport by 3 m CCCP was partially relieved with 3 m valinomycin in the presence of 200mm KCl, but the antibiotic was without effect when the cells were suspended in 200mm NaCl. The results are discussed in terms of current theories of the action of PMS, antimycin A, valinomycin, and uncouplers on the light-induced electron flow and photophosphorylation inR. rubrum.     

Comparison of the electrochemical proton gradient and phosphate potential maintained by Rhodospirillum rubrum chromatophores in the steady state.

A technique for the estimation of light-induced membrane potential in chromatophores is described. It is based on measurement of light-induced enhancement in fluorescence of 8-anilinonaphthalene sulfonic acid, which is calibrated by known K⁺ diffusion potentials. The electrochemical proton gradient (Δ_μH+^?) formed during lightinduced electron transport in Rhodospirillum rubrum chromatophores amounts to 250 mV, which is almost equally distributed between the membrane potential and the pH gradient as measured by changes in the fluorescence of anilinonaphthalene sulfonate and 9-amino acridine. Addition of the permeant anion, NaSCN, or of NH₄Cl reduces the overall Δ_μH+^? by less than 20% but changes its distribution between the pH gradient and the membrane potential so that with NaSCN it is composed mainly of the first and with NH₄Cl mainly of the second. Initiation of phosphorylation causes a drop of about 50 mV in the measured Δ_μH+^?. In the absence of salts, the drop is observed in both components, although two-thirds of it are reflected in the membrane potential. In the presence of NaSCN or NH₄Cl the 50-mV drop is exclusively recorded in the pH gradient or in the membrane potential, respectively. The steady-state phosphate potential maintained during electron transport was found to change in parallel to the Δ_μH+^?, but exceeded it by 60 to 80 mV when based on a stoichiometry of two protons translocated per ATP synthesized.     

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP⁺ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP⁺ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH₂) to NADP⁺. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH₂ as the electron donor, NADP⁺ had no effect on the light-induced redox changes of cytochromes: with or without NADP⁺, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP⁺ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b₅₅₉. Cytochrome b₅₅₉ appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP⁺) for the electron flow from water.     

Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles.

Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na⁺ extrusion through an antiport system, and the ability of K⁺ and Cl^? to act as counterions to the electrogenic movement of H⁺. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K⁺. The vesicle membrane is poorly permeable to K⁺, as shown by: lack of a K⁺ diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K⁺, and direct measurements of the slow rate of K⁺ influx during illumination. The finding that the rate of K⁺ uptake is a linear function of external K⁺ concentration between 0 and 1 m is inconsistent with the existence of a specific K⁺ permeation mechanism in these vesicles. Since at external K⁺ concentrations < 1.4 m the extrusion of Na⁺ during illumination proceeds much more rapidly than K⁺ influx, it must be concluded that the vesicles also lose Cl^? and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K⁺ is increased, either by increasing the external K⁺ concentration or by adding valinomycin.     

Effects of Alkalinization and ATPase Inhibition on Stromal Free Mg2+ Concentration in Spinach Chloroplasts

Our earlier studies indicate that stromal alkalinization is essential for light-induced increase in free Mg²⁺ concentration ([Mg²⁺]) in chloroplast. Stromal [Mg²⁺] was increased by dark incubation of chloroplasts in the K⁺-gluconate medium (pH 8.0), or by NH₄Cl. These results indicate that stromal alkalinization can induce an increase in stromal [Mg²⁺] without illumination. Some inhibitors of envelope proton-translocating ATPase activity involved in H⁺ efflux inhibited the alkalinization-induced increase in [Mg²⁺].     

The effects of tetraphenylboron on energy-linked reactions in spinach chloroplasts

1. The inhibitory action of tetraphenylboron, a lipid-soluble anion, on the proton uptake, the photophosphorylation and the light-induced quenching of the fluorescence of 9-aminoacridine by spinach chloroplasts was studied.2. The inhibition of the three processes by tetraphenylboron was transient; the proton uptake was affected to a much smaller extent than either the photophosphorylation or the fluorescence quenching.3. The inhibitory effects of tetraphenylboron on the proton uptake and the fluorescence quenching of 9-aminoacridine were qualitatively the same in CF₁-depleted chloroplasts, that were recoupled with N,N′-dicyclohexylcarbodiimide (DCCD).4. The reversal of the fluorescence quenching of 9-aminoacridine upon addition of tetraphenylboron in the light was found to be very fast, being completed within the response time of the apparatus.5. The presence of tetraalkylammonium salts in the incubation medium prevented the inhibitory effect of tetraphenylboron.6. Tetraphenylboron disappeared from the chloroplast suspension in a light-dependent irreversible way; in the dark no ‘ptake’ of tetraphenylboron could be detected.7. The effects of tetraphenylboron may be explained by the presence of groups with a high affinity for tetraphenylboron in the membrane; these groups become protonated upon illumination of the chloroplasts.     

