Characteristics of the Quenching of 9-Aminoacridine Fluorescence by Liposomes Made from Plant Lipids

Several laboratories have determined the surface charge density of membranes utilizing methods based on vesicle-induced quenching of the fluorescence of 9-aminoacridine and its relief by other cations. However, the computational methods by which surface charge density were calculated have not been verified in a model system. In this study, the quenching of 9-aminoacridine fluorescence by liposomes made from varying amounts of digalactosyldiacylglyceride and phosphatidic acid and relief of quenching by salts was examined. Quenching of 9-aminoacridine fluorescence increased with increasing amounts of phosphatidic acid added, independent of the composition of the added liposomes. In certain instances, the computational methods did not yield the surface charge density of the liposomes expected from their composition. However, when the effects of background ionic strength on surface potential were considered, there was a positive correlation between expected and calculated values. Therefore, the data support the contention that changes in the fluorescence of 9-aminoacridine can be used to calculate surface charge density of membranes. Received: 29 November 1999/Revised: 31 July 2000     

The involvement of the electrical double layer in the quenching of 9-aminoacridine fluorescence by negatively charged surfaces

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.     

The involvement of the electrical double layer in the quenching of 9-aminoacridine fluorescence by negatively charged surfaces.

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.     

9-Aminoacridine as a fluorescent probe of the electrical diffuse layer associated with the membranes of plant mitochondria.

1. Mitochondria from Jerusalem artichoke (Helianthus tuberosus) tubers and Arum maculatum spadices caused a quenching of the fluorescence of 9-aminoacridine when mixed in a low-cation medium (approximately 1 mM-K+) and addition of chelators further decreased the fluorescence. Salts released the quenching of the 9-aminoacridine fluorescence and the efficiency of the release appeared to be mainly dependent on the valency of the cation (C3+ greater than C2+ greater than C+). 2. The results are consistent with the theory of charge screening and demonstrate that 9-aminoacridine is a convenient probe of the behaviour of cations on the membranes of mitochondria and in the diffuse layer associated with these membranes. 3. The concentration of salt required to achieve half-maximal release of quenching of 9-aminoacridine fluorescence was proportional to the concentration of mitochondria in the solution and theoretical considerations show this effect to be inherent in the Gouy-Chapman theory. 4. 9-Aminoacridine was removed from the bulk of the solution by the mitochondria to a far greater extent than was Na+ or K+, which is suggested to be due to the formation of bi- and poly-valent cations by aggregation of 9-aminoacridine molecules in the diffuse layer. This would have implications for the use of 9-aminoacridine to determine delta pH across membranes. 5. Jerusalem-artichoke mitochondria removed from 9-aminoacridine and Ca2+ from the bulk of the solution and required more ions to screen the membranes than did an equal concentration (mg of protein/ml) of Arum mitochondria, indicating that Jerusalem-artichoke mitochondria contain more negative charges per mg of protein.     

9-Aminoacridine fluorescence changes as a measure of surface charge density of the thylakoid membrane

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).     

Effects of trypsin and cations on chloroplast membranes

A mild tryptic digestion of chloroplast membranes eliminates the effects of saturating concentrations of cations (3 to 5 millimolar MgCl₂) on chlorophyll fluorescence yield, membrane stacking, and photosystem II photochemical efficiency in spinach. At the same time, the negative surface potential of the membranes is increased (by trypsin) as revealed by studies with 9-aminoacridine. High concentrations of cations (25 to 100 millimolar MgCl₂) added after trypsin digestion are effective in restoring high fluorescence yields and membrane stacking. High concentrations of cations added after trypsin treatment do not increase the photosystem II efficiency. It is concluded that the “diffuse electrical layer” hypothesis of Barber et al. (Barber J, J Mills, A Love, 1977 FEBS Lett 74: 174-181) satisfactorily explains the effect of trypsin in eliminating the influence of saturating concentrations of cations on chlorophyll fluorescence yield and membrane stacking. However, the effect on photosystem II photochemical efficiency seems to require another mechanism.     

A specific role for Ca2+ in the oxidation of exogenous NADH by Jerusalem-artichoke (Helianthus tuberosus) mitochondria.

1. The addition of chelators to a suspension of mitochondria in a low-cation medium containing 9-aminoacridine caused a decrease in 9-aminoacridine fluorescence. The chelators removed bivalent cations from the membranes and allowed more 9-aminoacridine to move into the diffuse layer. The relative effect of EGTA and EDTA on the fluorescence suggested that the mitochondria are isolated with about equal amounts of Ca2+ and Mg2+ on the membranes. 2. The removal of the bivalent ions by chelators resulted in the inhibition of NADH oxidation. The inhibition could not be removed by adding sufficient decamethylenebistrimethylammonium ion (DM2+) to screen the fixed charges on the membranes and restore the fluorescence of 9-aminoacridine. This observation suggests that bivalent metal ions have a specific role in the oxidation of NADH. 3. Ca2+ and not Mg2+ reversed the inhibition of NADH oxidation caused by EGTA, whereas both reversed the inhibition caused by EDTA. This suggests that Ca2+ plays a specific role and that Mg2+ reverses the inhibition caused by EDTA by displacing the bound calcium from the chelator. 4. The results are interpreted as showing that Ca2+ plays a specific role in the oxidation of external NADH in addition to its ability to screen electrostatically or bind to the fixed charges associated with the surface of the membrane.     

