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1.
2.
W.S. Chow  J. Barber 《BBA》1980,589(2):346-352
1. When suspended in a low cation-containing medium, chloroplast thylakoid membranes and carboxymethyl-cellulose particles quench the fluorescence from 9-aminoacridine (Searle, G.F.W. and Barber, J. (1978) Biochim. Biophys. Acta 502, 309–320).2. Relief of this quenching is achieved by adding cations to the suspension medium with the order of effectiveness being C3+ > C2+ > C+, indicating that the fluorescence acts as an indicator of the surface electrical potential.3. Using the Gouy-Chapman theory, the differential effect of divalent (methyl viologen) and monovalent (K+) cations has been used to calculate surface charge densities.4. The calculations indicate that the surface charge density on the thylakoids significantly increases when cations are added to the low cation-containing medium. Under the same conditions the surface charge density of glutaralde-hyde-fixed thylakoids and carboxymethyl-cellulose particles remained essentially constant.5. It is argued that the 9-aminoacridine technique is able to probe localized areas on the membrane surface and that the variability of the surface charge density of untreated thylakoids may be due to redistribution of charges associated with membrane stacking as suggested by Barber and Chow (Barber, J. and Chow, W.S. (1979) FEBS Lett. 105, 5–10).  相似文献   

3.
Low concentrations (~ 3 mm) of salts of monovalent cations such as Na+, K+, and tetraethylammonium were found to decrease the turbidity of chloroplast suspensions. The turbidity changes (Δ540) had the same kinetics, salt concentration dependence, and pH dependence as the monovalent cation-induced decreases in chlorophyll a fluorescence (9), suggesting that structural changes are the cause of the associated increases in spillover. Electron microscopy revealed that the grana are stacked when spillover is inhibited (in the absence of salts or the presence of divalent cations) and that monovalent cations cause the grana to unstack, thereby promoting spillover.  相似文献   

4.
G.F.W. Searle  J. Barber 《BBA》1978,502(2):309-320
The addition of 9-aminoacridine monohydrochloride to carboxymethyl-cellulose particles or azolectin liposomes suspended in a low cation medium results in a quenching of its fluorescence. This quenching can be released on the addition of cations. The effectiveness of cations is related only to their valency in the series of salts tested, being monovalent < divalent < trivalent, and is independent of the associated anions. These results indicate an electrical rather than a chemical effect, and the relative effectiveness of the various cations can be predicted by the application of classical electrical double layer theory. Fluorescence quenching can also be released on protonation of the fixed negatively charged ionisable groups, and the quenching release curve follows the ionisation curve of these groups.We postulate that when 9-aminoacridine molecules are in the electrical diffuse layer adjacent to the charged surface their fluorescence is quenched, probably due to aggregate formation. As cations are added the 9-aminoacridine concentration at the surface falls as it is displaced into the bulk solution, where it shows a high fluorescence yield with a fluorescence lifetime of 16.3 ns. The fluorescence quenching is associated with an absorbance decrease, which is pronounced with carboxymethyl-cellulose particles and can probably be attributed to self-shielding.The negative charges carried by lipoprotein membranes are primarily due to carboxyl and phosphate groups. Therefore these results with carboxymethyl-cellulose (carboxyl) and azolectin (phosphate) support our earlier suggestion that 9-aminoacridine may be used to probe the electrical double layer associated with negatively charged biological membranes.  相似文献   

5.
Diatoms show a special organisation of their plastid membranes, such that their thylakoids span the entire plastid in bands of three. While in higher plants the interaction of the light harvesting complex II and photosystem II with divalent cations (especially Mg2+) was found to take part in the interplay of electrostatic attraction and repulsion in grana membrane appression, for diatoms the key players in maintaining proper membrane distances were not identified so far. In this work, we investigated the changes in the thylakoid architecture of Thalassiosira pseudonana in reaction to different salts by using circular dichroism and fluorescence spectroscopy in combination with other techniques. We show that divalent cations have an important influence on optimal pigment organisation and thus also on maintaining membrane appression. Thereby, monovalent cations are far less effective. The concentration needed is in a physiological range and fits well with the values obtained for higher plant grana stacking, despite the fact that strict protein segregation as seen in higher plant grana is missing.  相似文献   