The effect of diaminodurene on the delayed light and the carotenoid band shift in Rhodopseudomonas spheroides

1. When 2,3,5,6-tetramethyl-p-phenylenediamine (diaminodurene), which is an activator of cyclic electron flow, was added to chromatophores isolated from the photosynthetic bacterium, Rhodopseudomonas spheroides, it caused a large increase in the emission of delayed light measured at 5–10 ms after excitation. This increase was pH dependent, and ranged from 5–100 times the control intensity. Substances that counteract light-induced proton uptake, such as ammonium salts, amines and nigericin, caused a further increase in the delayed light emission. These compounds also markedly slowed a characteristic decline of the delayed light that occurs during sustained illumination. This decline in the delayed light may be related to the quenching of prompt fluorescence that is seen in the presence of diaminodurene. Substances, like valinomycin, that dissipate the membrane potential, almost completely abolish the diaminodurene-catalyzed increase in the delayed light.     

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H+, K+ and Na+ in phospholipid vesicles

Rates of M⁺/H⁺ exchange (M⁺=K⁺, Na⁺) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M⁺ and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=ΔpH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M⁺ dissociation constant, K_M, of the M⁺ bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H⁺ bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M⁺ and an anion) by forming 1:2 complexes with valinomycin–M⁺ or nonactin–M⁺. (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K_M increases. The decreases/increases in K_M mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.     

Determination of ΔpH in cholinergic synaptic vesicles: Its effect on storage and release of acetylcholine

The interior of purified cholinergic Torpedo vesicles is acidic, pH_in = 5.2 at external pH = 7.4. The internal pH changes linearily as a function of external pH yielding ΔpH = 2.0 and 2.5 at pH_out = 6.3 and 9.1 respectively. The proton translocator carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) + the ionophore valinomycin dissipate the proton gradient across the vesicular membrane and concurrently induce acetylcholine release from vesicles suspended in K⁺ buffer. The effect of FCCP + valinomycin is not sensitive to external pH values between 6.3 and 9.1 and is diminished at lower external pH. The possible role of intravesicular pH and of the proton electrochemical gradient in the storage of acetylcholine within cholinergic vesicles is discussed.     

Light dependence of the decay of the proton gradient in broken chloroplasts

The initial rates and steady-state values of proton uptake by broken chloroplasts have been measured as functions of light intensity at various concentrations of chlorophyll, pyocyanine, supporting electrolyte, buffer, as well as pH and temperature. Kinetic analysis of the data shows that the rate of decay of proton gradient due to backward leakage depends on light intensity. Under steady illumination, the decay constant k_L is equal to k_D + mR₀, where R₀ is the initial rate of proton uptake which is a function of light intensity, k_D is the decay constant in the dark and m is a parameter which is independent of light intensity. Treatment of chloroplasts with lysolecithin, neutral detergent, 2,4-dinitrophenol, or valinomycin in the presence of K⁺ increases k_D without affecting m. Treatment with N,N′-dicyclohexylcarbodiimide or adenylyl imidodiphosphate under appropriate conditions decreases m without affecting k_D. Treatment with glutaraldehyde makes k_L independent of light intensity and hence m = 0. These results suggest that the light-dependent part (mR₀) of k_L is due to leakage of protons through the coupling factor (CF₁-CF₀) complex which can open or close depending on light intensity and that the light-independent part (k_D) of the decay constant k_L is due to proton leakage elsewhere.     

The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components

The effects of serum components and amino acids on the uptake and cytotoxicity of NiCl₂ were examined in cultured Chinese hamster ovary (CHO) cells. CHO cells maintained in a minimal salts/glucose medium accumulated 10-fold more⁶³Ni than did cells maintained in complete medium supplemented with 10% fetal bovine serum. Cell-surface binding of⁶³Ni appeared to account for the majority of this increased accumulation of cell-associated nickel observed in the simple maintenance medium since such increases were reduced 70% by trypsin treatment. The addition of the Ni²⁺-binding amino acids cysteine or histidine to the salts/glucose medium markedly decreased⁶³Ni accumulations, an effect not observed following addition of any of several amino acids that do not bind Ni²⁺. Supplementation of the salts/glucose medium with fetal bovine serum decreased in a concentration dependent fashion both the⁶³Ni²⁺ uptake and cell detachment caused by Ni²⁺, while dialyzed (amino acid-free) serum was 3–5-fold less effective than undialyzed serum at reducing⁶³Ni²⁺ uptake and similarly exhibited only a slight protective effect against nickel-induced cytotoxicity. Supplementation of dialyzed serum with cysteine at levels approximating those in whole serum partially restored its inhibitory activity toward nickel uptake by cells and restored completely its inhibition of nickel's cytotoxicity, indicating the predominant role of specific amino acids over serum proteins in regulating the uptake and subsequent cytotoxicity of Ni²⁺. Addition of cysteine to the salts/glucose medium during a 2 h exposure of cells to either 100 μM HgCl₂ or 1 mM NiCl₂ masked the cytotoxic effects of these metal ions. These results demonstrate the importance of extracellular small molecular weight metal ion chelators in altering the biological effects of metal ions at the level of metal uptake.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Factors affecting the development of the capacity for ATP formation in isolated chloroplasts