High-temperature damage and acclimation of the photosynthetic apparatus

The influence of mono- (K⁺) and divalent (Mg²⁺) cations and protons (pH) on the temperature sensitivity of thylakoid membranes was investigated in three groups of young bean plants (control, heat-acclimated and non-acclimated). Thylakoid-membrane function was monitored by second and millisecond delayed fluorescence and 9-aminoacridine fluorescence quenching. It was established that metal ions at investigated concentrations decreased the thermostability of the photosynthetic parameters — an increase of MgSO₄ concentration from 0.1 to 20 mM decreased the temperature of their half-inactivation (T50) by 13°C. At the same time the pH dependence of the thermal stability of these parameters showed a maximum at pH 5.5–6.5. The half-inactivation temperatures of those photosynthetic parameters connected with the ability of the thylakoid membrane to form light-induced proton gradients increased by 6–7°C in the heat-acclimated plants compared with the control. It was assumed that the temperature inactivation of photosynthetic electron transfer and the energization of the thylakoid membrane was determined both by the thermoinduced dissociation of the light-harvesting chlorophyll a/b protein complex from PSII, leading to destruction of the excitation energy transfer to the reaction centres, and by the thermal denaturation of the membrane-protein components. The rate of these processes was probably controlled by the size of the negative surface charge and the viscosity of the thylakoid membrane.Abbreviations 9-AA 9-aminoacridine - DF delayed fluorescence - LHCP light-harvesting chlorophyll a/b protein complex - PSI (II) photosystem I (II) - T50 temperature of 50% inhibition of photosynthetic parameter - Tricine N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl] glycine     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack. These observations, in addition to a slight increase of charge density of the surface—as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies—and partial unstacking of the membranes—as monitored by digitonin method and 540 nm light scattering changes—after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.     

Monitoring of membrane-bound divalent cations in plant mitochondria using chlorotetracycline fluorescence

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.     

Effect of polyelectrolytes on serine proteinase secretion by Bacillus subtilis

Addition of polycations with molecular masses of 5-40 kDa as well as Na+, stimulated serine proteinase secretion by Bacillus subtilis cells. Polyanions and higher-molecular-mass polycations (100-200 kDa) were inefficient. The enzyme yields in the presence of polycations or Na+ were equal in magnitude. The results indicate that the cations, apparently counteracting the negative surface charge of the bacterial plasma membrane, cause the desorption of the serine (alkaline) proteinase. The synthesis of the proteinase is inferred to be stopped as the enzyme is bound to the outer surface of the plasma membrane. The desorption of the enzyme thus induces the synthesis of the new portions of proteinase.     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack.These observations, in addition to a slight increase of charge density of the surface-as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies-and partial unstacking of the membranes-as monitored by digitonin method and 540 nm light scattering changes-after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.Abbreviations ATP adenosine triphosphate - 9-AA 9-aminoacridine - Chl chlorophyll - EDTA ethylenediaminetetraacetate - GDA glutaraldehyde - Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - LHC light-harvesting chlorophyll a/b complex PS photosystem     

嗜碱性芽孢杆菌PB92碱性蛋白酶分别在大肠杆菌和枯草芽孢杆菌中表达

从Bacillus alcalophillus PB92中扩增出碱性蛋白酶基因Mapr,Mapr分别插入到大肠杆菌载体pET-22b( )和枯草芽孢杆菌载体pWB980中构建成重组分泌型表达载体pET22b( )-Mapr、pWB980-Mapr。碱性蛋白酶基因分别在大肠杆菌宿主BL21和枯草芽孢杆菌DB104中得到表达。SDS-PAGE分析,重组蛋白酶的分子量为28kD。在大肠杆菌,所得酶活为231U/ml,而在枯草芽孢杆菌,其酶活为1563U/ml。大概是由于碱性蛋白酶在枯草芽孢杆菌折叠成熟机制与大肠杆菌的不同造成的。     

The role of inhibitors in the fluorescent staining of benign naevus and malignant melanoma cells with 9-amino acridine and acridine orange

Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells.     

The Role of Inhibitors in the Fluorescent Staining of Benign Naevus and Malignant Melanoma Cells with 9-Amino Acridine and Acridine Orange

Abstract

Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells.     