6.
We studied the effects of a variety of cations on chlorophyll fluorescence yield of broken chloroplasts prepared under carefully controlled ionic conditions. In the absence of light-induced electron transport and associated proton pumping, two types of cation-induced chlorophyll fluorescence changes could be distinguished in broken chloroplasts. These are termed "reversible" and "irreversible" fluorescence yield changes. Reversible fluorescence yield changes are characterized by antagonistic effects of monovalent and divalent cations and are prevented by the presence of 5 mM Mg2+ in the suspending media. Reversible-type fluorescence yield changes show little or no dependence on the structure, lipid solubility, or coordination number of the cation, but depend strictly on the net positive charge carried by the ion. It is proposed that these fluorescence changes are brought about through the interaction of monovalent or divalent cations with an electrical double layer at the interface of the outer surface of the thylakoid membrane and the surrounding aqueous solution. The results are interpreted in terms of the Gouy-Chapman theory of the diffuse double layer, indicating that the thylakoid outer surface bears an excess fixed negative charge density of about 2.5 muC/cm2, or approximately 1 negative charge per 640 A2 of membrane surface. Chlorophyll fluorescence quenching in isolated broken chloroplasts suspended in media containing 5 mM MgCl2 is also observed on addition of certain polyvalent cations to the medium. This type of cation-induced fluorescence change appears to be largely irreversible and may occur through specific binding of the cation to the thylakoid as a result of the high electrostatic attraction exerted by the negatively charged membrane surface.  相似文献   

7.
G.F.W. Searle  J. Barber 《BBA》1979,545(3):508-518
The amphipathic fluorescence probe, 2-p-toluidinonaphthalene-6-sulphonate has been used to investigate the surface electrical properties of chloroplast thylakoid membranes. The fluorescence yield of 2-p-toluidinonaphthalene-6-sulphonate in aqueous solution increases on addition of hypotonically shocked chloroplast, and the emission maximum shifts towards the blue to 440 nm, although the emission spectrum is somewhat distorted by chloroplast pigment absorption.The intensity of 2-p-toluidinonaphthalene-6-sulphonate fluorescence is further increased on adding salts to the membrane suspension, and changes of >100% are routinely observed. Similar observations have also been made with soya bean phospholipid (azolectin) liposomes. The magnitude of the fluorescence increase is dependent on membrane concentration, being more pronounced at high surface area/suspending volume ratios. The effect of salt addition appears to be that of shielding the fixed negative charges on the membrane surface, thus increasing the fraction of 2-p-toluidinonaphthalene-6-sulphonate molecules at the surface, where the 2-p-toluidinonaphthalene-6-sulphonate has a higher fluorescence yield than in free aqueous solution. This concept is supported by the fact that the effectiveness of salts in increasing 2-p-toluidinonaphthalene-6-sulphonate fluorescence is as predicted by classical electrical double layer theory: governed mainly by the charge carried by the cation with an order of effectiveness C3+ > C2+ > C+, and not by the chemical nature of the cation or by the nature of its co-ion.It has been argued that the chlorophyll fluorescence yield, controlled by the cation composition of the suspending medium follows the total diffusible positive charge density at the thylakoid membrane surface (Barber, J., Mills, J. and Love, A. (1977) Febs. Lett. 74, 174–181). Although the cation induced 2-p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yield changes show similar characteristics, there are also distinct differences between the two phenomena particularly when cations are added to chloroplasts initially suspended in a virtually cation-free medium. Therefore it is concluded that although both 2-p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yields are governed by the electrical properties of the thylakoid membrane surface, the mechanism controlling their cation sensitivity is not the same.  相似文献   

8.
The addition of 9-aminoacridine monohydrochloride to carboxymethyl-cellulose particles or azolectin liposomes suspended in a low cation medium results in a quenching of its fluorescence. This quenching can be released on the addition of cations. The effectiveness of cations is related only to their valency in the series of salts tested, being monovalent less than divalent less than trivalent, and is independent of the associated anions. These results indicate an electrical rather than a chemical effect, and the relative effectiveness of the various cations can be predicted by the application of classical electrical double layer theory. Fluorescence quenching can also be released on protonation of the fixed negatively charged ionisable groups, and the quenching release curve follows the ionisation curve of these groups. We postulate that when 9-aminoacridine molecules are in the electrical diffuse layer adjacent to the charged surface their fluorescence is quenched, probably due to aggregate formation. As cations are added the 9-aminoacridine concentration at the surface falls as it is displaced into the bulk solution, where it shows a high fluorescence yield with a fluorescence lifetime of 16.3 ns. The fluorescence quenching is associated with an absorbance decrease, which is pronounced with carboxymethyl-cellulose particles and can probably be attributed to self-shielding. The negative charges carried by lipoprotein membranes are primarily due to carboxyl and phosphate groups. Therefore these results with carboxymethyl-cellulose (carboxyl) and azolectin (phosphate) support our earlier suggestion that 9-aminoacridine may be used to probe the electrical double layer associated with negatively charged biological membranes.  相似文献   