Full development of the capacity for ATP formation in isolated thylakoid membranes coincides with the beginning of illumination. Indeed, the yield of ATP per ms of illumination is about twice as great during the first 15 ms of high-intensity illumination as it is thereafter. The presence of valinomycin and K⁺ prevents the formation of a membrane potential (as indicated by the obliteration of most of the change in absorbance at 518 nm) and at the same time delays the formation of the capacity for ATP synthesis for many milliseconds. Presumably, phosphorylation is initially dependent on a rapidly formed membrane potential, whereas after a delay a ΔpH sufficient to drive ATP formation forms. The actual duration of this delay depends on the phosphoryl group transfer potential (i.e., ΔG_ATP) of the ATP-synthesizing reaction. If the delay in the presence of valinomycin and K⁺ represents the time required to develop a ΔpH capable of driving phosphorylation by itself, then the effect of ΔG_ATP on the duration of the delay suggests that the onset of phosphorylation is determined by the magnitude of the electrochemical potential of protons and not by factors affecting the activation of the coupling factor enzyme. The initial ATP formation, which is almost entirely dependent on the electrical potential, should not be affected by the electrically neutral exchange of cations catalyzed by nigericin. When the external pH is 7.0 this seems to be true, since the ATP synthesis which is initially sensitive to valinomycin and K⁺ is largely insensitive to nigericin and K⁺. However, when the external pH is 8.0 the response to nigericin is exactly the opposite and the ATP formation which is sensitive to valinomycin is also abolished by nigericin. These data suggest that there may be either an energetic requirement for both a ΔpH and membrane potential at alkaline pH or a non-energetic requirement for a minimum proton activity in the initiation of phosphorylation.     

Neoaplectana carpocapsae: Nematode accumulations on chemical and bacterial gradients

That infective juveniles of the nematode Neoaplectana carpocapsae accumulated specifically around certain host insect larvae was previously reported by us. In this work, the nematode's behavior was tested on defined chemical and bacterial gradients to determine whether these stimuli could cause the phenomenon. Nematode accumulations occurred around the peaks of certain gradients and, with NaCl, the initial accumulation rate was directly proportional to concentration up to 15 mM. Since the nematode did not respond to K-acetate, K and acetate salts were used to analyze responses to different ions. Maximal accumulations were observed with 60 mM Na⁺, 6 mM Mg²⁺, 0.75–7.5 mM Ca²⁺, and 6 mM CO₃^2?. Accumulations to concentrations of Cl^?, basic pH, and gram-negative bacteria were also observed. Acidic pH, 2.5, and 7.5 mM NH₄⁺, repelled nematodes.     

Photophosphorylation as a function of illumination time. I. Effects of permeant cations and permeant anions

(1) Very brief periods of illumination do not initiate photophosphorylation in isolated chloroplast lamellae. The time of illumination required before any phosphorylation can be detected is inversely proportional to the light intensity. At very high intensities, phosphorylation is initiated after illumination for about 4 ms.(2) There is no similar delay in the initiation of electron transport. The rate of electron transport is very high at first but declines at about the time the capacity for ATP synthesis develops. When the chloroplasts are uncoupled with gramicidin the high initial rate persists.(3) Various ions which permeate the thylakoid membrane (K⁺ or Rb⁺ in the presence of valinomycin, SCN^?, I^?, or ClO₄^?) markedly increase the time of illumination required to initiate phosphorylation. Potassium ions in the presence of valinomycin increase the delay to a maximum of about 50 ms whereas thiocyanate ions increase the delay to a maximum of about 25 ms. The effects of K⁺ with valinomycin and the effect of SCN^? are not additive. Permeant ions and combinations of permeant ions have little or no effect on phosphorylation during continuous illumination.(4) The reason for the threshold in the light requirement and the reason for the effect of permeant ions thereon are both obscure. However, it could be argued that the energy for phosphorylation initially resides in an electric potential gradient which is abolished by migration of ions in the field, leaving a more slowly developing proton concentration gradient as the main driving force for phosphorylation during continuous illumination. If so, the threshold in the presence of permeant ions should depend on internal hydrogen ion buffering.     

The effect of charged lipids on bacteriorhodopsin membrane reconstitution and its photochemical activities

Bacteriorhodopsin (BR) was reconstituted into artificial lipid membrane containing various charged lipid compositions. The proton pumping activity of BR under flash and continuous illumination, proton permeability across membrane, as well as the decay kinetics of the photocycle intermediate M₄₁₂ were studied. The results showed that lipid charges would significantly affect the orientation of BR inserted into lipid membranes. In liposomes containing anionic lipids, BRs were more likely to take natural orientation as in living cells. In neutral or positively charged liposomes, most BRs were reversely assembled, assuming an inside out orientation. Moreover, the lipids charges also affect BR’s M intermediate kinetics, especially the slow component in M intermediate decay. The half-life M_412s increased significantly in BRs in liposomes containing cationic lipids, while decreased in those in anionic liposomes.