9-Amino-acridine as a probe of the electrical double layer associated with the chloroplast thylakoid membranes

Chloroplasts washed with monovalent cations are found to quench 9-amino-acridine fluorescence after resuspension in a cation-free medium. This quenching occurs in the absence of a high energy state and can be reversed by the addition of salts. The effectiveness of these salts is related to the charge carried by the cations and appears to be essentially independent of the associated anions. The order of effectiveness is polyvalent > divalent > monovalent, and virtually no variation is found within the groups of monovalent cations and divalent cations tested. Furthermore, choline and lysine are as effective as alkali metal cations, and lysyl-lysine is almost as effective as alkaline earth metal cations. These results are consistent with an effect mediated by the electrical double layer at the membrane surface rather than chemical bonding, and can be qualitatively explained in terms of the Gouy-Chapman theory.It appears that 9-amino-acridine acts as a diffusible monovalent cation which increases its fluorescence when displaced from the diffuse layer adjacent to the negatively charged membrane surface. The 9-amino-acridine fluorescence changes have been experimentally correlated with the cation-induced chlorophyll a fluorescence changes also observed with isolated chloroplasts.     

9-Aminoacridine- and tetraethylammonium-induced reduction of the potassium permeability in pancreatic B-cells. Effects on insulin release and electrical properties.

The effects of 9-aminoacridine and tetraethylammonium on insulin release and rubidium efflux from perifused rat islets were investigated and correlated with their effects on the electrical properties of mouse B cells studied with microelectrode techniques. 9-Aminoacridine (0.05--1 mmol/l) and tetraethylammonium (2--40 mmol/l) produced a dose-dependent, reversible potentiation of glucose-stimulated insulin release. This effect was rapid, affected both phases of secretion and was maximum in the presence of 6 mmol/l glucose, but no longer significant at 20 mmol/l glucose. It was unaltered by atropine or propanolol, and abolished by mannoheptulose or omission of extracellular calcium. 9-Aminoacridine, but not tetraethylammonium, also induced insulin release in the absence of glucose stimulation. Neither drug modified glucose metabolism in islet cells and only 9-aminoacridine increased 45Ca2+ uptake. In the presence of 0, 3 or 6 mmol/l glucose, but no longer at 20 mmol/l glucose, 9-aminoacridine and tetraethylammonium reduced the rate of 86Rb+ efflux from the islets. Both drugs also slightly reduced 86Rb+ uptake by islet cells. In the presence of 11 mmol/l glucose, 9-aminoacridine reduced the amplitude and the duration of the polarization phases between the bursts of electrical activity; concomitantly these periods of spike activity were markedly prolonged. At lower glucose concentrations (3 or 7 mmol/l), 9-aminoacridine progressively depolarized B cells and induced electrical activity in otherwise silent cells. Tetraethylammonium also suppressed the repolarization phases between the bursts of spikes in the presence of a stimulating concentration of glucose. At low glucose, tetraethylammonium produced only a limited and not maintained depolarization. These results show that a reduction of the potassium permeability in pancreatic B cells potentiates the insulin-releasing effect of glucose and may even stimulate secretion. They also suggest that the initial depolarizing effect of glucose is due to a reduction of the potassium permeability, whereas the repolarization at the end of each burst of electrical activity is mediated, at least in part, by an increase in the potassium permeability of B cells.     

Effects of cations and polyamines on the aggregation and fusion of phosphatidylserine membranes

Effects of various metal cations and polyamines on aggregation and fusion of phosphatidylserine vesicles and their associated physicochemical properties (such as surface tension and vesicle electrophoretic mobility) have been studied. It was found that metal polycations and hydrogen ion caused an increase in the surface tension of a phosphatidylserine monolayer, whereas the polyamines and other monovalent cations did not increase the surface tension of the membrane appreciably. All cations used affected the vesicle mobility roughly in the order of the number of their valencies and linearly with respect to the logarithm of their concentrations of ions; vesicle surface charge densities are reduced by adsorption and screening of the counter ions depending on their valencies and concentrations. The degree of aggregation of lipid vesicles parallels somewhat that of the reduction of vesicle electrophoretic mobilities. However, the degree of membrane fusion induced by these cations parallels that of the increase in surface tension of the membranes induced by these cations.     

Inhibition of a cell surface protease after cisplatin chemotherapy.

The epithelial cells in squamous carcinoma and leukoplakia of the oral cavity possess the cell surface protease, guanidinobenzoatase (GB), in an active form. GB is closely similar to plasminogen activator, a protease associated with both transformed cells and tumour cells. The active centre of GB binds the fluorescent probe 9-aminoacridine (9-AA) enabling cells containing active GB to be visualised by fluorescent microscopy. It was observed that chemotherapy with cisplatin resulted in a marked decrease in cell surface GB activity and this decrease was due to the formation of an enzyme-inhibitor complex. One of the results of chemotherapy was shown to be the suppression of a cell surface protease which is known to be associated with migration and malignancy of cells in vivo.