9.
Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory.When a salt was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO4, MgCl2, CaCl2) were effective at concentrations lower than those of monovalent cation salts (NaCl, KCl, Na2SO4) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5–5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H+ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (?1.9 ± 0.5) · 10?3 elementary charge per Å2, and the surface potential of about ?100 mV in the presence of about 0.1 mM divalent cation or 5 mM monovalent cation were calculated.  相似文献   

10.
11.
Summary (1) When salts are added to buffered suspensions of membrane fragments containing the fluorochrome 1-anilino-8-naphthalenesulfonate (ANS), there is an increased fluorescence. This is caused by increased binding of the fluorochrome; the intrinsic fluorescence characteristics of the bound dye remain unaltered. These properties make ANS a sensitive and versatile indicator of ion association equilibria with membranes. (2) Alkali metal and alkylammonium cations bind to membranes in a unique manner. Cs+ binds most strongly to rat brain microsomal material, with the other alkali metals in the order Cs+>Rb+>K+>Na+>Li+. The reaction is endothermic and entropy driven. Monovalent cations are displaced by other monovalent cations. Divalent cations and some drugs (e. g., cocaine) displace monovalent cations more strongly. (3) Divalent cations bind to membranes (and to lecithin micelles) at four distinct sites, having apparent association constants between 50 and 0.2mm –1. The characteristics of the titration suggest that only one species of binding site is present at any one time, and open the possibility that structural transitions of the unassociated coordination sites may be induced by divalent cation binding. Divalent cation binding at the weakest site (like monovalent cation binding) is endothermic and entropy driven. At the next stronger site, the reaction is exothermic. Monovalent cations affect divalent cation binding by reducing the activity coefficient: they do not appear to displace divalent cations from their binding sites.  相似文献   

12.
G Lutze  W Liese 《FEBS letters》1974,42(1):54-56
Intact mitochondria are able to bind monovalent and divalent metal cations and to release protons in an energy-independent exchange process. Directly accessible binding sites exist in the outer membrane. They seem to be identical for monovalent and divalent metal ions. The inner membrane-matrix-fraction possesses exchange sites after ultrasonic disruption only for monovalent cations, but not for divalent cations.  相似文献   

13.
The nature of binding of metal cations to the glomerular basement membrane has been investigated using isolated bovine glomerular basement membrane. Highest-affinity binding for a number of ions is attributable to the glycosaminoglycans (mostly heparan sulfate) of the membrane. Some ions, such as divalent Mn, Ca and Ni, have specific binding sites on these polymers, while for others the ion-polyelectrolyte interaction is of a non-specific nature. Both structural and binding data indicate a linear charge density of close to unity for the heparan sulfate of the glomerular basement membrane, which at the ionic composition of the plasma filtrate corresponds to a polymer surface potential of about -45 mV. Several independent observations are better explained by a model of counter-ion condensation about the glycosaminoglycans than by conventional double layer theories. These include the valence dependence of ion binding, the sharp ejection of divalent ions at a critical concentration of La3+, and the relative insensitivity of 63Ni2+ binding to NaCl concentration in the neighbourhood of physiological ionic strength. In its interactions with metal ions, the glomerular basement membrane behaves like a dilute solution of polyelectrolytes. This conclusion has important consequences for the extent of charge reduction of the filtration barrier of the kidney, bathed as it is in an electrolyte solution of mainly monovalent salts.  相似文献   

14.
Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning alpha-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of "the cation" in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca2+, Mg2+ and Mn2+) and monovalent metal cations (Li+, Na+, K+ and Cs+), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation相似文献   

15.
Chlorotetracycline (CTC) shows a strongly enhanced fluorescence upon addition of mitochondria isolated from Jerusalem artichoke ( Helianthus tuberosus L.) tubers in a low-cation medium. This indicates the presence of membrane-bound divalent cations. The chelation by CTC of the membrane-bound divalent cations does not affect the oxidation of exogenous NADH significantly. The removal of the bound divalent cations using ethyleneglycol-bis-(β-aminoethylether)-N,N'-tetraacetic acid (EGTA) and EDTA causes an 80% decrease in CTC fluorescence. Titration of CTC fluorescence (a direct measure of bound divalent cations) and 9-aminoacridine fluorescence (a measure of surface potential) with EGTA and EDTA gives similar curves, although CTC fluorescence responds more slowly to the addition of chelators. The same bound divalent cations appear to be monitored by CTC fluorescence or by 9-aminoacridine fluorescence.  相似文献   

16.
《BBA》1986,851(2):322-326
We have used trivalent lanthanide metal cations in the buffering media of pea chloroplasts to probe the stacking arrangement of thylakoid membranes and the spatial distribution of chlorophyll-protein complexes of Photosystems I and II. Measurements of steady-state chlorophyll fluorescence emission spectra of pea chloroplasts at room temperature demonstrate that, within this tripositive valency group, the extent of membrane appression is a function of hydrated metal ionic radius. These results are in agreement with a recent investigation using monovalent and divalent metal cations (Karukstis, K.K. and Sauer, K. (1985) Biochim. Biophys. Acta 806, 374–389). In addition, the lanthanide cation concentration effective in producing the maximum chlorophyll fluorescence intensity upon grana formation is dependent on hydrated ionic size. The current investigation supports the proposed hypothesis that cation screening ability defines the extent of intermembrane separation as well as the extent of lateral distribution of chlorophyll-protein complexes.  相似文献   

17.
Shigeru Itoh 《BBA》1979,548(3):579-595
Salt- or pH-induced change of the rate of reduction of the phtooxidized membrane bound electron transfer components, P-700, by ionic and nonionic reductants added in the outer medium was studied in sonicated chloroplasts.

The rate with the negatively charged reductants increased with the increase of salt concentration at a neutral pH or with the decrease of medium pH. Salts of divalent cations were much more effective than those of monovalent cations. A trivalent cation was even more effective. The rate with a nonionic reductant was little affected by salts.

The change of the reduction rate was analyzed using the Gouy-Chapman theory, which explains the change of reduction rate by the changes of activities of ionic reductants at the charged membrane surface where the reaction takes place. This analysis gave more useful parameters and explained more satisfactorily the case with high-valence cation salts than the Brönsted type analysis. The values for the surface charge density and the surface potential of the membrane surface in the vicinity of P-700 estimated from the analysis were lower than those estimated for the surface in the vicinity of Photosystem II primary acceptor, suggesting the heterogeneity of the thylakoid surface.

The salt-induced surface potential change was shown to affect the activation energy of the reaction between P-700 and the ionic reagent.  相似文献   


18.
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   

19.
A. Telfer  J. Barber  A.T. Jagendorf 《BBA》1980,591(2):331-345
1. Increase in electron transport rate and the decay rate of the 518 nm absorption change, induced by EDTA treatment, is prevented by cations. The order of effectiveness is C3+ > C2+ > C+.2. In this respect methyl viologen is an effective divalent cation in addition to its action as an electron acceptor.3. Complete cation irreversible EDTA-induced uncoupling occurs in the dark in 2 min. Light greatly stimulates the rate of uncoupling by EDTA. It is concluded that the uncoupling is due to release of coupling factor I from the thylakoid membrane.4. Binding of purified coupling factor I to coupling factor I-depleted thylakoids can be achieved with any cation. The order of effectiveness is C3+ > C2+ > C+, reconstituted thylakoids are active in photophosphorylation regardless of the cation used for coupling factor I binding.5. The marked difference in the concentration requirements for cation effects on 9-aminoacridine fluorescence yield and for prevention of uncoupling by EDTA indicate that coupling factor I and its binding site have a lower surface charge density than the net surface charge density of the thylakoid membrane.6. It is concluded that coupling factor I binding only occurs when negative charges on coupling factor I and its binding site are electrostatically screened by cations.7. Previously reported examples of uncoupling by low ionic conditions are discussed in relation to the basic concepts of diffuse electrical layer theory.  相似文献   

20.
Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only approximately 10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587-595; Curtis & Bartel, RNA, 2001, this issue, pp. 546-552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.  相似文献   

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